Functional and morphological aspects of hypothalamic neurons.

PHYSIOLOGICAL

Vol.

REVIEWS

57, No. 3, July Printed in U.S.A.

1977

Functional and Morphological Aspects of Hypothalamic Neurons JAMES Departments

of Neurology of North

N.

HAYWARD

and Medicine and the Neurobiology Program, Carolina, Chapel Hill, North Carolina

.................................................... I. General Introduction A. Historical ........................................................... B. Identification of hypothalamic neurons ................................ ............................................. C. Hypothalamic pathways. D. Autonomic aspects of hypothalamic function ........................... ............. E. Electrophysiological strategies for analysis of unit activity F. Summary ........................................................... II. Magnocellular Neuroendocrine Cells ..................................... A. Introduction ......................................................... ...................................................... B. Neurosecretion .................................. C. Electrical membrane characteristics D. Morphological and functional cell types ............................... ..................................................... E. Osmosensitivity F. Blood volume and vasopressin release ................................. ........................................... G. Neural input and behavior ..................... H. Pharmacological actions and putative transmitters I. Summary ........................................................... III. Parvocellular Neuroendocrine Cells ...................................... A. Introduction ......................................................... B. Antidromically identified single hypothalamic neurons ................. .................... C. Identified and nonidentified regulator interneurons D. Summary ........................................................... .................................... IV. Nonendocrine Hypothalamic Neurons ............................ A. Neurons associated with thermoregulation B. Neurons associated with feeding ...................................... C. Neurons associated with cardiovascular function ....................... .............................. D. Behavior and hypothalamic unit activity .................................................. V. General Considerations A. Classification of hypothalamic neurons ................................ .................................... B. Peptidergic hypothalamic neurons

I. GENERAL

University

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INTRODUCTION

A . His torical On the basis of a wide variety of clinical and pathological studies, late nineteenth century scientists had reason to suspect that the hypothalamus was the coordinating center for various patterns of endocrine, behavioral, and autonomic adjustments. This concept had been consolidated into accepted 574

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dogma by the crescendo of basic and clinical research investigations on the hypothalamic neurons in the early years of the next century. By 1939 (1.34)) the hypothalamus and the preoptic regions, usually considered functional and anatomical continua, were known as areas important for controlling pituitary gland secretion, thermoregulation, feeding and drinking, and cardiovascular activity and for determining the behavioral state of the organism. Using light microscopy, anatomists recognized the major nuclear groupsnucleus supraopticus, nucleus paraventricularis, ventromedial nucleus, and others. They saw the rich network of ascending and descending fibers linking hypothalamic neurons to the adjoining forebrain areas, limbic system, midbrain, thalamus, and the pituitary gland (see Fig. 1). Summarizing their studies of neurosecretion in fish and mammalian magnocellular neurons, Scharrer and Scharrer (361) concluded that the intracytoplasmic granules they found in these hypothalamic neurons indicated glandular function. Applying the Gomori staining tech niques to these neurons, Bargmann (12) discovered stainable material that, under Palay’s electron microscope (325)) proved to be membrane-bound 150-nM secretory granules or vesicles. Cross and Green (67) approached the study of these magnocellular neurons electrophysiologically, demonstrating that cells in the vicinity of the supraoptic nucleus exhibi ted extracellularly measured spi ke potentials identical to those found in other central nervous sy stem neurons and responsi ve to osmotic and sensory stimuli. Refining this electrical approach further, Kandel (205) recorded electrical membrane potentials in the magnocellular neuroendocrine cells of the goldfish preoptic nucleus. He demonstrated that electrical stimulation of the pituitary stalk yielded antidromically conducted potentials. Hayward (162), extending this approach, stained the goldfish neuroendocrine cells intracellularly with Procion yellow, and Yagi et al. (441) demonstrated antidromically conducted potentials in the rat. Fluorescence histochemistry by Fuxe and Hokfelt (135) revealed fibers of the noradrenergic, dopaminergic, and serotonergic type arising in brainstem nuclei, the locus coeruleus, substanti a nigra, and raphe nuclei, respectively. These fibers then passed rostrally along the m .edial forebrain bundle to the hypothalamus (see Fig ). 1) . Recently, the immunohistochemical studies of several workers (451) have demonstrated the full ramification of vasopressin-, oxytocin-, and neurophysin-containing cell bodies and processes in the magnocellular neuroendocrine cell nuclei and pathways. Lying in the arcuate nucleus, ventromedial nucleus, suprachiasmatic nucleus, and elsewhere in the medial basal hypothalamus, parvocellular neuroendocrine cells are equally responsive to immunocytochemical techniques. Aided by the development of Palkovits and coworkers of the punch-biopsy method (48 9 194)) detailed chemical analysi .s of the regional distribution of monoamines, synthetic enzymes, and hypothalamic hormones is currently available. B. Identification

of Hypothalamic

Neurons

The broad range of activities vital to self-preservation and propagation the species required of the hypothalamic neurons demands a complex

of cir-


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MIDbRAIN NE DA 5Hj PERiAQ ACH I GREY FIG. 1. Hypothalamic regions and their connections. Cross-hatched region indicates magnocellular neuroendocrine cells, their unmyelinated axons passing through the internal zone of the median eminence and the supraopticohypophyseal tract to the terminal endings in the posterior pituitary gland. Solid black region indicates parvocellular neuroendocrine cells. Their unmyelinated fibers pass to the external zone of the median eminence, where hypophyseotrophic hormones are released and carried to the anterior pituitary via the portal vessels. Chemically specific midbrain neurons (NE, noradrenergic; DA, dopaminergic; 5-HT, serotonergic; ACH, cholinergic) send fibers rostrally along the medial forebrain bundle to the lateral hypothalamic area, lateral preoptic area (LPO), and amygdala, septum, and other regions of the limbic system. A descending olfactory-limbic system of fibers passes along the medial forebrain bundle to interact with hypothalamic and midbrain neurons. A medial system of ascending and descending fibers arises in the periaqueductal grey matter of the midbrain and courses along the periventricular system of the medial hypothalamus and medial preoptic area, with descending components arising in these regions. The third ventricle (III) is associated with the circumventricular organs: the organum vasculosum of the lamina terminalis, the subfornical organ, the median eminence, and the neurohypophysis. Abbreviations: AP, anterior pituitary gland; DA, dopaminergic neurons; LHA, lateral hypothalamus; LPO, lateral preoptic area; ME, median eminence; MH, medial hypothalamus; MPO, medial preoptic area; MgC, magnocellular neuroendocrine cells; MFB, medial forebrain bundle; NE, noradrenergic neurons; OVLT, organum vasculosum of the lamina terminalis; PORTAL, primary plexus of the portal vessels; PERIAQ. GREY, periaqueductal grey matter; PP, posterior pituitary gland; PV, periventricular fiber system; PVC, parvocellular neuroendocrine cells; SFO, subfornical organ; SOHT, supraopticohypophyseal tract; III, third ventricle; 5-HT, serotonergic neurons. (Based on references cited in sect. I.)

cuitry. The integrative action of hypothalamic neuronal circuits coordinates autonomic, endocrine, and behavioral patterns for the regulation of anterior and posterior pituitary glandular secretions, thermoregulation, feeding and drinking, cardiovascular function, and certain types of motivation and learn-


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ing. Over the years, investigative techniques have leaped ahead to develop and refine our knowledge of individual neurons and the neuronal pathways that integrate cell activity. The continuing importance of light (134, 207, 239, 242, 338, 395) and electron-microscopic (143,344) studies, as well as those that emphasize specialized staining and marking procedures (30, 135, 266, 337, 375, 4511, has been demonstrated. The techniques involving terminal bouton degeneration (127, 241, 449) and injection of marker proteins into pituitary (372), retina (269), and hypothalamus (373, 394) have been used successfully to elucidate the connectivity of hypothalamic endocrine and nonendocrine neurons. Electrical or chemical stimuli elicit neuroendocrine, thermoregularesponses from vario us hypotha .I.amic tory, feeding, drinking, and behavioral sites. Osmotic or thermal stimuli applied to local areas can bring forth specialized osmoreceptor or thermoreceptor activity. The electrophysiological techniques of the electroencephalogram 1EEG (359)], evoked potentials (28, 71), slow-wave potentials (28, 71), and unit activity (28, 61, 62, 71) have been used for studying single and multiple hypothalamic neurons and their connections. The field of hypothalamic research is now highly active, as shown by the large number of reviews of some of the traditional research approaches (47, 128, 134, 135, 158, 207, 223, 224, 238, 239, 242, 337, 365, 375). Since this review stresses single- and multiple-unit activity, the important primary papers are reviewed. Certain specialized reviews on neuroendocrine control (7,26,28,61, 62, 65, 66, 71, 100, 128, 135, 159, 160, 163, 184, 202, 223, 224, 237, 238, 272, 273, 352, 355, 378, 387, 402, 404,424, 445, 451), thermoregulation (34, 112, 113, 150, 161, 164, 179, 180, 182, 183,253, 365), feeding and drinking (119, 271, 303), the limbic system (116, 159), circumventricular organs (432)) neural cardiovascular control (233, 297,371), and neuropharmacology (182,235, 253,365) also are cited. Anatomical studies that highlight recent information about the chemical nature of hypothalamic neurons and the pathways that ascend and descend in this area (30,48,135,266,338,375,395,451) are important sources. This review is a selective commentary on studies of structure and function of single hypothalamic neurons that provide evidence for specialized activities in autonomic, endocrine, or behavioral areas. The emphasis is on the integration of these activities with each other and with the overall function of the organism. In order to deal with the large body of material that has been available since Cross and Green (67) initiated this area of research in 1959, it is both necessary and convenient to divide the subject of hypothalamic neurons into traditional, if artificial, sections -for example, “endocrine” and “nonendocrine.” Although a single “endocrine” neuron may be activated in a variety of “nonendocrine” situations, its primary role remains “endocrine.” Therefore the emphasis here is on the great value of electrophysiological studies of single cells that are strongly supported by pertinent anatomical and chemical data. This review should present to the interested reader an appreciation of how specialized the activities of the hypothalamic neurons are and how necessarily integrative their function is within the central nervous system.


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C. Hypothalamik

N. HAYWARD

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Pathways

Hypothalamic neurons lie among neural, cerebrospinal, and blood-borne pathways where they receive synaptic input and direct stimuli (see Fig. 1). 1. Neural

pathways

The ascending neural pathways include the monoaminergic (30, 135, 375), cholinergic (375), and nonchemically defined components of the medial forebrain bundle and the periventricular system of Schutz (see Fig. 1). The punch technique of Palkovits and co-workers determined the discrete local levels of the various monoamines and acetylcholine in the regions of the hypothalamus (48). The descending neural pathways derive from olfactory and limbic structures (septum, hippocampus, and amygdala) and arrive at the hypothalamus and preoptic area over the medial forebrain bundle, the fornix, and associated pathways (see Fig. 1). A rich and poorly understood intrahypothalamic pathway exists for interconnections among various neurons (241, 266, 395, 449) via axon collaterals (162, 346-348) and axodendritic interplay (241, 395; see Fig. 1). 2. Cerebrospinal

fluid

The neurons of the hypothalamus connect to the various circumventricular organs, such as the organum vasculosum of the lamina terminalis, the subfornical organ, the median eminence, and others (431; see Fig. 1). The ependyma of the third ventricle is specialized into tanycytes that have low cuboidal, nonciliated ventricular surfaces and long, extensive subependymal end-feet connected to the zona externa capillaries of the primary portal plexus of the median eminence (223, 43 1). Luteinizing-hormone-releasing hormone, vasopressin, and neurophysin, secretory products of neuroendocrine cells, are elevated in the cerebrospinal fluid. Knigge and Silverman (223) suggested that cerebrospinal fluid substances may influence hypothalamic neuronal and pituitary function. kndersson (6) suggested that the cerebrospinal fluid level of sodium ions was detected by a periventricular sensor and information transmitted to magnocellular neuroendocrine cells for release of vasopressin and to lateral hypothalamic neurons for initiation of drinking. 3. Blood-borne

stimuli

The blood-borne levels of sodium, water, steroid and peptide hormones, glucose, oxygen, carbon dioxide, and temperature play an important role in the feedback regulation of hypothalamic neurons. Regulating excitation or inhibition of magnocellular peptidergic neurons, the osmotic pressure of the carotid blood is a prime factor for the posterior pituitary release of vasopres-


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sin. In the classical feedback loop the level of cortisol in the carotid blood determines the level of cortisol in the hypothalamus. This level of cortisol determines the release of corticotrophin-releasing factor into the primary plexus of the portal vessels. Corticotrophin-releasing factor in turn percolates down the stalk to the adenohypophysis and determines the rate of release of adrenocorticotrophic hormone from the anterior pituitary cells. The adrenocorticotrophin levels in systemic blood determine the release of cortisol from the adrenal cortex. These various osmotic, steroid, and other blood-borne chemicals must cross the blood-brain barrier or stimulate the circumventricular organs in order to act on the appropriate hypothalamic neurons (202,237). Temperature of the arterial blood circulating through the preoptic-anterior hypothalamus determines the temperature of the brain (161). Neurons in the preoptic-anterior hypothalamus are selectivelv sensitive to changes in local temperature and provide a monitor of arterialblood temperature. When central integration for thermoregulation occurs in the preoptic-anterior hypothalamus, the input is from cutaneous and other deep body and brain thermoreceptors and the output is vasomotor, shivering, respiratory, and behavioral responses. Glucose levels in the arterial blood circulate through the ventromedial nucleus of the hypothalamus to provide a stimulus for the postulated glucoreceptors (303) that may be involved in feeding and satiety. D. Autonomic

Aspects

1. Endocrine

effecters

of Hypothalamic

Function

Action potentials of magnocellular neuroendocrine cells are transmitted from cell somata in the hypothalamus to the terminals in the posterior pituitary, where the influx of calcium into the nerve endings leads to exocytosis, namely, the extrusion of neurosecretory vesicle contents. Vasopressin, oxytocin, and neurophysin flow from the unmyelinated axons to the extracellular space of the posterior pituitary gland and thence into the systemic circulation (80, 238; see sect. II and Table 1). A similar process presumably occurs in parvocellular neuroendocrine cells with release of hypophyseotrophic hormones. at nerve terminals in the zona externa of the median eminence at the primary plexus of the portal capillaries. Releasing and inhibiting hormones (luteinizing-hormone-releasing hormone, thyrotropinreleasing hormone, somatostatin, corticotrophin-releasing factor, prolactininhibiting factor, growth-hormone-releasing factor; see Table 1) percolate down the portal vessels to the secondary plexus in the adenohypophysis. Here action on these pituitary cells results in synthesis and release of anterior pituitary trophic hormones (adrenocorticotrophin, luteinizing hormone, follicle-stimulating hormone, thyrotrophin, growth hormone, and prolactin) into the systemic circulation (see Table 1 and sect. III).


JAMES

580 TABLE

1. Hypothalamic

Hormone

Chemical

I. Neurohypophyseal Arginine (lysine)

N. HAYWARD

peptidergic structure

hormones Nonapeptide

vasopressin

Q&me

neurons

Cell

Body

Magnocell

Location

ular

:

supraoptic, paraventricular nuclei, internuclear Parvocellular:

Projection

Median

Target

Pathways

eminence,

Nonapeptide

Magnorell ular supraoptic, paraventricular nuclei, internuclear

II. Hypophyseotrophic Thyrotropinreleasing hormone

hormones Tripeptide

Median neural

Periventricular in medial anterior hypothalamus, suprachiasmatic and arcuate

Somatostatin

(SRIF)

Tetradecapeptide

Periventricular in median anterior hypothalamus

area preoptic,

of

breast

ejection; smooth muscle of uterus and blood vessels; ? neuromodulator

organs, in brain Decapeptide

Myoepithelial tissue-milk

Median eminence, circumventricular

(TRH)

Luteinizinghormone-releasing hormone (LHRH)

eminence, lobe

zone

Unknown

and

smooth muscle gut and blood vessels; ? neuromodulator

zone.

:

Organ Action

Renal tubule and collecting ductwater balance;

neural lobe, amygdala, thalamus

suprachiasmatic nucleus Oxytocin

57

Anterior release

widespread

Median eminence, circumventricular organs: neural

pituitary:

thyrotrophin prolactin

lobe

Anterior pituitary: release luteinizing and folliclestimulating hormones

nuclei area preoptic

Median eminence, circumventricular organs, ventromedian,

Anterior

pituitary: inhibit release growth hormone and thy rotrophin

of

arcuate. and premammillary nuclei lobe

2. Nonendocrine

and

neural

effecters

a) Thermoregulation. Maintenance of body temperature depends on skin and brain thermal input. The resultant autonomic signals discharge over sympathetic and motor pathways for cutaneous vasomotor, sweat gland, respiratory heat loss (panting), and shivering changes. An appropriate behavioral repertoire facilitates heat gain or heat loss for regulation of body temperature (see sect. IvA). b) Feeding. Maintenance of body weight depends on the metering of energy output and food intake with the hypothalamus as one of the sensors of blood levels of glucose, fatty acids, and food intake. When an imbalance between intake and output occurs, the resultant autonomic effecters summate in the search for and the ingestion of food (see sect. IvB). c) CardiovascuZar regulation. Maintenance of arterial blood pressure, blood volume, and peripheral vasomotor tone during changes in behavior depends in part on vascular, afferent, pressor-sensitive stimuli and central


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nervous receptors for detection of pressure changes. The resultant autonomic signals - discharged over sympathetic, parasympathetic, and endocrine pathways -regulate heart rate, cardiac output, peripheral vasomotor tone, and salt and water retention. The appropriate regulation of blood pressure, blood volume, and peripheral blood flow follows (see sect. IvC). E. Electrophysiological 1. Technical a)

Single-Unit

Strategies

for Analysis

of Unit Activity

approaches activity.

I. EXTRACELLULAR

RECORDING.

As

a lOgiCa

eXten-

sion of their studies elsewhere in the nervous system, Cross and Green (67) studied the single cell in the hypothalamus of acutely prepared, urethananesthetized rabbits by using an electrolytically sharpened steel dental broach with a tip diameter of 1 pm. Recorded potentials originated primarily in the cell soma. When marked with electrolytically deposited iron stained with Prussian blue, these cells were shown to be in and around the supraoptic nucleus. Despite the traumatic effects of acute surgical operation and the depressant effects of general anesthesia, this technique, modified to either metal or glass electrodes, has been used to study a wide variety of nonantidromically identified hypothalamic neurons purported to be involved in endocrine and nonendocrine functions. Recently Cross (62) suggested that this technique of study of nonantidromically identified hypothalamic neurons might have reached its limit of productivity. The method, nevertheless, remains a sound approach to the study of single hypothalamic neurons, having been recently extended to include cultured preoptic (188, 228) and tuberal (137, 138) hypothalamic neurons and deafferented hypothalamic islands (63, 64, 68, 388). When Cross and Green (67) tried to stimulate the supraopticohypophyseal tract in the median eminence in order to identify supraoptic neurons antidromically, they failed, perhaps because they could not stimulate this tract in isolation in the posterior pituitary gland. Subsequently, Kandel (205) in the goldfish and Yagi et al. (441) in the rat stimulated the pituitary gland in order to drive antidromic potentials in the hypothalamus. They were able to identify magnocellular neuroendocrine cells physiologically. Many subsequent researchers have used this technique of antidromically identified neurons to study magnocellular neuroendocrine cells (see sect. XI). The criteria for antidromic invasion of the perikaryon of neurons include invariance of latency, high-frequency following, and collision of antidromic and orthodromic responses. The collision test, introduced by Paintal (324) to identify muscle afferent nerve fibers, was adapted for use in magnocellular neuroendocrine cells by Dyball (go), for study of parvocellular neuroendocrine cells by Makara et al. (2X$, for study of preoptic hypothalamic neurons by Dyer and Cross (102), and for use in other preoptic neurons associated with thermoregulation by Eisenman (113). These workers faced the same problems of current


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spread and inexact localization of the antidromic responses that confronted Cross and Green (67) and that Fuller and Schlag (133) discussed quite openly in their recent paper. In order to localize these neurons precisely, researchers probably should insist on the collision technique with test stimuli of progressively greater strengths (133) and a specific cell-marking technique similar to the fluorescent dye method with Procion yellow (162; see sect. II, 05). In order to correlate unit activity with behavior and to avoid the trauma of acute surgery and the depressant effects of anesthesia, Hellon (175) adapted the head-mounted microdrive for use with an unanesthetized preparation. While simultaneously heating and cooling the recording field with a water-driven thermode, he drove l- to Z-pm tungsten microelectrodes into the preoptic-ante rior hypothalamus of the chronic rabbit to record from thermosensitive neurons. Subsequently, other workers utilizing similar techniques moved microelectrodes into the hypothalamus to study various aspects of identificahypothalamic firing patterns. For a more precise and physiological tion of the recorded hypothalamic neuron , Vincent et al. (414,415) introduced the method of antidromic identification of supraoptic neurons in the chronic monkey by combining the microdrive-driven microelectrode method with pituitary gland stimulation. This method of study of magnocellular neuroendocrine cells allowed analysis of drinking (414), osmotic (9, 10, 166, 168), and other behavioral stimuli (165, 167, 169). II) INTRACELLULAR REcoRDIh’G. The techniques of intracellular recording with measurement of the resting membrane potential, postsynaptic potentials, and axosomatic spike potentials were first applied to the hypothalamus of the goldfish by Kandel (205), with antidromic identification of magnocellular neuroendocrine cells in the preoptic nucleus. Other workers have subsequently applied these techniques of intracellular recording to the goldfish (162), in vitro bullfrog preoptic nucleus (188, 228), mammalian hypothalamus, and cultured puppy supraoptic nucleus (351, 356, 357). III> MARKING OF SINGLE CELLS. Precise techniques for marking physiologically identified magnocellular and parvocellular neuroendocrine cells include the Prussian blue reaction (67), pontamine sky blue 6B (178), and the fluorescent dye Procion yellow (99, 162). The latter technique of intracellular injection of a marker dye provides the only precise method presently available for accurate marking of a single studied neuron (162). Such an absolute intracellular marking technique will be required when investigators attempt to separate vasopressinergic and oxytocinergic magnocellular neuroendocrine cells. As discussed in detail in section II, these chemically specific peptidergic neurons lie side by side in various regions of the hypothalamus, both with axons projecting to the posterior pituitary gland and therefore both antidromically identified. By the combined use of intracellular Procion yellow marking and subsequent immunohistochemical staining for the specific peptide, vasopressin or oxytocin (432,451,453), future workers can study the physiological characteristics of each of these neuroendocrine cells. Such an approach could be applied to any of the peptidergic neurons of the brain. IV)MICROIONTOPHORESIS OFCHEMICALS. Bloomandco-workers (35)introduced the method of application of chemicals onto the membranes of noniden-


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tified hypothalamic neurons for study of spike discharge. Subsequent improvements have involved the study of antidromically identified neurons (84) and the use of a variety of putative transmitters, hypothalamic hormones, glucose, fatty acids, and other substances on mammalian and invertebrate neurons (13). The problems and limitations of these techniques have recently been discussed extensively (235). b)Multiple-unit activity. I>SINGLEAND MULTIPLE-UNIT ACTIVITY. There are advantages to studying single units in correlation with behavioral activity if the method used avoids the trauma of operation, the depression of anesthesia, and the weight and complexity of a cranial-mounted microdrive in a small animal. In 1958 Strumwasser (384) introduced chronically implanted microwires (tip diam. 80 ,um) for recording the potentials of multipleunit activity in the mesencephalic reticular formation of hibernating ground squirrels. This method was then used for hypothalamic recordings by Naka and Kido (286). On the basis of spike-train wave-form analysis and histological study, Olds (307 > esti .mated th at his blunt nichrome wire m .acrom .icroelectrodes (tip diam. 82 Pm) recorded st imultaneously from 9 to 25 large neurons in the rat hypothalamic regions under study. He emphasized that although these sequential spikes were not derived from a single cell many, if not all, were of similar size and thought to be homogeneous in function (78, 307). However, recent data indicate that even in the most homogeneous-appearing nuclei, such as the supraoptic and paraventricular nuclei, adjacent neurons may be quite different chemically and functionally (see sect. IID). Under these conditions, such a multiple-unit technique would be unable to separate these two neuronal types. II) INTEGRATED MULTIPLE-UNIT ACTIVITY. Because recording single- and multiple-unit activity required careful placement of electrodes during operation with physiological testing, because many of the microwires failed to yield recordable single spikes, and because many of those microwires that did yield single- and multiple-unit activity initially soon lost it, Buchwald and coworkers (49, 50, 430) developed a different approach, which Johnson et al. studies. Incoming signals from large and mw adapted for hypothalamic small neurons, from axons, and from other sources were “integrated” by passa @ through appropriate fil .ters. Some grOUPS (62 100,307) questioned the exact so urce and . nature of the sign .a1 and . wheth ,er it represented activity of local somata, fibers, nearby fibers, or noise in the recording system (100). In view of the complex neuropil in the hypothalamus (266,395), it is rem arkable that signals from such heterogeneous sources as large and small cell somata and fibers, located close to and at a distance from the macromicroelectrodes, could be expected to correlate precisely with other physiological parameters, such as blood hormone levels, sleep-waking activity, and brain temperature changes. 9

2. Analytic

techniques

a) Correlation of hypothalamic

of spike trains

unit

activity

with other events. A major goal in the study is to determine the relationship between the


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electrical events and autonomic, endocrine, and behavioral activity. Cross and Green (67) used the technique of intracarotid injections of hypertonic solutions in the study of hypothalamic neurons and found positive correlations. A more complex type of study involved applying an osmotic stimulus, then attempting to correlate the unit response to the vasopressin or oxytocin output from the neurohypophysis (90). It remained difficult to relate the activity of a single element of a hypothalamic group of neurons with the collective activity of that particular group- the summated output of vasopressin, for example. This difficulty resulted partly from limited knowledge of the chemical nature of the individual cells and partly from the variable responses of each unit to the i mpact of imposed, sometimes excessive, unphysiologi cal stimuli. b) Data analysis. The methods for analyzing the activity of single hypothalamic cells have progressed from the initial use of photographic recording of spike trains at the time of recording (67) to the use of magnetic tape for capturing unit spike-train activity and analyzing these spike trains statistically by digital computer with histograms of varying order (129, 319). At present many investigators utilize digital computers for data analysis, spiketrain statistics, histograms of various orders, and cross correlation of spiketrain data with other parameters. F. Summary The hypothalamus is a small region of the diencephalon lying on either side of the third ventricle, connected to the hypophysis below by a neurovascular stalk and to the adjacent forebrain and hindbrain by periventricular and medial forebrain bundles. Input pathways include those chemically specific cholinergic and monoaminergic ascending fibers, olfactory-limbic forebrain descending fibers, connections to the circumventricular organs and cerebrospinal fluid, and a rich arterial blood supply. The hypothalamus contains final common neuroendocrine pathways to the hypophysis and is concerned with regulation and integration of autonomic functions with behavior. There is increasing evidence that peptidergic, cholinergic, and aminergic neurons play an important role in hypothalamic function. In this review I shall examine structural and functional aspects of hypothalamic neurons with particular emphasis on single- and multiple-unit activity related to the chemical nature of these neurons. II.

MAGNOCELLULAR

NEUROENDOCRINE

CELLS

A. Introduction Magnocellular neuroendocrine cells are hypothalamic neurosecretory neurons that have large Gomori-positive somata lying in the supraoptic and paraventricular nuclei and along the paraventricular-neurohypophyseal


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tract of Greving [ internuclear zone (167,453)]. In mammals these cells evolve from the single, paired, midline preoptic nucleus, pars magnocellularis, of fishes and amphibians (134, 158, 162, 205). Studies of neurosecretory activity in central cholinergic and monoaminergic neurons suggest that “neurosecretion” may be a general property of many neuronal assemblies. The term “magnocellular neuroendocrine cell” defines, specifically, one of two major classes of neurosecretory, endocrine hypothalamic neurons, its inclusiveness allowing discussion of data across species and across traditional anatomical boundaries in the hypothalamic magnocellular zone (supraoptic and paraventricular nuclei, internuclear zone). Research data indicate that only the unmyelinated magnocellular axons project down the pituitary stalk, ending in the median eminence and the posterior pituitary gland (152; see Fig. 2). These axons carry neurosecretory products to the neurohemally situated capillary beds, where release of hormones and “carrier” proteins into the bloodstream can occur (223,238; see Fig. 2). Electrical stimulation of the posterior pituitary should activate these magnocellular axons exclusively. Such pituitary gland stimulation not only yields direct antidromic invasion of the neuroendocrine somata, but also results in a transsynaptic activation of cells connected by axon collaterals (14,83,167,205,229,291,415). In mammals these neuroendocrine cells synthesize, transport, and release the neurohypophyseal peptides vasopressin and oxytocin for regulation of water balance and for milk ejection. Many of the basic principles of neurosecretion have been developed in the study of these hypothalamic neurons (12,80,238,361; see Table 1 and Fig. 2). B. Neurosecretion 1. Intracytoplasmic

granules

and exocytosis

Scharrer and Scharrer (361) first recognized the secretory nature of the magnocellular neuroendocrine cell in fishes, mammals, and insects. Bargmann (12) further consolidated the neurosecretory hypothesis by finding stainable intracytoplasmic granules. Later work demonstrated that these were electron-dense, membrane-bound, 150-nm secretory granules, vasopressin and oxytocin 1mol wt 1000 (238)]. Both of these small peptides and the larger neurophysins lmol wt 10,000 (424, 453)] are transported as membranebound vesicles by axoplasmic flow to the posterior pituitary gland, where they are released by the action-potential, secretion-coupled, calcium-dependent process called exocytosis (80). 2. Neu rosecre tory impulse In the process of neurohypophyseal hormone release, the action potential is of prime interest. Harris (152) first showed the capacity of the electrically stimulated pituitary stalk to release the antidiuretic hormone. Subsequently,


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MAGNOCELLULAR NEUROENDOCRINE

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CELLS

BARORECEPTOR, NIPPLE GENITAL AFFERENTS

IRECEPTOR

FIG. 2. Hypothalamic magnocellular neuroendocrine cells and their connections. The two types of chemically specific peptidergic neurons, vasopressinergic and oxytocinergic, lie scattered in the supraoptic (NSO) and paraventricular (NPV) nuclei and in the internuclear zone (not shown). Th eir vesicle-laden neurosecretory axons descend through the hypothalamus and traverse the internal zone of the median eminence to give off collaterals that terminate on the capillaries of the primary portal plexus. The main supraopticohypophyseal tract axons sweep into the posterior pituitary gland, where they terminate on capillaries. The hormones, vasopressin and oxytocin, are released into the portal and systemic bloodstream under the influence of specific osmotic, volumetric, and neurobehavioral stimuli. The osmoreceptor neurons of Verney, which lie in the internuclear zone, detect the osmolality of the carotid arterial blood and provide excitatory input to the vasopressinergic neuroendocrine cells. Other excitatory input for vasopressinergic neuroendocrine cells arises from afferents of cranial nerves IX and X, from baroreceptors, chemoreceptors, and atria1 volume receptors. Excitatory input for oxytocinergic neurons derives from afferents in the nipple of the mammary gland and in the genital tract. For further details see text. Abbreviations: AC,, anterior commissure; AP, anterior pituitary gland; NPV, paraventricular nucleus; NSO, supraoptic nucleus; OC, optic chiasm; OT, oxytocin; PP, posterior pituitary gland; PV, portal vessels; VP, vasopressin; n VP, vasopressinergic neuron; 0 OT, oxytocinergic neuron; A OSMORECEPTOR, osmoreceptor neuron of Verney. (Based on references cited in sect. II.)


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Kandel (205) in the goldfish and Yagi et al. (441) in the rat demonstrated the capacity of neurohypophyseal axons to transmit an antidromic impulse (see sect. II, 03) to the hypothalamic magnocellular cells with conduction velocities of 0.4-1.0 m/s. Ishikawa et al. (190) recorded orthodromic potentials in the pituitary stalk of the cat at conduction velocities of 0.6-1.4 m/s. The latter group demonstrated that both tonic action potentials and phasic-burst action potentials could be evoked when the nipple, uterus, and supraoptic nucleus were stimulated. Electrical activity in the neural lobe increased .or decreased with intracarotid infusions of hypertonic NaCl and distilled water, respectively (190, 450). 3. Secretory . products

in relation

to electrical

activity

In the lactating rabbit in vivo, the milk-ejection response to electrical stimulation of the supraoptic-neurohypophyseal tract was critically dependent on the frequency of stimulation. The response was unobtainable at frequencies below 25-30 cycles/s (153). In Locke’s solution, in vitro hormone release depended on the stimuli number and frequency (82). Identical numbers of stimuli were progressively less effective as the frequency of stimulation increased above 350 cycles/s. The amplitude of the compound action potential decreased as a function of frequency. Abolition of the compound action potential by addition of tetrodotoxin to the incubation media also blocked hormonal release evoked by electrical stimulation of the stalk in vitro (82). In tetr o d ot oxin-treated neural lobes, the resting release of hormone continued. Despite the absence of conducted action potentials (82), excess potassium was still effective in eliciting graded secretory responses. When parallel in vitro experiments were conducted in low-sodium medium, the hormone release was dependent on the frequency of the electrical stimulation, despite experimental conditions that precluded action-potential generation (299). These results suggest that sustained changes of the axonal membrane potential and the action-potential shape caused high-frequency depression of hormone release and compound action potential. In the urethan-anesthetized rat, Dyball (90) found that intracarotid injections of hypertonic NaCl accelerated antidromically identified supraoptic and paraventricular units, releasing oxytocin (B-fold) and vasopressin (2fold). The time course of these events was not consistent, however, with a simple relationship between action-potential activity and hormone secretion. Unit activity peaked at 30 s in response to intracarotid hypertonic NaCl, while oxytocin secretion peaked at 60 s and vasopressin peaked at 180 s. Control levels of these bioassayable hormones were unusually high: the oxytocin released was 100 times normal and the vasopressin was 200 times normal. With the technical defects in this bioassay corrected, this experimental design would present thought-provoking results. Further quantitative studies comparing hormone levels and unit activity are needed to verify the efficacy of the anesthesia-modified, as opposed to the


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conditioned and unanesthetized, preparation. These initial data indicate a close relationship between the electrical activity of unmyelinated axons of the magnocellular neuroendocrine cells and hormone release. C. Electrical

Membrane

Characteristics

Intracellular recordings were used by Kandel (205) and Hayward (162) to investigate goldfish hypothalamic neuroendocrine cells. The cells showed resting potentials of 47-50 mV and action potentials of up to 117 mV. Action potentials of long duration (3.9 ms) occurred in two steps: long-lasting hyperpolarizing afterpotentials and orthodromic driving from olfactory input. Total neuron (input) resistance was measured at 3.3 x lo7 (2; total neuron time constant was 42 ms. Orthodromic volleys, produced by olfactory tract stimulation, generated graded excitatory postsynaptic potentials. Antidromic volleys - that is, pituitary stimulation-produced inhibitory postsynaptic potentials. These findings suggested the presence of inhibitory recurrent collaterals in the goldfish (205). Koizumi and Yamashita (229) described similar resting membrane potentials and action potentials in the cat and dog. Stimulation of the septal area and the midbrain reticular formation produced excitatory postsynaptic potentials of short duration. Long-lasting inhibitory postsynaptic potentials followed. Hyperpolarization was always longer than the preceding excitatory postsynaptic potentials and its duration was generally 80 ms. Antidromic excitation from stimulating the pituitary area produced inhibitory postsynaptic potentials lasting 100 ms. These findings suggest there are inhibitory recurrent collaterals in the cat (229). Bullfrog magnocellular endocrine neurons in vitro revealed similar resting membrane potentials and action potentials (188, 228). In addition to showing evidence for inhibitory recurrent collaterals, electrophysiological changes indicative of excitatory recurrent collaterals were found (188,228). The data derived from these investigations into the electrical membrane properties and postsynaptic potentials of the magnocellular neuroendocrine cell revealed that these secretory neurons had electrical characteristics that paralleled those found in nonsecretory neurons. D. Morphological 1. Structural

and Functional

Cell Types

aspects

Partly because histologists find staining large neuroendocrine secretory cells with Golgi silver difficult, there have been limited analyses of the detailed anatomical structure of the somata, dendrites, and axons. Leontovich (240), using a classical but perhaps incomplete Golgi silver staining, found that the neurons of the puppy supraoptic and paraventricular nuclei appeared


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undifferentiated, neuroblastic in shape, and with few dendrites projecting from simple bipolar neurons. There was no evidence of recurrent collaterals or axonal bran .ching. In a more complete SllV ‘er impregna tion of the supraoptic neurons of the m .onkey, LuQui and Fox (254) found two POPUl.ations of cells, graded in size. The large supraoptic neurons had irregular somatic membranes, somatic spines, and moderately branched dendritic trunks arising from the cell body. There were occasional dendritic spines arising from dentrites. One axon emerged from a conical elevation either on the soma or from a dendrite with the axon directed toward the supraoptic-hypophyseal tract. Smaller neurons had short, beaded axons that ended a short distance from their point of origin. In her light- and electron-microscopic analyses of the rat’s supraoptic nucleus, Rechardt (344) found two distinct types of neurosecretory cells, dark and light, depending on the number of free ribosomes available. Dehydration further increased the ribosome content of the dark cells. Rechardt (344), Leranth et al. (241), and LuQui and Fox (254) saw four types of synapses in the rat’s supraoptic nucleus: axoaxonic, axodendritic, axospinal, and axosomatic. In a search for the origin of these synaptic endings, Palkovits and coworkers (241, 449) applied quantitative electron microscopy to the supraoptic nucleus of the rat. They found 596 synaptic terminal boutons per neuron, twothirds originating intranuclearly or in the immediate vicinity and one-third arising outside the supraoptic nucleus. Of these intranuclear afferents, the source was either hypothetical recurrent collaterals from the magnocellular neuroendocrine cell axons or projections from hypothetical interneurons. These latter may possibly correlate with the smaller neurons of LuQui and Fox (254) or with the light cells of Rechardt (344). Thirty-three percent of the extranuclear afferents arise from the lower brainstem, 21% from the medial basal hypothalam us, 14% from the amygdala, 14% from the septum, 9% from the hippocampus, and 17 % from the olfactory tubercle and rostra1 cortical regions. l

2. Vasopressinergic

and oxytocinergic

.a

neurons

Knowledge of the chemical nature of the secretory neurons of the supraoptic and paraventricular nuclei is essential to the analysis of the functional organization of the magnocellular neuroendocrine cells. Earlier workers recognized the heterogeneity of cell types in these nuclei, but most favored the nuclear hypothesis. This latter interpretation indicated that the cells of the supraoptic nucleus produced vasopressin and the cells of the paraventricular nucleus produced oxytocin (238). Studies in the Brattleboro rat, an animal with hereditary diabetes insipidus, revealed the probability that two neuron types lay scattered throughout the supraoptic and paraventricular nuclei. Since hereditary diabetes insipidus is a genetic disease state linked to a deficient vasopressin production but with oxytocin production intact, this was extended to the magnocellular neuroendocrine cell types, one defective


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for vasopressin secretion, the other a normal oxytocin secretor (407). With the advent of immunohistochemical techniques, Swaab et al. (391-393) and Vandesande and Dierickx (410-412) stained two neuron types specifically: one vasopressinergic, the other oxytocinergic (see Fig. 2 and Table 1). These were scattered in roughly equal numbers throughout the supraoptic and paraventricular nuclei, with the oxytocinergic neurons located preferentially in the rostra1 portions of these nuclei. For the first time, these data indicated the chemical nature of the magnocellular neuroendocrine cells, the validity of the cellular hypothesis (one cell, one hormone) as opposed to the nuclear hypothesis (one nucleus, one hormone), adding a necessary clarification to the earlier confusion (90). This immunohistochemical separation of vasopressinergic and oxytocinergic neurons opens the way for physiological studies on chemically specific, peptidergic hypothalamic neurons, a promising goal for the future. Using similar immunohistochemical techniques but different antibodies to oxytocin and vasopressin, other workers found both hormones in a single neuron (451). The objections to such data were that for any neuron to produce two chemical substances, and transport and release them from posterior pituitary nerve terminals, would present complex organizational problems of stimulus-response specificity. Perhaps such puzzling results can be attributed to vasopressin and oxytocin cross-reactions with vasopressin and oxytocin antisera, respectively. Future studies with affinity chromatography perhaps may clarify such data.

3. Spontaneous

firing

patterns

a) Nonantidromically identified ceZZs. In the acutely prepared, anesthetized mammal, some workers found that nonantidromically identified single cells in the supraoptic and paraventricular nuclei either discharged irregularly and continuously or were silent (43, 44, 66, 67, 118, 189, 203, 227, 388). Cross and Green (67) and Joynt (203) found that nonidentified cells in and near the supraoptic and paraventricular nuclei were poorly responsive to natural sensory stimuli. Brooks and co-workers (43-45, 189, 227, 388) found nonantidromically identified cells in and near the supraoptic and paraventricular nuclei to be quite responsive to electrical stimulation of peripheral nerves and central brain sites. Studying the unanesthetized monkey, Hayward and Vincent (174) found that nonantidromically identified single supraoptic neurons fired continuously in an irregular manner or remained silent. These cells were poorly responsive to nonnoxious natural sensory stimuli. Divergent views concerning the sensory responsivity of nonantidromically identified magnocellular neuroendocrine cells from these regions may be explained by the fact that different groups - Brooks and co-workers on the one hand and Cross and Green (67), Joynt (203), and Hayward and Vincent (174) on the other-were probably reporting on totally different cells. In addition, basic variations in experimental design might contribute to


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discrepancies in the neurophysiological definitions of supraoptic and paraventricular neurons. b) Antidromically identified neurons. The majority of studies performed on antidromically identified supraoptic and paraventricular neurons, whether in the acutely prepared, anesthetized mammal (66,81,83,84,90,92, 95, 154, 249, 250, 292, 302, 390, 419, 421, 422, 448) or in the chronically prepared, unanesthetized mammal (9, 10, 165-168, 414, 415), revealed three types of spontaneous firing patterns: silent (3-lo%), continuously active (6577%), and phasic or burster (20-25%). The burster, a discharge pattern unique to the magnocellular neuroendocrine cells, often went unrecognized in the earlier studies. It has since become evident that this single-cell hypothalamic discharge pattern, experimental design or animal preparations notwithstanding, represents a physiological mystery. The burster may represent an intrinsic pacemaker, such as that found in invertebrates (13, 16, 187). It may instead represent the end product of inhibition or excitation by some local or distant influence extrinsically applied, such as local recurrent collateral facilitatory influence, involving vasopressin as the transmitter (187). Whether such burster activity labels cells as vasopressin secretors (81, 154) has not been established. The role the burster plays in the “efficient” release of vasopressin or oxytocin from the nerve terminals in the posterior pituitary remains undetermined at present.

4. Recurrent

facilitation

and inhibition:

neuroendocrine

Renshaw

cells

Stimulation of the pituitary gland or stalk in anesthetized or unanesthetized preparations revealed evidence of recurrent inhibition (83, 167,205,217, 229, 290, 291, 301, 302, 366, 414, 415, 448) and recurrent facilitation (188, 228) in antidromically identified magnocellular neuroendocrine cells. The duration of facilitation and inhibition varied with the type of preparation and nature of the anesthesia. Whether an interneuron, that is, a neuroendocrine Renshaw cell (417) of one or more types, was involved in the recurrent facilitation and inhibition remains obscure. The anatomical studies of Palkovits’ group (241, 449), indicating that 66% of the synaptic boutons on the supraoptic neurons arise from intranuclear elements, favored a recurrent collateral or an interneuron population of cells as the origin. Smaller Golgistained cells in the supraoptic also tended toward the neuroendocrine Renshaw cell definition. Koizumi et al. (229) recorded from supraoptic and paraventricular cells that responded to antidromic stimulation by brief discharges (5-7 spikes) at 500-800 spikes/s. These magnocellular neuroendocrine cells resembled spinal cord Renshaw cells. An appropriate anatomical marking technique would demonstrate conclusively the presence of Golgi type II interneurons within the supraoptic and paraventricular nuclei. Recurrent magnocellular neuroendocrine collaterals still await firm anatomical definition.


592

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5. Procion-yellow-stained

N. HAYWARD

neuroendocrine

Volume

57

cells

Procion yellow (Imperial Chemicals, Inc.) iontophoresis is a promising technique for resolving the identification of cell types and of recurrent collaterals and interneurons in the mammalian supraoptic and paraventricular regions (162). Antidromically identified magnocellular neuroendocrine goldfish preoptic cells were studied with intracellular recording and intracellular iontophoresis of Procion yellow fluorescent dye. By the spread of the dye throughout the cytoplasm into dendrites and axons, Hayward (162) localized antidromically identified preoptic neurons in the magnocellular portion of the nucleus. An elaborate dendritic tree, establishing the presence of bifurcate and trifurcate axons, was photographed and three morphological cell types were outlined. Cell type I was a large (37~pm) multipolar neuron, 48 ,um from the ependyma, its fine “dendrites” having multiple branched processes that projected into the lateral hypothalamus and within the preoptic nucleus. Cell type II was a large (31~pm) multipolar neuron, 24 ,urn from the ependyma, with a coarse dendrite to the ependyma and fine dendrites within the preoptic and with limited axonal branching. Cell type III was a small (M-pm) multipolar neuron, 46 ,um from the ependyma, with fine dendritic processes distributed within the preoptic and with limited axonal branching. Applied to mammalian magnocellular neuroendocrine cells, a marking technique with Procion yellow (162) or horseradish peroxidase (372,373,394) should provide a means of establishing the presence or absence of recurrent collaterals and interneurons. The physiological data of recurrent inhibition (83,167,205,217, 229, 290, 291, 301, 302,414, 415) and facilitation (188, 228>, the high-frequency responses (229), and the anatomical data of 66% intranuclear origin for terminal boutons (241, 449) suggest this necessary culminating identification procedure. Furthermore, the combined use of immunohistochemical(391-393, 410-412, 451) and Procion yellow (162) techniques promises to open a new era in physiological studies of chemically defined peptidergic neurons. E. Osmosensitivity 1. Osmoreceptors

of Verney

In his classical Croonian lecture on the antidiuretic hormone, Verney (413) described the main physiological stimuli that determined the release of vasopressin from the neurohypophysis. These stimuli were 1) osmotic pressure of the carotid arterial blood, 2) volume of blood in the vascular system, and 3) the behavioral state of the mammal. His osmometric hypothesis states that the “osmoreceptors,” neural elements in the hypothalamus, detect the osmotic pressure of carotid arterial blood and initiate release of vasopressin from the neurohypophysis during hyperosmolal states. At present, we know more about this osmometric hypothesis than about the osmoreceptors (130, 131, 204). The latter may be supraoptic neurons (67, 203, 413, 423, 425),


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separate cells in the perinuclear zone synaptically connected to the vasopressinergic neurons (25, 41, 42, 157, 174, 415, 417, 447; see Fig. 2), or elements such as sodium detectors that are located in the vicinity of the third ventricle (6) and lie outside or inside the blood-brain barrier. On the basis of short-term and long-term intracarotid infusions of hypertonic solutions in the conscious dog, Verney (413) concluded that sodium salts and sucrose, substances unable to penetrate the hypothetical hypothalamic osmoreceptors, cause dehydration and volume reduction of these receptors with consequent release of antidiuretic hormone. When he found that intracarotid hypertonic urea did not produce an antidiuretic response, Verney (340) had no reason at that time to consider the blood-brain barrier. He therefore concluded that urea was freely diffusible through the osmoreceptor membrane. Recent studies indicate that the blood-brain barrier may be impermeable to low concentrations of urea (306, 340), thus negating Verney’s hypothesis. However, since infusions of intracarotid urea in high concentrations (2 M) may damage the blood-brain barrier (340), making it permeable to urea and other substances, Verney’s osmoreceptor hypothesis remains viable (see Fig. 2). By studying the nonantidromically identified single hypothalamic cells in the chronically prepared, unanesthetized monkey, Hayward and Vincent (174, 417) tried to resolve some of the conflicts surrounding Verney’s osmoreceptors and the blood-brain barrier. On the basis of anatomical location and discharge patterns elicited by osmotic and arousing sensory stimuli, two osmosensitive cell groups emerged, “specific” and “nonspecific” (174, 417). Fifty percent of the osmosensitive cells were specific, responding to an intracarotid injection of hypertonic NaCl, generally not responding to nonnoxious arousing sensory stimuli, and lying in or near the supraoptic nucleus. The specific cells divided into subtypes. Twenty percent of the supraoptic cells were biphasic, with acceleration followed by inhibition; 30% of the cells in the perinuclear zone of the supraoptic nucleus were monophasic, limited to either acceleration or inhibition. Fifty percent of the osmosensitive cells were nonspecific, responding to intracarotid injections of hypertonic NaCl and to mildly arousing sensory stimuli and showing the monophasic excitation or inhibition response. Hayward and Vincent (174) concluded that the specific biphasic osmosensitive supraoptic neurons were the magnocellular neuroendocrine cells; the specific monophasic osmosensitive perinuclear cells were the osmoreceptors of Verney; the nonspecific monophasic perinuclear-zone osmosensitive neurons were the osmoreceptors related to drinking and behavior. In addition, they suggested that Verney’s osmoreceptors were separate neurons lying adjacent to the supraoptic, with osmotic input across the capillaries of the blood-brain barrier, connecting to the magnocellular neuroendocrine cells by excitatory axosomatic synapses. Other osmoreceptors (32, 259, 359) in the area may be involved in the nonendocrine behavioral effects stemming from such intracarotid osmotic stimuli as drinking and arousal. Antidromically identified magnocellular neuroendocrine cells in the supraoptic and internuclear zone revealed further pattern subtleties (165-168).


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In the unanesthetized monkey, magnocellular neuroendocrine cell data fell into one of three spontaneous, functional categories: silent, continuously active, and burster. Hayward and Jennings (166, 168) showed that, with appropriate NaCl loading, some silent cells shifted transiently to continuously active, some continuously active cells burst briefly, and a few burster cells reached hyperburster states. Antidromically identified magnocellular neuroendocrine cells showed specific biphasic osmosensitive responses to intracarotid hypertonic NaCl (166, 168). Intracarotid infusions of hypertonic n-glucose (1.2-l .5 M) produced a specific biphasic osmosensitive response (166). The same magnocellular neuroendocrine cell required an osmolality level 2-3 times higher than the intracarotid n-glucose infusion to produce a biphasic osmosensitive response equivalent to that produced by hypertonic NaCl (166). Other workers reported intracarotid NaCl to be 2-3 times more effective, osmole for osmole, than intracarotid n-glucose for driving nonantidromically identified supraoptic neurons (67, 203, 227) and for producing shifts in hypothalamic steady potentials (28; see Fig. 2). 2. Intraventricular

sodium

ion detectors

Five-second intracarotid injections of hypertonic NaCl (168) and hypertonic n-glucose (166) in the unanesthetized monkey produced an immediate (l- to 5-s delay) increased firing rate in identified and nonidentified magnocellular neuroendocrine cells. Such a rate of stimulation, however, may have been too rapid to have been detected by a slow change in the sodium ions in the cerebrospinal fluid (6, 120). The effect might have been a rapid osmotic one across the endothelial barrier of the rich capillary bed of these hypothalamic neurons. Although these results with short-term intracarotid infusions suggested an osmoreceptor mechanism in Verney’s sense (413), the results of long-term infusion by Verney (413) and Ericksson et al. (120) seemed to favor a basically different mechanism, such as a sodium ion detector in the vicinity of the third ventricle (6). Whether unit recording accompanied by rapid or slow infusions of hypertonic NaCl would release vasopressin from the neurohypophysis under these conditions is unknown. Until simultaneous measurements of vasopressin and hypothalamic unit activity can be made under physiological conditions, the exact mechanisms will be difficult to determine.

F. Blood

Volume

and Vasopressin

Release

Verney (413) found that hemorrhage in the unanesthetized dog produced antidiuretic hormone release. Share (371) determined that the reflex paths for hypovolemic vasopressin release involved two vascular elements: stretch receptors in the wall of the left atrium, arterial baroreceptors mainly in the aortic arch and carotid sinuses. Sjostrand (378) found that the rat laryngeal communicans vagal branch was critical for these afferent pathways. In the pentobarbital-anesthetized cat, Barker et al. (14) found that electrical stimu-


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lation of vagus or carotidr:sinus nerves produced long-latency (129 ms and 83 ms, respectively) excitatory responses in antidromically identified supraoptic and paraventricular cells. They found excitatory stimuli from carotid sinus and vagus nerves converging on single magnocellular neuroendocrine cells. In a study of nonantidromically identified single cells in the supraoptic and paraventricular nuclei of the allobarbital-urethan-anesthetized cat, Menninger and Frazier (262) found that left atria1 balloon inflation increased firing in three cells and decreased in six. These data, derived from few nonidentified neurons, were difficult to interpret (see Fig. 2). Testing the effects of hemorrhage in the urethan-anesthetized rat on antidromically identified, single paraventricular cells, Wakerley et al. (422) and Metoki (263) found bursters with accelerated firing, lengthened burst durations, and shortened interburst intervals. With blood replacement these firing patterns reversed. These workers suggested burster magnocellular neuroendocrine neurons should be identified as vasopressinergic. In a similar study Dreifuss et al. (81,154) examined the effects of bilateral common carotid occlusion on the firing of antidromically identified supraoptic bursters. Of these bursters 91% began bursts of activity, alternating with silence less than 10 s after carotid occlusion. Most randomly firing neurons were inhibited or unaffected and during occlusion the interspike intervals were significantly shorter. The impression was that burster neurons were secreting vasopressin (see Fig. 2). Hemorrhage releases vasopressin predominantly, but it also releases small amounts of oxytocin (121). Some oxytocin may be released with bilateral carotid occlusion (56). These kinds of studies tend to support bioassay, physiological, and chemical data that indicate a parallelism between magnocellular neuroendocrine cell activity and neurophysiological stimuli such as hemorrhage and carotid occlusion. Ultimately, simultaneous measurement of physiological levels of plasma vasopressin and oxytocin will allow separation of vasopressinergic and oxytocinergic neurons. G. Neural

Input

1. Hypothalamic

and Behavior island

deafferentation

When investigators (272) isolated supraoptic nuclei from all neural connections except that to the hypophysis, vasopressin release was unchanged as long as blood osmolality was maintained. Electrical recordings from nonantidromically identified single supraoptic neurons in the deafferented hypothalamus revealed lengthy increased cell discharge (388). These deafferented supraoptic neurons continued to respond, however, to intracarotid infusions of hypertonic glucose (388). When antidromically identified paraventricular neurons were deafferented, however, spontaneous discharge rates reduced and oxytocin secretion stopped (93). These and other data indicate the importance of extranuclear influences for the spontaneous and stimulated release of mammalian vasopressin and oxytocin.


596 2. Circadian

JAMES

rh .ythrns:

sleep-waking

N. HAYWARD

activity .

The behavioral state of a mammal must be considered whenever the neurohypophysis releases vasopressin or oxytocin. The paradoxical antidiuresis of sleep occurs despite brain cooling and increased pulmonary blood volume, events associated with diuresis in waking states. Vasopressin circadian rhythms show peak secretion at midnight and low at noon (139, 404). “Continuously active” supraoptic neurons fired irregularly during waking and with periodic discharge in sleep (165). During brief periods of higher voltage EEG, these cells showed accelerated discharge, 10 spikes/s. At lower voltage EEG, cells showed 2 spikes/s with 1 burst every 10 s (165). The relationship of the magnocellular neuroendocrine cell sleep bursts to vasopressin or oxytocin release is unknown. 3. Nociceptor

stim rrli

Pain, a powerful behavioral determinant, causes the posterior pituitary to release vasopressin. When Verney (413) applied electrical stimuli subcutaneously to his conscious dog to the “point of resentment,” he attributed the prolonged diuresis to “emotional stress.” Others, stimulating central pain pathways (352)--midbrain and limbic sites suspected of motivational and affective linkage -caused the neurohypophysis to release antidiuretic hormone (159, 172). When natural, nonnoxious, mildly arousing stimuli like light, touch, and sound were given (l74), unanesthetized monkeys retained the stable discharge rates from nonidentified, “specific biphasic” osmosensitive supraoptic neurons. Later, antidromically identified magnocellular neuroendocrine neurons were found to react similarly (165). The reactions appeared to be conditioned and habituated, verifying Verney’s conclusion (413) that emotional stress is important whenever cutaneous stimuli activate supraoptic nociceptors (165).

Electrical stimulation of the medial and basolateral amygdala, the diagonal band of Broca, olfactory tubercle, ventral amygdalohypothalamic pathway, uncus of hippocampus, medial forebrain bundle, periaqueductal grey, ventral tegmental area of Tsai, central tegmental tract, and midbrain reticular formation ( 172, 173) produced vasopressin-dependent antidiuretic responses in unanesthetized monkeys. Other workers found similar results (65, 352) in these central nervous sites. Zaborszky et al. (449) confirmed these physiological studies in their quantitative electron-microscopic study of the synaptic input to rat supraoptic afferents. They found that 33?% of the afferents came from the brainstem, 2% from the medial basal hypothalamus, 15%# from the septum, 9cT(lfrom the hippocampus, 22% from the olfactory tubercle, plus the basal forebrain. Their conclusion was that the limbic system and the lower brainstem modulate magnocellular neuroendocrine neuronal activity.


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Single-pulse electrical stimulation of the septal area, midbrain reticular formation, central grey, anterior commissure, and hippocampus produced orthodromic excitation in antidromically identified single supraoptic and paraventricular neurons in the anesthetized cat and dog (229). Intracellular recordings of these magnocellular neuroendocrine cells showed that septal area and midbrain reticular formation stimulation produced short-duration excitatory postsynaptic potentials followed by long-lasting inhibitory postc potentials. Hyperpolarization, generally of 80 ms duration, was synapti always longer than the preceding excitatory postsynaptic potentials (229). Other workers confirmed the orthodromic excitatory effects of single-pulse electrical stimulation of septum (206,291,447), amygdala (291), and midbrain reticular formation (206). Electrical stimulation with pulse trains yielded inhibitory responses from septum (206, 389, 447) and excitatory effects from midbrain reticular formation (206, 447) in antidromically identified magnocellular neuroendocrine neurons. Intracarotid injection of hypertonic NaCl produced an excitatory-inhibitory sequence in antidromically identified supraoptic neurons in the anesthetized cat (206, 447). When the septal or midbrain reticular formation was repeatedly stimulated electrically during osmotic stimulation, the osmotic excitatory phase was inhibited or enhanced, respectively (206, 447). The results suggest that neural input greatly alters the responses of supraoptic cells to osmotic stimuli. The latter act on neuroendocrine cells through osmoreceptors, while stimuli from the septal and midbrain reticular formation may act directly on magnocellular neuroendocrine cells (206,447). These data are consistent with the concept of a separate group of osmoreceptor-neural elements in the hypothalamus (174, 414, 415, 417).

5. Drinking Potentially a significant behavioral state for vasopressin release, drinking should modify supraoptic unit activity in the unanesthetized mammal. In the dehydrated monkey, plasma vasopressin dropped quickly to normal during 4 h of drinking while osmolality remained elevated (171). In the waking monkey, Vincent et al. (414) found antidromically identified single supraoptic units were inhibited during drinking. In order to determine the importance of peripheral taste receptors versus central osmoreceptors in drinking-related vasopressin changes mediated by magnocellular neuroendocrine cells, Emmers (118) studied the pentobarbital-anesthetized cat. He found that nonantidromically identified supraoptic units were phasically inhibited when the gustatory nucleus of the ventrobasal thalamus was stimulated. Osmotic stress activated supraoptic and paraventricular nuclei and the lateral hypothalamus. Although destroying the gustatory thalamic nucleus, denervating the tongue, and sectioning the spinal cord did not change supraoptic or paraventricular responses, these maneuvers did markedly reduce lateral hypothalamic osmotic responses (118). Emmers (118) confirmed the gustatory-inhibitory supraoptic effects that Vincent et al. (414) found, suggesting


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further that there might be a direct ventrobasal thalamic-supraoptic pathway, as opposed to an indirect gustatory-excitatory path way through the lateral hypothalamu .s to the supraoptic nucleus. Whether gustatory stimuli reach the supraoptic through the thalamus or more directly along the pons and amygdala (300) remains unexplored. For example, rats drinking 2% NaCl had accelerated magnocellular neuroendocrine unit responses (97). At present, the complexities of the role of the lateral hypo thalamus in drinking requ ire more investigation and understanding (446). 6. Suckling,

coitus,

and parturition

Beh avioral states related to reproduction and the milk .-ejection reflex release oxytoci .n from the m agnocellular neuroendocrine neurons (402). Brooks et al. (44) stimulated the nipple of the postpartum, anesthetized cat by gentle suction and uterine distension to increase the nonantidromically identified paraventricular unit discharge and evoke the milk-ejection response. Wakerley, Lincoln, and Hill (248, 419-421) recorded from antidromically identified single paraventricular and supraoptic magnocellular neuroendocrine cells in anesthetized rats during milk ejection evoked by natural suckling. In response to the stimulus, 58% of the paraventricular units and 48% of the supraoptic developed the biphasic response: l- to 4-s burst discharge (3080 spikes/s) followed by a period of silence (7-56 s>. Milk ejected lo-20 s after the burst. These responses came from the silent, continuously active, and burster patterns of spontaneously firing activity (249, 250, 419). Evidence suggests that all oxytocinergic magnocellular neuroendocrine cells fire synchronously, with the number of suckling pups controlling the occurrence and ejection of the milk (250). By increasing intraductal mammary pressure abruptly, by injecting sal ine, and by adding or removing a PUP7 Lincoln and Wakerley (250) triggered magnocellu lar neuroendocrine ccl .l acti .vity and the milk-ejection response. The amount of oxytocin released and the size of the suckling unit response correlated closely. These data indicate the, presence of supraoptic and paraventricular oxytocinergic neuroendocrine cells, with the peripheral nipple receptors acting as the finely tuned control of afferent input (see Fig. 2 and Table 1). In studies of the rabbit’s milk-ejection reflex, Novin and Durham (301) observed that urethan blocked antidromic facilitation but not antidromic inhibition at the paraventricular nucleus. They also noted that operational stress activated a central monoamine inhibitory system that seemed to block ascending excitatory afferents (milk-ejection pathway) at the nucleus. In a study of the relation of vaginal distension to the activation of antidromically identified paraventricular units in the freely moving rabbit, Findlay (128) found that most cells were nonspecific, responding identically to vaginal stimulation and other rousing stimuli. One cell, however, did accelerate specifically to vaginal distension. In the estrogen-primed, propranololtreated, urethan-anesthetized, lactating rat Dreifuss et al. (87a) found vagi-


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NEURONS

599

active (38%), and bursnal distension accelerated silent (44%), continuously ter (37%) supraoptic neurons and induced milk ejection. These data indicate probable activation of oxytocinergic neurons from vaginal distension with the pattern of firing not indicative of chemical cell type (see Fig. 2). 7. Somatic

input

pathways

Brooks and co-workers (43, 189, 227, 388) found that osmosensitive, nonantidromically identified single units in the vicinity of the supraoptic and paraventricular nuclei responded to electrical stimulation of peripheral motor nerves. Other workers (67, 165, 167, 174, 203) did not find supraoptic neurons highly responsive to nonnoxious natural sensory stimuli. Some of these results may have depended on the presence of interneurons within the supraoptic nucleus, the presence of nonsecretory neurons in the vicinity, and the use of repetitive trains of stimuli (229). Hata and Miura (156), in support of the observations of Suda et al. (388) that supraoptic neurons were inhibited by cerebellar effects, found that electrical stimulation of the cerebellar fastigial nucleus blocked the release of antidiuretic hormone when the carotid was occluded bilaterally. Beardwell et al. (24) found that vigorous exercise produced a marked rise in plasma vasopressin concentrations that was out of proportion to the rise in plasma osmolality. These data support the need for careful observation in the study of the relationships between the motor system and vasopressin physiology of the magnocellular neuroendocrine cells.

H. Pharmacological

Actions

and Putative

Transmitters

1. Anesthesia General anesthetics such as urethan release high levels of vasopressin and oxytocin into the blood (91, 96, 154) from neuroendocrine cell nerve terminals. Hayward and Vincent (174) found that urethan anesthesia depressed the spontaneous activity of nonidentified supraoptic neurons and blocked the osmosensitivity of these hypothalamic cells. Novin and Durham (301) noted that urethan could block antidromic facilitation but not antidromic inhibition at the paraventricular nucleus. The stress of operation activated a central monoamine inhibitory system that seemed to block ascending excitatory afferents at the nucleus. In the acute rat Dyball and McPhail (96) found firing rates of the magnocellular neuroendocrine cells were lower with pentobarbital than with urethan, tribromoethanol, chloral hydrate, or ethanol. Further systematic studies evaluating the role of anesthetics generally used in the laboratory in the release of vasopressin and oxytocin are needed.


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2. Microiontophoresis Bloom et al. (35) obtained excitatory and inhibitory responses after microiontophoresis of norepinephrine, acetylcholine, and serotonin onto the surface of a few nonantidromically identified supraoptic and paraventricular neurons in the anesthetized cat. Barker, Crayton, and Nicoll (15) then demonstrated in the urethan-anesthetized cat the norepinephrine p-adrenergic depression, acetylcholine muscarinic depression, and nicotinic excitation of antidromically identified supraoptic units. With the exception of the muscarinic inhibitory effects of acetylcholine, Moss et al. (274, 275, 278) and Dreifuss and Kelly (84) confirmed these results. Sakai et al. (356) reproduced all the findings of Barker et al. (15) in organ-cultured puppy supraoptic neuroendocrine cells. The acetylcholine dose-response curve was bell shaped, with spiking initiated at lower concentrations and spike frequency reduced at higher concentrations. The nicotine antagonist dihydro-P-erythroidine decreased acetylcholine spiking whereas the muscarinic receptor blocker atropine increased supraoptic neuroendocrine cell spiking (356). These studies indicated that supraoptic neuroendocrine cell membranes had excitatory nicotinic (15, 84, 274, 275, 278) and inhibitory muscarinic cholinergic receptors (15,366) and inhibitory P-adrenergic receptors (14, 15,274,275,278,356). Nicotine releases vasopressin definitively (170). After systematically eliminating such putative transmitters for recurrent collateral inhibition as acetylcholine, glycine, gamma-aminobutyric acid (GABA), and norepinephrine, Nicoll and Barker (294) concluded that vasopressin was the transmitter involved. Moss et al. (275) obtained equivocal effects with vasopressin. They found that oxytocin excited most paraventricular units but had no effect on nonantidromically identified paraventricular and supraoptic cells. Dyball (92) cast further doubt on vasopressin as the synapse transmitter for recurrent collateral inhibition when he demonstrated the presence of recurrent inhibition in diabetes insipidus rats with known vasopressin deficiency. The inhibitory synaptic transmitter for recurrent inhibition and the transmitter for recurrent collateral facilitation remain unknown (188, 228, 229). The presence of a peptide factor in molluscan ganglia (13, 187) that can modulate the bursting pacemaker potential in molluscan endocrine cells is presumptive evidence for the role that vasopressin and oxytocin may play in the mammalian neuroendocrine system. Induced bursting pacemaker potential resulted from long-term changes in membrane properties, whereas conventional neurotransmitters produced transient conductance changes, presumably enhancing neurosecretory activity (16, 187) For such long-term membrane and cell-firing effects of vasopressin, oxytocin, and the molluscan peptide, it is not surprising that some experiments yield inconclusive or contradictory results. Future experiments may be designed to examine the possibility that synchronized magnocellular neuroendocrine cell-firing patterns may emanate from recurrent collateral s (241) that impinge on adjacent


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1977

HYPOTHALAMIC

NEURONS

601

vasopressinergic or oxytocinergic neurons. The resulting release of vasopressin or oxytocin, respectively, and the consequent long-term changes in membrane properties should lead to bursting-pattern activity and enhanced hormone release in synchronous pulses. Angiotensin II, a peptide im .portant in regulation of water balance and .ood pressure and volume (37l), accelerated supraopti c neuroendocrine cell discharge after direct microiontophoretic application in the pentobarbitalanesthetized cat (294). In organ-cultured puppy supraoptic neuroendocrine cells, Sakai et al. (356) elicited concentration-dependent spiking activity by superfusion with angiotensin II. Specific angiotensin antagonists (cysteine-8angiotensin II and sarcosine-l-isoleucine-8-angiotensin II) blocked this activity. Spiking initiated by glutamate or nicotine superfusion was not blocked. Supraoptic magnocellular neuroendocrine cell membranes therefore must contain specific angiotensin II receptors. When infused into the third ventricle or directly into the supraoptic nucleus, histamine, normally present in large amounts in the hypothala .mus, . produced antidiuretic hormone release from the neurohypophysis (145) . Mlcroiontophoresis of histamine on unidentified (144) hypothalamic neurons or on antidromically identified supraoptic neuroendocrine cells (145) in the pentobarbital-anesthetized cat accelerated a majority of neurons. Metiamide, an antagonist against histamine H, receptors, did not change base-line firing patterns in antidromically identified supraoptic neurons. These studies suggest that supraoptic neuroendocrine cell membranes contain specific histamine receptors. 3. Blood-borne

pharmacological

agents

In order to determine the effects of varying amounts of estrogen and progesterone on the firing patterns of antidromically identified paraventricular nucleus neurons, Negoro et al. (292) examined urethan-anesthetized rats at different points in the reproductive cycle, after ovariectomy, with and without estrogen and progesterone. Cell-firing rates were higher during proestrus, estrus, full-term pregnancy, parturition, and lactation and in ovariectomized, estrogen-treated rats. When estrogenized rats were given progesterone, the estrogen-increased paraventricular neuroendocrine cell activity was significantly depressed 4 h after administration (292). Eight hours later, the firing rate had recovered completely. Relating vaginal distension to paraventricular unit activation in urethan-anesthetized rats, Negoro et al. (293) found that estrogen lowered and progesterone elevated the activation threshold. These results indicated that steroids, like estrogen and progesterone, produced long-term effects on magnocellular neuroendocrine cell-firing properties. Mechanisms similar to those described above for peptides and molluscan neurons (13, 16) and membrane properties may be involved. More precise microiontophoresis and intracellular data are needed to further the examination of such changes.


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I. Summary The magnocellular neuroendocrine cells in the supraoptic and paraventricular nuclei and the internuclear zone synthesize, transport, and release the neurohypophyseal hormones vasopressin and oxytocin, in addition to carrier proteins (the neurophysins), for the regulation of water balance and milk ejection. The rate of neurohypophyseal hormone release is dependent on the intensity of action-potential firing in the magnocellular neuroendocrine cells. As least two types of neurons, vasopressinergic and oxytocinergic, coexisting in the supraoptic and paraventricular nuclei and the internuclear zone, have been characterized by their spontaneous activity patterns as silent, continuously active, and burster. Whether these functional states of activity are related to the chemical cell type is unclear. Recurrent facilitation and recurrent inhibition are present in the magnocellular neuroendocrine cells, but the anatomical and chemical nature of these electrically stimulated synaptic events is unknown. The cell membrane of the magnocellular neuroendocrine cells has excitatory receptors for nicotinic cholinergic and angiotensin II putative synaptic transmitters, along with inhibitory receptors for muscarinic cholinergic and p-adrenergic types. Driven by osmotic, volumetric, circadian, nociceptor, limbic-midbrain, drinking, suckling, and vaginal distension stimuli, magnocellular neuroendocrine cells provide important support for self-preservation of the organism and for propagation of the species.

III.

PARVOCELLULAR

NEUROENDOCRINE

CELLS

A. Introduction 1. General Magnocellular neuroendocrine cells control posterior pituitary secretions; the parvocellular neuroendocrine cells control anterior pituitary hormonal release. Located in the hypophyseotrophic zone (146)) parvocellular neuroendocrine cells have smaller, anatomically less well-defined cell bodies with unmyelinated axons that project into the zona externa of the median eminence and terminate on the capillaries of the hypophyseal portal vessels. According to the hypothesis of portal vessel chemotransmitter regulation of the anterior pituitary, the hypophyseotrophic hormones are synthesized in the hypothalamic parvocellular neuroendocrine cells, transported along the nerve fibers, released into the extracellular spaces close to the primary portal capillary plexus, and carried to the anterior pituitary along the long and short portal vessels (140, 223). The median eminence, a neurohemal organ, therefore is the final common neural pathway to the glandular pituitary (223). A confluent capillary bed exists among the parts of the neurohypophy-


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NEURONS

603

sis, which include the median eminence, infundibular stalk, and infundibular process (323). This recently discovered structure fosters the possibility that releasing and neurohypophyseal hormones in the anterior pituitary, posterior pituitary, and median eminence will interact (323, 452; see Fig. 3). 2. Median

eminence

The anatomical site of the interface between the brain and the anterior pituitary, the median eminence, distributes releasing and inhibiting hormones to the glandular pituitary over a regional portal circulation (223). The unmyelinated fibers of the supraopticohypophyseal tract traverse the median eminence in the internal zone, passing to the posterior pituitary gland. The vessels of the pituitary portal plexus invaginate into the outer palisade zone, where they meet the terminals of the tuberoinfundibular tract (parvocellular endings), the endings of the monoamine@ and cholinergic neurons, and the end-feet of the ependymal tanycyctes (223). Tanycyte processes stream through the median eminence .from the floor of the third ventricle to portal vessels. These modified ependymal cells may be involved in chemical transport from the cerebral spinal fluid to the pituitary (223) or the reverse (323). In the median eminence the nerve terminals contain both dense-core and clear vesicles, abut in the perivascular space, may form axoaxonic contacts and make “synaptoid” contacts with ependymal cells. Theoretically, electrical stimulation of the median eminence can stimulate the parvocellular and magnocellular neuroendocrine fibers and the terminals of the monoaminergic (30, 135) and cholinergic (376) neurons. The absence of a physical barrier to current spread, such as the diaphragma sellae, however, and the short distance (0.5-2.5 mm) between the site of stimulation and the remainder of the hypophyseotrophic zone make selective stimulation of parvocellular neuroendocrine cell axon terminals technically demanding (see Fig. 3). 3. Chemical

and morphological

localization

of hypophyseotrophic

hormones

Three hypophyseotrophic (releasing and inhibiting) hormones have been chemically identified, synthesized and localized in the hypothalamus: thyrotrophin-releasing hormone (TRH), luteinizing-hormone-releasing hormone (LHRH), and somatostatin (SRIF) (345; see Table 1). The simple cyclic tripeptide TRH (pyro-Glu-His-Pro-NH,) induces thyroid-stimulating hormone (TSH) release (345). Within the hypothalamus, TRH is found concentrated in the median eminence, with lesser amounts in the ventromedial-arcuate, periventricular , and dorsomedial nuclei (48). The location of the cell bodies that synthesize TRH remains unknown; TRH-like activity is also present in significant amounts in the spinal cord, brainstem, mesencephalon, preoptic area, septum, basal ganglia, cerebral cortex (481, and circumventricular organs (222).


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57

CELLS

n LRH . SOMATOSTATIN @DA (3 TRH, etc. A STEROID-PEPTIDE RECEPTORS

ANT PITUITARY HORMONES FIG. 3. Hypothalamic parvocellular neuroendocrine cells and their connections. The two histochemically identified, chemically specific peptidergic neurons, LHRH and somatostatin, lie scattered in the periventricular regions of the preoptic area and the medial basal hypothalamus. The hypothalamic dopaminergic neurons lie in the arcuate nucleus. The locations of the cells of origin of thyrotropin-releasing hormone and other nonchemically identified hypophyseotrophic hormones (corticotrophin-releasing factor, prolactin-inhibiting factor, growth-hormone-releasing factor, etc.) are not histochemically identified. The vesicle-laden neurosecretory axons of these peptidergic and aminergic neurons descend through the hypothalamus to end on the capillaries of the primary portal plexus (PV) in the external zone of the median eminence. The hormones (LHRH, somatostatin, TRH) and others are released into the portal bloodstream under the influence of a variety of stimuli. The hypothetical hypothalamic steroid or peptide receptor neurons detect blood levels of circulating pituitary gland and target gland hormones and, via synaptic action on the hypophyseotrophic neurons, provide positive and negative feedback. See text for further details. Abbreviations: AC, anterior commissure; AP, anterior pituitary gland; ARC, arcuate nucleus; OC, optic chiasm; PP, posterior pituitary gland; PV, portal vessels; RH, hypophyseotrophic releasing and inhibiting hormones; n LHRH, luteinizing-hormone-releasing hormone-producing neurons; 0 SOMATOSTATIN, somatostatin-producing neurons; @ DA, dopaminergic neuron; G) TRH, etc., thyrotropin-releasing hormone and other hypophyseotrophic factor-producing neurons; A STEROID-PEPTIDE RECEPTORS, neurons responsive to steroids and peptides. (Based on references cited in sect. III.)


July

1977

HYPOTHALAMIC

605

NEURONS

Indicative of its role as a gonadotrophin-releasing hormone (GNRH), the simple linear decapeptide LHRH (pyro-Glu-His-Trp-Ser-Try-Gly-Leu-ArgPro-Gly-NH,) causes the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in a wide variety of mammalian species (345). The LHRH concentration is high in the median eminence, the arcuate nucleus and the organum vasculosum of the lamina terminalis and other circumventricular organs, with smaller amounts in the ventromedial, supraoptic and suprachiasmatic nuclei (48,220, 222,326). Located in the arcuate nucleus and the preoptic-anterior hypothalamus, the cell bodies that synthesize LHRH have nerve terminals ending in the median eminence (11, 18, 19, 369, 370, 377,451,454). Immunocytochemical methods have shown LHRH fibers in the median eminence to be located rostral, ventral, and lateral to SRIF fibers (221, 222). The simple cyclic tetradecapeptide somatostatin (H-Ala-Gly-Cys-LysAsn-Phe-Phe-Trp-Lys-Thr-Thr-Phe-Thr-Ser-Cys-OH) inhibits the release of basal and stimulated growth hormone (GH), the secretion of TRH-induced TSH, and the secretion of insulin and glucagon by pancreatic islet cells (345). Somatostatin was found in the highest concentrations in the median eminence, with high levels in the arcuate, periventricular, ventral premammillary, and ventromedial nuclei and in other circumventricular organs (48, 327). High levels of SRIF were found throughout the brain, pancreas, and stomach (48). Although immunocytochemical techniques have not clearly located SRIF in nerve cell bodies, this hypophyseotrophic hormone was found in nerve fibers in the median eminence and caudal, dorsal, and medial to LHRH fibers (221). Other hypophyseotrophic factors such as corticotrophin-releasing factor (CRF), prolactin-inhibiting factor (PIF), prolactin-releasing factor (PRF), and growth-hormone-releasing factor (GHRF) remain chemically unidentified at present. Information is limited regarding the location of nerve cell bodies synthesizing these factors (345). B. Antidromically

Identified

Single

Hypothalamic

Neurons

1. Introduction In the complex relationships between the brain and the pituitary and endocrine glands, the hypothalamus reacts to several types of hormonal feedback: the long feedback of the gonadal hormones estrogen and progesterone, the short feedback of the pituitary hormones LH and FSH, and the ultrashort feedback of the hypophyseotrophic hormone LHRH. In addition to these classical neuroendocrine roles, the releasing hormones may function as coordinators of diverse aspects of hypothalamic function. There is histochemical evidence (451) suggesting that neurons that secrete releasing hormones into the hypophyseal portal vessels also have collateral branches that synapse


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on other hypothalamic and limbic system cells (273). In this way a single neuron or group of neurons in the hypothalamus could influence the behavior of the animal and the secretion of its pituitary hormones.

2. Tuberoinfundibular

hypophyseotrophic

neurons

The parvocellular neuroendocrine cells are small, slow-firing hypothalamic cells in the acute1 .y prepared, urethan-anesthetized m .ammal . Without antidromic stimu lation the single-unit would be biased recording technique toward the larger, faster firing nonendocrine neurons. In a strategy designed to study these neuroendocrine cells, Makara et al. (255) stimulated the median eminence stalk junction electrically in urethan-anesthetized rats. They found antidromically identified single neurons located histologically (dye marking) in the hypophyseotrophic zone, anterior periventricular, and dorsal premammillary nuclei; 20% of these cells were spontaneously active and 80% were silent, wi th axon .a1 conduction velocities below 0.5 m/s in the Cfiber range. The median eminence stimulation site was 0.5 mm from the hypothalamus, with all cells found within 2.5 mm of this point. Other workers confirmed these studies (210, 213, 214, 276, 347, 348, 358, 444). Some of the difficulties involved in this study of these putative parvocellular neuroendocrine cells relate to possible current spread beyond the median eminence to axons en passage, the presence of other axon terminals in the area, and finally to the difficult task of identifying the various hypophyseotrophic neuron types, i.e., LHRH, TRH, SRIF, CRF, PIF, PRF, and GHRF. Other characteristics of these antidromically identified, tuberoinfundibcells include a recurrent collateral system ular parvocellular neuroendocrine (347, 443, 444) with GABA as a possible synaptic neurotransmitter (444) for inhibitory effects and unknown transmitters for excitatory effects (444). These parvocellular neuroendocr ‘ine cells may also have axon collaterals that extend to the median eminence, to adjacent cells, to the anterior hypothalamus, to the preoptic-anterior hypothalamus (155, 256, 347), and to the thalamic nucleus medialis dorsalis (444). Identified neuroendocrine cells in the arcuate nucleus presented two distinct pools of monoaminergic-sensitive neurons: those excited by norepinephrine and those excited by dopamine (276). Identified arcuate neurons showed a high level of unit activity in proestrous and castrated rats, where LH secretion is high (442). Estrogen produces accelerated cell discharge in identified arcuate nucleus units (213, 442), as does microiontophoresis of LH, FSH, and LHRH onto membranes of antidromically identified arcuate units of the urethan-anesthetized rat (213). During the estrous cycle, the responsivity of tuberoinfundibular neurons to LHRH fluctuates; a larger percentage of units respond in proestrus than in diestrus-1 (213). These initial studies on antidromically identified tuberoinfundibular neurons in the arcuate nucleus support the concept of the cells’ involvement in secretion and feedback regulation of pituitary gonadotrophin.


July

1977

C. Identified

HYPOTHALAMIC

and Nonidentified

Regulatory

NEURONS

607

Interneurons

1. Gonadotrophin a) Preoptic-anterior hypothalamus neurons. In order to select those medial preoptic-anterior hypothalamic neurons that might be involved in anterior pituitary regulation rather than sexual behavior, temperature regulation, and other activities, Dyer and co-workers (98, 102, 103, 105 117) stimulated the ventromedial-arcuate region electrically while recording from single preoptic-anterior hypothalamus cells. Their antidromically identified neurons for this region (type A, 41%) fired slowly at 1.2 spikes/s (20% silent), with a mean conduction velocity of 0.3m/s. These neurons could be driven antidromically and orthodromically by corticomedial amygdala stimulation (105, 117). Th eir nonantidromically identified preoptic-anterior hypothalamic cells were either orthodromically excited or inhibited by arcuate-ventromedial stimulation (type B cells, 32%) or were not affected by such stimulation (type C cells, 27%) (98). These nonantidromically identified preoptic-anterior hypothalamic neurons were also driven by antidromic and orthodromic corticomedial amygdaloid stimulation (105). The major problem with this experimental design is current spread, as evidenced by milk-ejection responses, and the indeterminate nature of the axons stimulated. The terminals, which may be in the arcuate-ventromedial nuclei or en passage elsewhere, certainly could be involved in functions other than neuroendocrine. Recognition of the preoptic-anterior hypothalamus influences on cyclic release of LHRH and LH caused Dyer (98) to examine the proestrous activities of the neurons in this area. The nonidentified neurons showed accelerated proestro us acti vity, but the antidromically identified cells did not change firin .g rates. Cells projecting to the arcuate-ventromedial nuclei (type A) received significantly more synaptic input from the amygdala in males than in either normal females or neonatally castrated males (105). Female rats neonatally treated with testosterone occupied an intermediate position. In normal females and neonatally castrated males, neurons that were not influenced by arcuate-ventromedial stimulation fired twice as fast as the same type of cell fired in normal males and females treated with testosterone. The sex-dependent properties of two categories of preoptic-anterior hypothalamic neurons were related to endocrine status. The male type was induced by exposing the brain to androgen in the critical perinatal period (105). Attempting to determine the effect of microinjecting a known LH-releasing agent like ferrous ions on the preoptic-anterior hypothalamus cells, Dyer and Burnet (101) recorded from nonantidromically identified cells in the proestrous, urethan-anesthetized rat. The uniform result of the maneuver was either depressed activity or no response. These workers concluded that some mechanism other than activation of preoptic-anterior hypothalamic neurons must be involved in LH release after deposition of iron. Dyball et al. (94) and Whitehead et al. (435) found antidromically identi-


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fied and nonantidromically identified cells consistently inhibited by norepinephrine and dopamine. Acetylcholine excited some preoptic-anterior hypothalamus neurons (94) but had no effect on other similarly located cells (435). To test the presence of short-feedback and ultrashort-feedback loops of hypophyseotrophic and anterior pituitary hormones on preoptic-anterior hypothalamus neurons, Kawakami and Sakuma (213) used iontophoresis of LH and FSH, with excitatory effects. On these same cells LRH had little effect. Microiontophoresis of TRH (7 of 17) and LRH (4 of 12) but not oxytocin (0 of 8) onto preoptic-anterior hypothalamus neurons inhibited spontaneous activity in the urethan-anesthetized rat (94). Despite the limitations inherent in b) Genital and sensory stimuli. recording from hypothalamic nonantidromically identified single cells or multiple-unit activity (62, 100, 307), these approaches can establish certain general principles for future more precise study. In the anesthetized or freely moving mammal, single- and multiple-unit activity shows characteristic regional patterns of spontaneous activity and responsiveness to genital and somatosensory stimuli. Depending on the stage of the estrous cycle and the circulating levels of pituitary hormones (2, 31 51, 54, 70, 88, 89, 106, 147, 208, 209, 211, 215, 231, 245, 261, 277, 336, 341, 416, 442, 455), changes in both spontaneous and evoked activity were also found. c) HypothaZamic isLand. Whether these hypothalamic regional unit patterns depend on local or generalized effects of anesthesia, on hormonal activity, on the level of arousal, or on other factors has been the subject of several studies. Cross and co-workers (63, 64, 68) isolated the recording zone, the hypothalamic island, from the remainder of the brain. They found an increased firing rate of hypothalamic units, suggesting that there was a tonic extrahypothalamic inhibition in the usually urethan-anesthetized rat (64). Despite its profound alteration of metabolism, urethan had no effect on hypothalamic unit activity, whereas fast-acting barbiturates (methohexitone) markedly depressed the firing rates of island cells (64). Neurons of the hypothalamus concentrate progesterone d) Progesterone: (357a): In their search for electrophysiological correlates of progesterone action, Cross and co-workers progesterone in (17 7 70) fou .nd that intravenous propylene glycol blocked the exci .tatory effect of cervical probing on lateral hypothalamic single-unit activity in the urethan-anesthetized rat. Since they considered this a specific effect on hypothalamic units, they did not monitor the EEG. When Sawyer and co-workers (27, 231) and Lincoln and co-workers (245-247) repeated these experiments, measuring the EEG along with singleand multiple-unit activity, they found that cervical probing produced EEG arousal and parallel nonspecific changes in most hypothalamic units of the unanesthetized rat. Progesterone induced high-voltage, slow synchronized EEG and simultaneously blocked both EEG arousal and hypothalamic unit acceleration. These workers (27, 231, 245-247) concluded that there was no evidence of a selective action of progesterone on the hypothalamus. Lincoln (245) and Haller and Barraclough (147) did find anterior and ventromedial hypothalamic single-unit activity that responded to cervical probing without


July

1977

HYPOTHALAMIC

NEURONS

609

arousal. Terasawa and Sawyer (398) found that integrated multiple-unit activity in the arcuate nucleus-median eminence responded specifically with a diurnal variation to subcutaneous progesterone in the ovariectomized, estrogen-primed, urethan-anesthetized rat. It may be concluded that, while nonspecific arousal can modify hypothalamic units, there are cells responsive specifically to genital stimuli. Progesterone in propylene glycol can nonspecifically depress the brain, but certain hypothalamic units are specifically responsive to this ovarian steroid and perhaps involved in its feedback action. e) Estrogen. Neurons of the hypothalamus concentrate estrogen (216, 261, 387). Looking for electrophysiological correlates to estrogen action, several groups found single- and multiple-unit activity reciprocally affected by estrogen in the preoptic-anterior hypothalamus as opposed to the posteriorlateral hypothalamus and mesencephalon (236). High levels of endogenous or exogenous estrogen produced depressed single-unit activity in the preopticanterior hypothalamus and increased similar activity in the lateral hypothalamus of the urethan-anesthetized rat (209, 243, 247, 440). Low-level estrogen and ovariectomy increased preoptic-anterior hypothalamus and decreased lateral hypothalamus single-unit activity (209, 247). While estrogen increased the percentage of units inhibited by somatosensory and genital stimuli in the lateral and anterior hypothalamus, it decreased the number responding in the lateral septal area (247). In contrast to septal, thalamic, and cingulate cortical single-unit activity (106, 277), single units in the preoptic-anterior hypothalamus of the rat showed accelerated firing patterns on the day of proestrus. In the deermouse the responsiveness of hypothalamic neurons to vaginal stimuli depended on the estrogen-dependent EEG after reaction. In the paralyzed cat, estrogen increased the anterior-medial hypothalamic multiple-unit response to somatosensory and genital stimuli, but the hormone depressed such responses in the mesencephalic reticular formation (2). In the chloralose-anesthetized cat, Ratner et al. (341) found that estrogen depressed the effects of genital stimulation on posterior hypothalamic single units but had no effect on the anterior hypothalamic units. Despite the diverse experimental methods used and the varied results, it is clear that estrogen can modify hypothalamic neuronal activity. Until more uniform neuronal populations are studied under appropriate conditions more system .atical 1Y 7 however, reproducible data are likely to be unavailable for future study f) Luteinizing hormone. In the search for the electrophysiological correlates of pituitary hormone action, hypothalamic neuron responses that correlate in time with hormone release have been found. Kawakami and Saito (211, 212) found that nonantidromically identified single-unit activity in the hypothalamus of the paralyzed cat increased or decreased firing rates and changed patterns of discharge when intravenous LH and oxytocin were administered. Pseudopregnancy induced by cervical stimulation in the freely moving rat was associated with biphasic multiple-unit changes in the arcuate-median eminence (inhibitory-excitatory) and the preoptic-anterior hypothalamus (excitatory-inhibitory) from day 1 to day 4 (208). Blake and 7


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Sawyer (31) simultaneously measured increased preoptic-lateral and basal medial hypothalamic integrated multiple-unit activity and LH release after genital stimulation in the proestrous, urethan-anesthetized rat. The rise in integrated multiple-unit activity began in both regions in 20 min, reached maximal levels in 12 h, and returned to base line in 3 h. These data are consistent with the hypothesis that the reflex discharge of LH in the proestrous rat involves preoptic activation of the basal hypothalamus. In the acute and chronic rat, Wuttke (439) found that cervical stimulation produced a biphasic neuronal effect after the initial elevation of medial preoptic, integrated multiple-uni .t activity, and LH rel .ease. Subsequently, integrated multiple-unit activity in the medial preop tic was depressed while LH remained elevated. Infusion of LH resulted in decreased integrated multiple-unit activity in the premammillary area and decreased it in the anterior hypothalamus as well. Infused LH increased integrated multipleunit activity in the premammillary area and decreased it in the anterior hypothalamus. Simultaneous measurement of LH after vaginal stimulation revealed elevation of both single-unit activity and LH in the unanesthetized rabbit (88, 89,416). These results indicate that mechanisms of LH release and feedback may influence hypothalamic unit activity in the mammal. g) EZectrochemicaZ stimdi. In order to study the release or inhibition of LHRH from the hypothalamus in response to anodal direct-current electrochemical stimulation to the brain, Sawyer and co-workers (136, 397) measured plasma LH, ovulation, and the integrated multiple-unit activity in the arcuate nucleus. Electrical activation in the medial preoptic area in female rats resulted in increased activity in the arcuate nucleus immediately, with peak activity in 10 min and for a total duration of 30-60 min (397). This increased arcuate integrated multiple-unit activity correlated with the activation of pituitary LH release and was followed, some hours later, by ovulation (397). Electrochemical activation of the ventral hippocampus in female rats caused increased integrated multiple-unit activity in the arcuate nucleus (136). This increased electrical activity, which began after 5 min, peaked in l2 h, and lasted 2-7 h. Pituitary LH was inhibited and ovulation blocked (136). Since increased arcuate integrated multiple-unit activity indicated LH release and ovulation with medial preoptic stimulation and inhibition of LH release and blockade of ovulation with ventral hippocampus stimulation, these authors were faced with a problem. They explained these apparently contradictory results by assuming the presence of two opposing pools of neurons in the arcuate nucleus. One pool responds after medial preoptic stimulation with increased activity and associated LH release (excitatory pool); the second pool responds after hippocampal stimulation with increased activity and depressed LH release (inhibitory pool). These data clearly demonstrate the limitations of the integrated multiple-unit technique. The researcher must discriminate among a variety of active individual neurons, since only the active neurons provide data. With this methodology the inhibited cell is unmeasurable (see Table 1 and Fig. 3). A dilemma arises with integrated multiple-unit activity. Does increased integrated multiple-unit activity measure cell soma, axon, or some other


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1977

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NEURONS

611

parameter (lOl)? Dyer and Burnet (101) tried to examine the locally increased integrated multiple-unit activity resulting from electrochemical stimulation in the medial preoptic area, as Columbo et al. (60) had attempted. Injecting ferrous ions into the medial preoptic area resulted in LH release, ovulation, and local destruction of neurons. When they recorded from single neurons and applied ferrous ions by microiontrophoresis, 24 of 29 cells were inhibited (101). They concluded that cell damage caused the elevated LH after medial preoptic electrochemical stimulation. Disinhibition of medial basal LHRH neurons or spread of LHRH from the damaged medial preoptic LHRH neurons to the medial basal hypothalamus by diffusion were the likely mechanisms invoked by these authors for the LH release and subsequent ovulation (101). When Sawyer and co-workers (58,59) exa .mined in tegra ted multiple-unit activ ity in the medial preoptic area and the ventromedial hypothalamus during potassium chloride-induced cortical spreading depression in the rat, they found depressed electrical activity 2-3 min after the chemical treatment, with recovery in E-20 min. Since their previous experiments indicated LH and prolactin release during such potassium chloride-induced cortical spreading depression, they argued that hypothalamic-depressed, integrated multiple-unit activity indicated inhibition of cortical or local hypothalamic neurons. Unfortunately, the lack of single-unit data can allow interpretation of integrated multiple-un .it acti vity accord .ing to the circumstance and therefore limit the value of the data. h) Luteinizing-hormone-releasing hormone. In order to establish whether LHRH has a direct effect on the electrical activity of hypothalamic neurons, Dyer and Dyball (104) studied nonantidromically identified singleunit activity in the preoptic-anterior hypothalamus. Four of 12 cells showed LHRH inhibition without effect on cerebral cortical units. In parallel studies in the urethan-anesthetized female rat, Renaud et al. (351) found inhibited cell discharge in 76% of the cerebellar cortical units and in 81% of the ventromedial nucleus hypothalamic units. Many LHRH-sensitive neurons in the cerebellar cortex and hypothalamus were also inhibited by thyrotropinreleasing hormone (TRH). These studies (104, 351) indicated that LHRH has a potent depressant action on the excitability of a certain population of neurons in several areas of the nervous system. The inhibitory effects on preoptic-anterior hypothalamic units found by Dyer and Dyball(104) contrast with the absence of LHRH effects on antidromically identified preopticanterior hypothalamic parvocellular neuroendocrine cells that Kawakami and Sakuma (213) found (see Table 1 and Fig. 3). The latter also showed excitation as well as inhibition to microiontophoresed LHRH in other parvocellular neuroendocrine cells. These data indicate that LHRH may function as both a hypothalamic hormone and as a synaptic neurotransmitter or neuromodulator in the regulation of gonadotrophic activity in the rat. 2. Adrenocorticotrophin a) Adrenocorticotrophin-steroid feedback. The secretion of corticotrophin-releasing factor, adrenocorticotrophin, and adrenal steroids is a complex


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phenomenon involving a number of control factors that include stress, circadian rhythms, and feedback mechanisms. The proposed negative-feedback effect of CRF, ACTH, and steroids acting through the central nervous system has been supported by evidence from a variety of experimental approaches (202, 232). In studies of the effects of steroids on brain electrical activity, Slusher et al. (379) found single cells in the posterior hypothalamus-midbrain tegmentum excited or inhibited within 10 min by intravenous or intracerebra1 injection of hydrocortisone in the paralyzed cat. In single cells in the anterior hypothalamus, 10 min after intravenous hydrocortisone in the pentobarbital-anesthetized cat, Feldman and Dafny (122) described acceleration of spontaneous activity but decreased responsiveness to peripheral sensory stimuli. Hypoxic stress produced short-term acceleration of single-unit activity in the lateral hypothalamic area and the preoptic-anterior hypothala mic area (69) and long-term inhibition of inte grated multiple-unit activity in the area (360) of arcuate-ventromedial nucleu .S and in the lateral hypothalamic excitatory effec ts of i ntravethe urethan-anesthetized rat Th e short-latency nous ACTH on arcuate-ventromedial nucleus integrated multiple-unit activity may represent an internal “short-loop” positive feedback; this would be an important mechanism for optimal stress response (360). This effect of ACTH was present both with and without adrenals (360). Intravenous dexamethasone produced an early (15 min) increase of arcuate-ventromedial nucleus integrated multiple-unit activity and a late decrease (35 min) of lateral hypothalamic area and dorsomedial hypothalamic integrated multiple-unit activity in the urethan-anesthetized rat (360). In order to localize more precisely the action site of the negative feedback of ACTH and steroids, Steiner et al. (354, 381, 383) examined the effects of chemical microiontophoresis on the firing patterns of over 300 nonantidromitally identified single hypothalamic and mesencephalic neurons in the chloralose-urethan-anesthetized rat. Dexamethasone phosphate depressed 57 cells (17%), activated 4 (la/,), and caused no change in the rate of discharge of 276 (82%). Steroid-sensitive cells were localized over wide areas in the hypothalamus and midbrain. No stero id-sensi tive neurons were found in the cortex, the dorsal hippocampus, or in the th alamus. Steroid-sensitive neurons were predominantly activated by microiontophoretically applied acetylcholine, whereas inhibition occurred with norepinephrine and dopamine. Locally delivered ACTH activated steroid-sensitive neurons. These results suggest that ACTH has a positive-feedback effect, corticosteroids a negative-feedback effect. These two effects may be modulated by the excitatory cholinergic and inhibitory monoaminergic influences of cells involved in regulating CRF secretion. Mandelbrod et al. (258) used cortisol sodium succinate in microiontophoresis to find that 177 (50%) of their tuberal hypothalamic, nonantidromitally identified single cells were steroid sensitive, 145 (41%) were inhibited, and 32 (9%) were facilitated. Yamada (446) found that microiontophoresis of betamethasone inhibited 11 of 22 prolactin-activated units and inhibited 4 of 8 prolactin-inhibited units in the hypothalamus. These results confirm the results of Steiner et al. (354, 381, 382).


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1977

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NEURONS

613

By using systemic administration in cats, earlier workers who studied the effects of corticosteroids on hypothalamic unit firing showed that cortisol primarily increased the rate of firing (122, 123, 379). Intravenous dexamethasone in the urethan-anesthetized rat increased or decreased the integrated multiple-unit activity in the zona incerta and dorsal-lateral hypothalamus without affecting the basal hypothalamus (360). In other studies in intact rats, intravenous cortisol or dexamethasone increased or decreased firing rates equally, depending on the recording area and the presence or absence of anesthesia (77, 79, 124, 125, 131, 331, 360). In studies of rats with diencephalic islands, intravenous administration of cortisol or dexamethasone yielded predominantly inhibitory responses from hypothalamic units (125,311). Data showing that corticoids are primarily inhibitory to island unit firing parallel the studies of Steiner et al. (354,381,382) and others (258,446). The excitatory effects of systemic corticoids in the intact animal possibly occur because of the action on remote neural structures synaptically connected to the hypothalamus. The net effect of steroid action depends on local inhibitory and remote excitatory effects as well as cholinergic and monoaminergic actions and the actions of other hormones. b) Circadian rhythms. Parallel circadian rhythms have been demonstrated at various levels of the brain of the intact rat. There is the pituitaryadrenal circuit, where 1) rhythms of plasma and adrenal corticosterone peak at the beginning of the dark period, 2) rhythms of plasma and pituitary ACTH peak earlier in the day, and 3) rhythms of the hypothalamic CRF content peak earlier than the corticosterone high (202). Searching for the basis for a 24-h rhythm of integrated multiple-unit activity of freely moving female rats, Terkel et al. (400) found that adrenalectomy abolished the nocturnal peak electrical activity in the arcuate-ventromedial nucleus and septum. These workers suggest that the circadian rhythm of hypothalamic integrated multiple-unit activity in the female rat may be related to the rhythm of ACTH secretion or hypothalamic CRF release. The trigger for CRF circadian rhythm probably depended in part on the retinohypothalamic pathways synapsing on the suprachiasmatic nucleus parvocellular neuroendocrine cells (269, 270), some of which synthesize vasopressin (451). The presence of vasopressin and neurophysin in the pituitary portal vessels (452) and the confluence of the capillary beds of the median eminence, infundibular stalk, and infundibular process (323) raised the possibility that vasopressin from the suprachiasmatic nucleus or elsewhere may be involved in ACTH release (269, 270, 452). 3. Suprachiasmatic

parvocellular

vasopressin

neurons

The suprachiasmatic nucleus is heterogeneous with parvocellular neurons, some producing vasopressin and neurophysin (412, 432, 451) and some, as Moore and Lenn (270) demonstrated, with input from the visual pathways in the rat. Five types of synapses were distinguished in suprachiasmatic neurons: two types of asymmetrical synapses (Gray type I, 33%) plus three


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types of symmetrical synapses 1Gray type II, 66% (l43)]. In protein-marker experiments with horseradish peroxidase, Swanson and Cowan (394) traced the efferent connections of suprachiasmatic neurons to cells of the periventricular area and on dendrites of the cells in the ventromedial, medial dorsal, and arcuate nuclear areas of the hypothalamus. Repetitive stimulation of the optic nerve or light acting on the eye augmented the firing of approximately half (42%) of the suprachiasmatic neurons (67 of 159 cells). Thirty-seven neurons (23%) showed clear inhibition by the same stimuli (298). Suprachiasmatic neurons showed short-duration oscillations at 3- to 5-min intervals. At other times the same neuron showed a steady low frequency of firing (298). Light, optic nerve stimulation, and suprachiasmatic stimulation inhibited the activity of cervical sympathetic nerves. Destruction of the suprachiasmatic nucleus resulted in the disappearance of circadian fluctuations of adrenal corticosterone levels in rats (269). These data suggest that suprachiasmatic neurons may be involved in light-mediated hypothalamic circadian rhythms important for endocrine and nonendocrine neurons. At present we do not know whether the vasopressinergic or the nonvasopressinergic neurons of the suprachiasmatic nucleus are involved in the relay of visual input to the hypothalamus. In the future a combination of cell recording, cell marking 1Procion yellow (162)], and immunohistochemical identification (451) techniques may clarify the function of the suprachiasmatic nucleus. 4. Thyrotropin Because of the wide distribution of thyrotropin-releasing hormone within the central nervous system (48; see Table 1 and Fig. 3) without specific localization within cell somata (451) and the induction of behavioral changes by systemic administration of TRH independent of the pituitary-thyroid axis (335), there is wide interest in this central nervous system peptide as a synaptic neuromodulator and as a hypophyseotrophic hormone. In support of a possible synaptic action, Dyer and Dyball (104) applied TRH microiontophoretically onto nonantidromically identified preoptic-anterior hypothalamic single neurons, and found 8 of 17 cells sensitive: 7 were inhibited, 1 excited. These TRH-responsive cells were not influenced by oxytocin, nor did TRH affect cerebral cortical single cells. Renaud et al. (349, 351) found 51% of the ventromedial nucleus neurons depressed by iontophoretically applied TRH with no excitation. In contrast with the work of Dyer and Dyball (104), Renaud’s group (349, 351) also found extrahypothalamic cell firing depressed in the cuneate nucleus (28%), cerebellar cortex (34%), and parietal cortex (47%). Twelve of 14 peptide-sensitive neurons (6 in the cerebellar cortex, 4 in the cerebral cortex, 4 in ventromedial nucleus) were depressed by both TRH and LRH (351). Coupled with the recent electrophysiological demonstration that axons of certain tuberoinfundibular neurons have multiple axon collaterals, one terminating on the portal vessels of the median eminence, others either terminating locally within the hypothalamus or in the extrahypothalamic regions (347,348,350), these data indicate a dual synaptic and hormonal


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1977

HYPOTHALAMIC

function for TRH. In future TRH by immunohistochemical functional role of this brain 5. Other pituitary

hormones,

NEURONS

615

studies the identification of the cells of origin of methods (451) will facilitate the analysis of the peptide. monoamines,

and prostaglandins

a) ProZactin. In a search for a short-loop feedback of prolactin on the brain, Clemens et al. (57) studied the effects of intravenous prolactin on the firing rates of single-unit activity in the hypothalamus of the unanesthetized rabbit. Of the 40 nonantidromically identified single units tested, 11 increased , 14 decreased , and I.5 showed no change. Responsive cells were located in the median dorsal, ventromedial, and arc uate regions and in the preoptic-anterior hypothalamus. Cross (62) questioned the specificity of these prolactin-induced responses. The marked variation of response time and response magnitude caused him to suggest that such nonreproducible unit data might be the product of spike-train analysis by window circuit and digital computer. Actually, much discontinuous unit data can be caused by the movement of the microelectrode tip toward or away from the cell under study. Mechanical irritation of the cell membrane can lead to acceleration or deceleration. A rising and falling spike height can lead to discontinuous recording by the window-circuit and computer-calculated histograms, thus distorting the data (129, 165, 168, 174, 319). Yamada’s prolactin microiontophoresis data (446) partly support the findings of Clemens et al. (57) of prolactin-sensitive neurons in the hypothalamus. Yamada (446) verified the findings of Clemens’ group (57) that many prolactin-activated neurons were present in the medial dorsal hypothalamus, while the prolactin-inhibited neurons were distributed diffusely from the arcuate nucleus to the zona incerta. In contradiction to the data of Clemens et al. (57), however, Yamada (446) found only a few prolactin-sensitive neurons in the preoptic-anterior hypothalamus and lateral hypothalamic regions. This difference may be explained by species variation (rabbit vs. rat), anesthesia effect (unanesthetized vs. urethan), or by assuming that most of the neurons found by Clemens et al. (57) in the preoptic-anterior hy -potha lamus and lateral hypothalamus could have been activated indirectly by prolactinsensitive neurons located in the medial dorsal hypothalamus or elsewhere. In addition to finding cortical cells unresponsive to prolactin, Yamada (446) discovered habenular nucleus units that were both excited and inhibited by prolactin. Approximately half the prolactin-sensitive neurons in the hypothalamus were inhibited by betamethasone and estrogen but unreactive to the microiontophoresis of TRH or oxytocin. These data support Clemens et al. (57) and indicate several possible feedback sites for prolactin in the diencephalon (446). b) Monoamines. The hypophyseotrophic system is influenced by the dopaminergic, noradrenergic, adrenergic, serotonergic, and histaminergic pathways that impinge on the basal medial hypothalamus (48). Studying the effects of exogenous amines on the electrical activity of the hypothalamus,


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Weiner et al. (433) administered monoamines into the third ventricle of estrogen-primed, ov ,ariectomized, urethan-an .esthetized female rats, while recording integrated multiple-unit activity in the median eminence, arcuate nucleus, and elsewhere in the hypothalamus and limbic system. Intraventricular epinephrine or norepinephrine (5 pg) caused LH release (234) and an increase of median eminence integrated multiple-unit activity over 6 min, followed by an hour-long decrease (433). Epinephrine was slightly more potent than norepinephrine in initiating this biphasic response. Dopamine was relatively ineffective for triggering either the initial increase or the secondary decrease in integrated multiple-unit activity. Biphasic responses were observed in the median eminence or in the border area separating the median eminence from the arcuate nucleus. Monoaminergic decreases in integrated multiple-unit activity were observed in the arcuate nucleus. Elsewhere in the hypothalamus or hippocampus integrated m ultiple-u .nit activity showed no consistent change. These data suggest that neurons containing epinephrine or norepinephrine with endings in the hypothalamus are influential in hormone-secreti-on control. When Fenske et al. (126) studied the effects of single-pulse electrical stimulation of the corticomedial amygdala, the mesencephalic raphe nuclei, and the medial basal hypothalamus on single, nonantidromically identified medial preoptic neurons in the urethan-anesthetized female rat, they found biphasic responses. Fifty-five percent of the medial preoptic cells were excited initially, followed by inhibition; 29% medial preoptic cells were inhibited initially, followed by excitation; 16% medial preoptic cells were unaffected. W ith each of these three Medial preoptic unit responses were consistent electrical stimulation sites. Pulse-train . stimulatio n of the amygdala or mesencephalon yielded medial preoptic facilitation at low frequency (10 Hz) and inhibition at high frequency (100 Hz). When intraventricular 5,6-dihydroxytryptamine or 6-hydroxydopamine depleted hypothalamic serotonin or catecholamin .es, respectively, these medial preoptic neuron responses did not alter with mesencephalic or medial basa 1 hypoth alamic stimuli. These data indicate that certain hypothalamic neuronal pools are not dependent on the presence of monoaminergic terminals for their interaction with limbic, mesencephalic, or other hypothalamic cells. c) ProstagZandins. Prostaglandins have been implicated in the release of adenohypophyseal hormones (237). It is not known, however, whether their action is on the brain, the pituitary gland, or both. In a study of 77 cells, 38 antidromically identified and 39 nonantidromically identified neurons in the preoptic-anterior hypothalamic and arcuate regions of the pentobarbitalurethan-anesthetized guinea pig, Poulain and Carette (334) found 64 sensitive neurons: 57 were excited and 7 inhibited by microiontophoresed prostaglandins (PGE*, 25 neurons; PGEaX, 52 neurons). The patterns of excitation by prostaglandins were 1) slow in onset and long-lasting, 27 cells; 2) rapid in onset, 11 cells; 3) strong but slow in onset, 4 cells; 4) active with modulated bursting activity, 15 cells. These data indicate that prostaglandins can exert powerful excitatory and inhibitory effects on parvocellular neuroendocrine


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cells and associated hypothalamic regulatory neurons that may be physiologically important for hypophyseotrophic hormone secretion and pituitary gland regulation. D. Summary Parvocellular neuroendocrine cells in the hypothalamus synthesize, transport, and release the hypophyseotrophic hormones - luteinizing releasing, thyrotrophin releasing, somatostatin, and others - into the primary plexus of the median eminence portal vessels for regulating the secretion of anterior pituitary trophic hormones. The exact anatomical locations of most of these parvocellular neuroendocrine cells are not well defined, with three exceptions. Luteinizing-releasing hormone has been located in cell bodies of the medial preoptic area, the supraoptic and arcuate nuclei, and the anterior hypothalamus. Dopamine, a potential hypophyseotrophic hormone, has been found in the cell bodies of the arcuate nucleus. Vasopressin, another potential hypophyseotrophic hormone, has been found in the cell bodies of the suprachiasmatic nucelus. Located in the arcuate and ventromedial nuclei and elsewhere in the medial basal hypothalamus as well as in the anterior periventricular and dorsal premammillary nuclei, those parvocellular neuroendocrine cells that have been antidromically identified are sensitive to putative transmitters, to hypophyseotrophic, pituitary trophic, and targetgland hormones, and to a wide range of sensory inputs. The nonantidromitally identified regulatory neurons are similarly sensitive. It has not been possible to determine whether a hypothalamic neuron under physiological study is involved, directly or indirectly, in the synthesis, transport, and release of a particular hypophyseotrophic hormone. Accordingly, these parvocellular neuroendocrine and regulatory neuronal data are inconclusive at the present time. IV.

NONENDOCRINE

A. Neurons

HYPOTHALAMIC

Associated

with

NEURONS

Thermoregulation

1. Introduction The hypothalamus, including the preoptic area, contains neuronal assemblies responsive to changes in local brain temperature and to neural input from spinal and peripheral thermoreceptors (112, 113, 149, 161, 180, 183). The integrative functions of the hypothalamus require inputs from a variety of sources, some of which are temperature receptors. Thermoregulatory effector mechanisms are triggered by shifts in arterial blood temperature and by changes in skin temperature, motor activity, level of behavioral arousal, and hormonal secretion. Therefore, the rise in temperature associated with exer-


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cise, the body temperature’s diurnal rhythm associated with sleeping and waking, and the hyperthermia that follows ovulation all represent biases superimposed on hypothalamic thermoregulatory neurons. Hypothalamic single-unit activity associated with thermoregulation is considered here (see Fig. 4). 2. Strategy

to identify

thermoregulatory

neurons

Attempting to determine the cellular basis for preoptic-anterior hypothalamus thermosensitivity and the nature of thermoregulatory mechanisms, a number of workers (see 112) have used the microelectrode technique for recording the activity of single, thermal ly responsive cells in the hypothalamus of the anesthetized or unanesthetized animal. Since the hypothalamus is a complex neuropil with many short neurons, multiple synapses, and few well-defined input and output pathways, the study of specific, anatomically and physiologically identified thermoregulatory neurons has not been possible. The major strategy has been to heat and cool the hypothalamus and to probe with an extracellular recording microelectrode those hypothalamic regions known to be thermally or electrically responsive or lesioned for thermal change. Once such thermally responsive neurons are identified, workers have attempted to classify them as thermodetectors, warm and cold interneurons (see 112). Further thermosensitive characterization involves thermally sensitive responses to peripheral thermal input, to central neural input, and to chemicals applied locally by microiontophoresis or administered intraventricularly or intravenously. At present there is no certainty that those neurons highly responsive to temperature change have a role in the transmission of thermal information or in thermoregulation of the intact animal (114). 3. Anatomy

of thermosensitive

neurons

a) Preoptic-anterior hypothalamus. In the earliest studies of single-unit activity in the preoptic-anterior hypothalamus thermosensitive zone, Hardy and co-workers (287, 288) examined in the urethan-anesthetized cat the firing-rate changes induced by heating and cooling preoptic and septal neurons, 2 mm either side of the midline, and found the neurons to be 80% thermally insensitive. About 20% had a positive temperature coefficient of their discharge frequency, responding with an increased firing rate on warming. All warm-sensitive cells had a Qlo of 5-15 (288). Similar results have been described in anesthetized dogs (151), anesthetized (36,52> and unanesthetized rabbits (175), and anesthetized rats and ground squirrels (37). Hardy’s group (151) first described cold-sensitive neurons with negative temperature coefficients in the anterior hypothalamus of chloralose-urethan-anesthetized dogs. There were 60% insensitive, 30% warm-sensitive, and 10% cold-sensitive neurons, with the three types seemingly randomly distributed in the explored


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NEURONS ASSOCIATED WITH THERMOREGULATION

A WARM DETECTOR n COLD DETECTOR

FIG. 4. Hypothalamic neurons a ssociated with thermoregulation. An e levated preoptic arterial blood temperature is detected by the preoptic warm detectors, which in turn activate the preoptic-anterior hypothalamic heat-loss neurons and inhibit (not shown) the preopticanterior hypothalamic heat production-conservation neurons. Simultaneous elevation of skin temperature is detected by cutaneous warm thermoreceptors, which further excite preopticanterior hypoth alamic heat-production-conservation neurons. Simultaneous elevation of cervical spinal cord arterial (not shown) blood temperature is detected by spi nal warm detectors with further excitation (not shown) of preoptic-anterior hypothalamic heat-loss neurons and inhibition of heat-production neurons. The preoptic-anterior hypothalamic heat-loss neurons activate the posterior hypothalamic heat-loss neurons. These in turn activate autonomic neurons for sweating, panting, behavioral heat-loss responses and cardiovascular neurons for cutaneous vasodilation. Reciprocal inhibitory connections (not shown) between these warmdetector, heat-loss neurons and the cold-detector, heat-production neurons provide for the inhibition of any heat production or for heat conservation activities such as piloerection, shivering, muscle activity, or behavioral state. During periods of a lowered preoptic arterial blood temperature and cold skin, the cold-thermodetector, heat-production set of neurons is activated and, reciprocally, the warm-detector, heat-loss set of neurons inhibited. The important input from limbic-midbrain circuits and chemically specific pathways is not shown. See text for further explanation. Abbreviations: AC, anterior commissure; AP, anterior pituitary gland; CV, cardiovascular effecters; HL, heat-loss effecters; HP, heat-production-conservation effecters; OC, optic chiasm; PP, posterior pituitary; n heat-loss interneurons; 0 heat-production-conservation interneurons; @ cardiovascular effector interneuron 23; A preoptic warmdetector neurons; n preoptic cold .-detector neurons. (Based on references cited in sect. 1VA.)


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area (151.). Similar results have been described in anesthetized cats (115, 267), anesthetized (36, 52) and unanesthetized rabbits (1751, and anesthetized rats and ground squirrels (37), as shown in Figure 4. In addition to these warm-sensitive and cold-sensitive neurons that show a linear or continuous relation between firing rate and local temperature over 8-lO”C, several groups have described four types of thermosensitive neurons with nonlinear or discontinuous temperature characteristics (37, 38, 115, 175, 267, 436). These neurons with a nonlinear response to temperature had a threshold temperature close to normal brain temperature for the species. Nonlinear thermal-firing relationships closely resembled the plots of the effector responses to hypothalamic heating and cooling. Eisenman and Jackson (115) proposed that the cells with positive linear slopes were the thermodetectors and that those cells with positive or negative nonlinear responses were interneurons This prosynaptically connected to the thermodetectors. posal was supported by their finding that, although the thermodetector group was relatively insensitive to barbiturates, the interneuron group was markedly depressed by barbiturates (I 15). Cell size, deduced from the duration of recording, is another possible method for classifying thermosensitive neurons in the preoptic-anterior hypothalamus. Based on the spontaneous firing rate and the duration of recording, Boulant and Bignall (37) found two groups of thermosensitive neurons, small and large, in the anesthetized or decerebrate rat and ground squirrel. One group with single units with higher spontaneous firing rates (>5 spikes/ s), held for longer than 90 min, had the greater proportion of thermosensitive neurons (53% warm sensitive, 16% cold sensitive, 31% insensitive). These Boulant and Bignall considered the small-cell thermodetectors. Other preoptic-anterior hypothalamic units with lower spontaneous firing rates (~5 spikes/s), and held for less than 75 min, had fewer thermosensitive cells (19% warm sensitive, 18% cold sensitive, 63% insensitive) of this type. Boulant and Bignall (37) considered these the large-cell nonthermodetectors. Whether cell damage biased the data from either of these cell groups is unknown. When Boulant and Bignall (38) examined the spontaneous firing rates and the local thermosensitivity of these thermodetector cells, they found changes that could not be correlated wih any of the measured temperatures. They concluded that undetected factors (blood glucose, endocrine& may influence thermoregulation by affecting preoptic-anterior hypothalamic neuronal activity. b) Posterior hypothalamus. The posterior hypothalamus is less thermosensitive than the preoptic-anterior hypothalamus (1831, though still important for the integration and transmission of thermoregulatory impulses (see Fig. 4). In the earliest single and multiple-unit activity in the hypothalamus, Birzis and Hemingway (29) interpreted the increase or decrease of posterior hypothala mic unit ac tivity to whole-body cooling or heati .ng, respectively 7 as indicative of efferent motor activity related to shivering. Cabanac et al. (52) reported the presence of thermosensitive neurons in the posterior hypothalamus of anesthetized rabbits. In a detailed analysis of posterior hypothalamic


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HYPOTHALAMIC

621

NEURONS

and tuberal thermosensitive neurons in anesthetized cats, Edinger and Eisenman (107) found fewer thermodetector (high Q,tJ, linear, continuous) cells in this region (7%) than they had previously found in the preoptic-anterior hypothalamus 122% (1191. Other thermosensitive and insensitive neuronal types were similar in these regions of the hypothalamus (112). In the urethananesthetized rabbit, Wunnenberg and Hardy (438) found 17% of their units responsive to local temperature change in contrast to 30-70% in the preopticanterior hypothalamus. These unit data suggest the posterior hypothalamus serves more as a center for integrative thermoregulation than for primary thermodetection (see Fig. 4).

4. Central

and peripheral

input

to thermosensitive

neurons

a) Thermal input: preoptic-anterior hypothalamus. Physiological data indicate that thermoregulatory responses can result whenever central hypothalamic, spinal cord, or peripheral cutaneous thermoreceptors or combinations of these (181) are activated. Investigations into the exact nature and sites of these interactions have utilized the microelectrode technique. In an early study, Murakami et al. (280) found that local hot or cold stimuli to the skin of the anesthetized dog failed to change preoptic-anterior hypothalamic unit firing patterns. When Wit and Wang (436) heated the whole body surface of the anesthetized cat, they found 16% of responsive preoptic-anterior hypothalamic neurons also responsive to elevated brain temperature. In the anesthetized rabbit, Hellon (176, 177) observed that 75% (6 of 8) of responsive preoptic-anterior hypothalamic neurons were also responsive to peripheral thermal stimulation and to shifts in hypothalamic temperature. Five of these units had the same general slope of the thermal response curves for both central and peripheral thermal stimuli. In the anesthetized rabbit, Guieu and Hardy (142) found 13 locally thermosensitive preoptic-anterior hypothalamic neurons of the nonlinear, warm-interneuron type also responsive to changes in spinal cord temperatures. Ten of these units had similar slopes of the thermal response curves for both spinal and preoptic-anterior hypothalamic temperatures. In the anesthetized rat and ground squirrel, Boulant and Bignall (39) found 75% (9 of 12) of the preoptic-anterior hypothalamic units responsive to both peripheral and central thermal stimulation, with most (7 of 9) having similar slopes of the thermal response curves for both local preoptic and peripheral cutaneous temperatures. In these studies, peripheral thermal stimulation that resulted in increased preoptic-anterior hypothalamic unit firing was usually associated with a decreased local thermosensitivity, suggesting a competititon between central and peripheral facilitatory inputs for neuronal excitation (39). In the urethan-anesthetized rat, Knox et al. (226) found a correlation between preoptic-anterior hypothalamic integrated multiple-unit activity and single-unit activity firing and changes induced in tail skin temperature. Reaves (342) confirmed the presence of dually thermosensitive preoptic-anterior hypothalamic neurons in the unan-


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esthetized rabbit when he found cells responsive to local preoptic-anterior hypothalamic temperature peripheral thermal stimulation (see and also to Fig. 4). In a systematic study of the neuronal integration of thermal information, Boulant and Hardy (40) observed the effect of spinal and skin thermal afferents on the firing rate and thermosensitivity of preoptic-anterior hypothalamic neurons in urethan-anesthetized rabbits. In warm-sensitive preoptic-anterior hypothalamic neurons the incidence of extrahypothalamic (spinal and cutaneous) thermal afferents increased among units having higher firing rates. There was no such correlation among the cold-sensitive preopticanterior hypothalamic units. These data suggest that peripheral thermal afferents may determine the level of firing rate in warm-sensitive units. With preoptic-anterior hypothalamic temperature at 38°C an increase in the firing rate usually resulted in a decreased preoptic thermosensitivity in the warmsensitive preoptic-anterior hypothalamic neurons, but an increased preoptic thermosensitivity in the cold-sensitive units (40). On the basis of these data, Boulant (36) proposed a neuronal model for thermoregulation in which peripheral temperatures should have relatively little influence on heat-loss responses, but should integrate with hypothalamic temperature in the control of heat-production responses. The heat-loss responses s hould be al .most enti rely controlled by hypothalamic temperature (36). This concept of unequal influences of peripheral temperature on heat-loss and heat-production responses has been demonstrated in dogs implanted with hypothalamic thermodes (183). b) Thermal input: posterior hypothalamus. In their study of the responses of posterior hypothalamic neurons in anesthetized rabbits to local, preoptic-anterior hypothalamic and spinal thermal stimulation, Wunnenberg and Hardy (438) found that most of these posterior hypothalamic thermosensitive types - warm-sensitive, cool-sensitive, linear, nonlinear thermodetector and interneuron-responded to thermal inputs from spinal cord and preoptic-anterior hypothalamus. These results differed strikingly from those obtained in the preoptic-anterior hypothalamus. There, interneuron units (warm-sensitive, cool-sensitive, nonlinear units) responded to spinal cord thermal stimuli (142), while thermodetector units (warm-sensitive, coolsensitive, linear units) did not respond. Nutik (304, 305) studied posterior hypothalamic neurons, finding them responsive to cutaneous and preoptic anterior hypothalamic thermal stimuli in the thiopental or locally anesthetized and paralyzed cat. c) Neural and humoral input and sleep-waking activity. In the anesthetized or unanesthetized mammal the influence of oscillating or rhythmic neural and humoral factors on thermoregulatory neurons has been investigated, but incompletely. Murakami et al. (280) applied noxious peripheral stimuli to the unanesthetized or anesthetized dog, finding acceleration or inhibition, respectively, of both warm-sensitive and insensitive preopticanterior hypothalamic neurons without alteration of temperature responsivity in these warm-sensitive neurons; EEG activity was not monitored in these studies.


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Cutaneous vasoconstriction, elevation of hypothalamic temperature, and acceleration of nonidentified, single hypothalamic neurons were observed during paradoxical [rapid-eye-movement (REM), fast-wave] sleep in the rabbit (129) and cat (328). The effects of sleep-waking activity on thermosensitive neurons are not known. Edinger and Eisenman (107) found two posterior hypothalamic neurons that fired at the extreme ends of the thermal range (warm-cool units), increasing their firing rates with both heating and cooling and speculated that these units might be part of a generalized alerting system responding to potentially harmful levels of central temperature. No EEG was monitored in these studies. In the rat and ground squirrel, Boulant and Bignall(38) described rapid, slow-fluctuating increase and decrease of spontaneous firing rate and thermosensitivity in single preoptic-anterior hypothalamic units. These workers saw no correlation with peripheral, deep-body, or brain temperatures. They speculated that blood glucose or hormonal levels might be involved. These studies lack EEG correlation. In the urethan-anesthetized cat, Eisenman (113) found that single electrical shock stimuli applied to the ventromedial mesencephalic reticular formation activated 62% of thermosensitive preoptic-anterior hypothalamic neurons. Seventy-three percent of responding units were inhibited and 27% facilitated. Midbrain stimuli affected only 48% of the thermally insensitive cells studied, with equal numbers facilitated and inhibited. The thermodetector type (high QIo, linear, continuous) neurons in the preoptic-anterior hypothalamus were among those thermosensitive cells facilitated and inhibited by midbrain and pontine stimulation. Conduction latencies indicated monosynaptic or oligosynaptic facilitatory pathway, as well as a more complex inhibitory pathway. These studies seem to indicate that ascending noradrenergic and serotoninergic pathways may be involved in facilitation and inhibition of preoptic-anterior hypothalamic thermosensitive neurons either directly or via interneuron activation. Since these same monoaminergic pathways are involved in the modulation of sleep-waking behavior and hypothalamic neuroendocrine secretion, the integration and interaction between preoptic-anterior hypothalamic thermoregulatory neurons and these other activities may occur at the level of the brainstem monoaminergic neurons (182). cl) ZnteruaZ coding of temperature. It is not known whether the mean rate of hypothalamic unit activity provides an adequate description of the functional role of a neuron. In the urethan-anesthetized cat, Eisenman (112) described thermodetector preoptic-anterior hypothalamic neurons that, upon heating, developed a bursting pattern. The accompanying bimodal distribution of interspike intervals and a mean firing rate were not representative of the much higher intraburst frequencies. In their analysis of periodic components of hypothalamic thermosensitive neurons, Jahns and Werner (196,197) described three classes of units: those with periodicities at all thermal states, those with correlograms with periodicities at only one or two thermal states, and those with no recognized periodicities at all. The warm-sensitive cells were characterized predominantly by a periodic correlogram. Nonthermosensitive cells predominantly showed a correlogram without periodicity at all temperatures or at one temperature phase only. Cold-sensitive cells usually


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did not reveal periodicity. These results indicate that a coupling exists between thermosensitivity, particularly the warm-sensitive cells, and the periodic behavior of the neuron. Just how much rhythmic oscillations become relevant indices of information transmission for thermoregulation remains to be studied. In their single- and multiple-unit activity study of rate-insensitive ( Qlo = 0.6-l .4) preoptic-anterior hypothalamic neurons in the unanesthetized rabbit, Reaves and Heath (343) found shifts from unimodal to bimodal distributions of interspike intervals with preoptic and cutaneous thermal stimuli. In parallel studies Reaves (342) found that a gain near 10.0 was characteristic of preoptic-anterior hypothalamic thermosensitive neurons in the unanesthetized rabbit and in other mammalian thermoregulatory processes. These studies of Eisenman (112), Jahns and Werner (196, 1971, and Reaves and Heath (342, 343) indicate that, in addition to firing rate, factors such as interspike interval distribution should be considered when determining thermosensitivity of preoptic-anterior hypothalamic neurons and included in models of thermoregulatory systems as a means of coding thermal information.

5. Pharmacology a) Biogenic amines. The monoaminergic and cholinergic neurons of the brainstem project rostrally to the diencephalon, with resulting high levels of these biogenic amines found in the hypothalamus (48). Intracerebroventricular microinjection of norepinephrine, serotonin, and acetylcholine suggests that these amines are involved in hypothalamic control of body temperature (182, 185). De pending on the species studied, different groups have obtained opposing results with these particular amines. The results of Myers and Yaksh (284) in the primate suggested that norepinephrine mediated heat loss and serotonin mediated heat production, but acetylcholine appeared to mediate both. In sheep, goats, and rabbits, Bligh et al. (33) obtained results that suggested that serotonin mediated heat loss, whereas norepinephrine seemed to be active in the mediation of reciprocal inhibition of both heat loss and production; acetylcholine appeared to facilitate heat production. Assessing the effects of intravenous, intracerebroventricular, or local (microiontophoretic) administration of the biogenic amines on preoptic-anterior hypothalamic thermosensitive and nonthermosensitive single neurons might resolve some of these discrepancies. Using chloralose-urethan-anesthetized dogs, Cunningham et al. (72) tried to correlate the thermal properties of preoptic-anterior hypothalamic neurons with their response to serotonin and epinephrine administered intravenously or by intraventricular injection. Biogenic amines depressed the activity of temperature-sensitive and temperature-insensitive preoptic-anterior hypothalamic units. This generalized depression does not match the proposed antagonistic role for serotonin and epinephrine in maintaining body temperature.


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Applying norepinephrine, serotonin, and acetylcholine directly to preoptic-anterior hypothalamic neurons microiontophoretically, Beckman and Eisenman (23 7 112) found similar results in cat and rat. Thermodetector (high Qlo, linear, continuous), warm-sensitive cells were affected by current flowing from the micropipette but very few responded to the amines. These results were interpreted as indicative of the lack of synaptic input for thermodetector neurons (see Fig. 4). Subsequently, Eisenman (113) showed these thermodetector neurons receiving facilitatory and inhibitory input from the ventrolatera1 mesencephalon and the midline raphe of pons. The warm-sensitive interneurons (positive slope, nonlinear, discontinuous) were accelerated by acetylcholine and inhibited by norepinephrine. The cold-sensitive interneurons (negative slope, nonlinear, discontinuous) were accelerated by norepinephrine but inhibited by serotonin. Murakami (279) found somewhat similar effects in the rat. These results are consistent with the concept of a transmitter role for these substances in thermoregulation. In the rat these findings are expected from the hyperthermic action of norepinephrine and acetylcholine and the hypothermic action of serotonin .. Paradoxically, preoptic-anterior hypothalamic thermosensitive neurons in cats showed the same responses as rats, yet these amines had opposite effects on body temperature (182). In subsequent cat studies, Jell (198, 199) examined the effects of norepinephrine, serotonin, and acetylcholine microiontophoresis on preoptic-anterior hypothalamic single neurons responsive to local hypothalamic (198) or facial cutaneous thermal stimuli (199). He found no consistent relationship between amine responses and responsiveness to either preoptic-anterior hypothalamic (198) or facial temperature (199). He concluded that his cat results tended to support the sheep, goat, and rabbit amine model of Bligh et al. (33) rather than the primate amine model of Myers and Yaksh (284). In the rabbit, intracerebroventricular injections of norepinephrine produced hyperthermia; injections of serotonin and acetylcholine led to hypothermia (182). The pharmacological evidence pointed to the involvement of norepinephrine in pathways controlling heat production and conservation, whereas serotonin and acetylch .oline activated heat loss (182 ). In the urethan-anesthetized rabbit, Hori and Nakayama (185) showed the majority of warm-responsive, single preoptic-anterior hypothalamic cells were accelerated by serotonin, inhibited by norepinephrine. Cold-sensitive preoptic-anterior hypothalamic units were depressed by serotonin and excited by norepinephrine, whereas acetylcholine was without influence on any thermosensitive rabbit neurons. These results are consistent with the body temperature changes produced by serotonin and norepinephrine injections into the rabbit hypothalamus (33, 182). Similar results were found in the rat (225). Whether these differences in amine response between single thermosensitive hypothalamic neurons and intracerebroventricular microinjection represent an anatomical-physiological variation among species, a methodological difference among experimenters, or the pharmacological nature of the test systems are problems as yet unsolved. The release of endogenous prostaglandins to produce fever and the anesthetic effects of agents to produce hypothermia may influence some of these results.


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b) Fever. Wit and Wang (437) studied single preoptic-anterior hypothalamic neurons in the urethan-anesthetized cat in order to determine the central site of pyrogen action. Intravenous bacterial endotoxin pyrogen produced a depression of the warm-thermosensitive neuronal response to an increase in brain temperature. These results, confirmed in the cat (111) and rabbit (52), indicate that bacterial endotoxic fever is probably caused bY reciprocal effects on thermal responsiveness of neurons in the preoptic-an terior hypothalamic thermoregulatory areas. The action of acetylsalicylate on fever was studied at the single, preopticanterior hypothalamic cell level in the urethan-anesthetized cat by Wit and Wang (437). Warm-sensitive preoptic-anterior hypothalamic neurons were depressed to local thermal stimuli by intravenous pyrogen. When intravenous or intracarotid acetylsalicylate followed, the cells’ thermal responsiveness returned toward the prepyrogen level within 30-70 min. These experiments suggest that antipyretic effects of systemic acetylsalicylate are the reversal of pyrogen suppression by warm-sensitive preoptic-anterior hypothalamic neurons. In order to validate these presumptions and to examine the pyretic-antipyretic antagonism, Schoener and Wang (364) studied the effects of microinjections of pyrogen and acetylsalicylate on preoptic-anterior hypothalamic thermosensitive neurons. They found that the microinjection of cat to preoptic-anterior hypothalamus, proximate leukocyte pyrogen into the warm-sensitive (thermoposit ive) or cold-sensiti .ve (thermonega tive) interne Urons, was followed by a prompt depression of the thermopositive unit firing rate and an elevation of all thermonegative unit discharges. Microinjecting acetylsalycilate alone into preoptic-anterior hypothalamus caused little change in thermosensitive neuronal discharge. When acetylsalicylate was given after pyrogen, however, thermopositive units were released from pyretie inhibition and thermonegative ones from excitation. These results indicate that the action of an antipyretic agent, acetylsalicylate, as well as that of leukocyte pyrogen, occurs within the preoptic-anterior hypothalamic area (364). Whether these effects are mediated by prostaglandins remains to be determined. Prostagl .andin #El, a potent pyrogenic agent when microinjected into the preoptic-anterior hypothalamus of rabbits (182)) was applied by microiontophoresis to preoptic-anterior hypothalamic single units in the urethan-anesthetized rabbit (383). Prostaglandin E, indiscriminately facilitated the action preoptic of 8--10% of warm-sensi tive, cold-sens litive, and therma lly insensitive neurons. There was no evidence that prostaglandin E 1 acted as an an .tagonist against the effect of norepinephrine on preoptic-anterior hypothalamic units. Jell and Sweatman (200) confirmed these negative results in the cat. Because E, as a of these experiments, no simple neuronal basis for prostaglandin fever-produci .ng agent in conscious rabbits can be predicted. These results suggest that antipyretic agents, such as acetylsalicylate, that can produce prostaglandin synthetase inhibition are not dependent on prostaglandins for their actions. Progesterone produces its pyrogen effect by mechanisms that are poorly


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understood. When administered either intramuscularly or intravenously, progesterone was followed by a depression of the firing rate of warm-sensitive (thermopositive) units and an elevation of discharge rates of cold-sensitive (thermonegative) preoptic-anterior hypothalamic neurons in the urethananesthetized rabbit (289). Thermally insensitive neurons were not affected by progesterone. It seems probable that progesterone (or its metabolites) has a direct or indirect action on preoptic thermosensitive neurons, producing a pyrogenlike upward shift of the set point of basal body temperature (289). 7. Summary Containing neurons sensitive to central and peripheral thermal input, the preoptic-anterior hypothalamus is considered to be the brain’s thermoregulatory center (see Fig. 4). Of the thermoregulatory neurons, the linear warm-sensitive and cold-sensitive neurons are considered by some to be thermodetectors, whereas the nonlinear warm-sensitive and cold-sensitive neurons are described as interneurons. The posterior hypothalamic thermosensitive neurons show primarily nonlinear interneuron curves (see Fig. 4). Cold- and warm-thermosensitive neurons are sensitive to cutaneous thermal input, nociceptor stimuli, and chemical influences, including biogenic amines, acetylcholine, pyrogen, acetylsalicylate, prostaglandins, and progesterone. The major challenge to further systematic study of hypothalamic thermoregulatory neurons remains the anatomical, chemical, and physiological identification of thermodetectors and thermosensitive interneurons and their input-output connections. All these thermoregulatory neurons described in this section were nonantidromically identified.

B. Neurons

Associated

with Feeding

1. Introduction Adjacent to the third ventricle, the medial region of the hypothalamus contains the highly cellular ventromedial nucleus with neurons and dendrites that extend into the more lateral hypothalamic area (266,395; see Figs. 1 and 5). In this area the widely dispersed cells lie among the fibers of the medial forebrain bundle, where the ascending monoaminergic and cholinergic pathways are also found (30, 48, 136, 266, 375, 395). Lesions in the ventromedial nucleus cause hyperphagia and lateral hypothalamic lesions cause aphagia (271, 303); electrical stimulation elicits feeding behavior from the lateral hypothalamus and feeding suppression from the ventromedial nucleus (271,303). On the basis of these and other studies the lateral hypothalamic area is designated the feeding center and the ventromedial nucleus the satiety center. Several types of hypothalamic chemostatic controls over food intake have been postulated: ventromedial nucleus glucoreceptors sensitive


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NEURONS ASSOCIATED WITH FEEDING

u LHA “APPETITE l VMH “SATIETY” A RECEPTORS

FIG. 5. Hypothalamic neurons associated with feeding behavior. An elevated arterial blood level of substrate (glucose, nonesterified fatty acids, amino acids, other) is detected by the ventromedial substrate detectors, which activate the ventromedial satiety neurons and inhibit the lateral hypothalamic feeding neurons. Simultaneous afferent input from the oral cavity (gustatory input), pharynx and upper gastrointestinal tract (food intake) and other visceral receptors provides additional excitatory input to ventromedial satiety neurons (oropharyngeal afferents) and inhibitory input to the lateral hypothalamic feeding neurons. Reciprocal pathways from ventromedial to lateral hypothalamic areas further reinforce the satiety or feeding state. The important input from limbic-midbrain circuits andchemically specific pathways is not shown. See text for further explanation. Abbreviations: AC, anterior commisure; AP, anterior pituitary gland; OC, optic chiasm; PP, posterior pituitary gland; n LHA APPETITE, lateral hypothalamic feeding interneurons; 0 VMH SATIETY, ventromedial hypothalamic satiety interneurons; A RECEPTORS, ventromedial hypothalamic substrate receptors. (Based on references cited in sect. IV&)

to blood glucose, ventromedial nucleus lipostatic receptors sensitive to body fat stores by detection of plasma free fatty acids, and a thermostat dependent on the specific dynamic action of food intake. Other inputs such as visceral, olfactory and gustatory sensory, limbic system, behavior, and drugs are also recognized as modulators of food intake (see Figs. 1 and 5). An important finding of these studies of feeding is the reciprocal effect various inputs have on ventromedial nucleus versus the effects produced on lateral hypothalamic neurons, suggesting that some integrative interaction


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may be involved between medial and lateral hypothalamic structures. Levels of glucose, free fatty acids, insulin, and the degree of stomach distension, as well as the stimuli from vision, smell, taste, and other sensory systems, may be important for eliciting feeding and satiation behavior. Important modulating influence for hypothalamic organization of feeding behavior is derived from the limbic system, in particular the amygdala (271, 303).

2. Glucostatic theory When Anand et al. (4) and Oomura et al. (314) examined the glucoreceptor hypothesis by recording from nonantidromically identified single hypothalamic cells in anesthetized cats, dogs, and rats, they found that while intravenous glucose excited ventromedial nucleus neurons (satiety center), it inhibited lateral hypothalamic units (feeding center) (321). After further multiple-unit activity study in the paralyzed rat, Marrazzi (260) and others (46) found excitatory-inhibitory, biphasic responses of ventromedial nucleus neurons after intracarotid hypertonic glucose. Using microiontophoresis of the ether-anesthetized rat, Oomura et al. (318) studied the direct effects of glucose on individual ventromedial and lateral hypothalamic cells. Approximately half the ventromedial nucleus cells were specifically excited by glucose (0.4 M) with no effect from NaCl (0.2 M). Two classes of lateral hypothalamic neurons were found: 1) osmosensitive neurons that hypertonic glucose and hypertonic NaCl excited or inhibited nonspecifically and 2) glucosesensitive neurons that hypertonic glucose but not hypertonic NaCl inhibited specifically. C ells in th .e thalamus and cortex did not respond to gl ucose. Subsequent1 .YY Oomura et al. (322 ) confirmed these results. When these glucose-sensitive lateral hypothalamic neurons were inhibited by glucose, intracellular recording revealed marked inhibition of spontaneous spike generation, accompanied by hyperpolarization of the membrane (320). These workers suggested that, when ouabain or sodium azide blocked this inhibition, the glucose-induced hyperpolarizations were the result of energy-dependent sodium pump activation. Chhina et al. (55) found further support for a direct hypothalamic effect of glucose in decerebrate, starved cats. Intra .venous glucose accelerated the slow, resting firing rate of single, nonantidromitally identified ventromedial nucleus neurons, while it inhibited the faster, control discharge of lateral hypothalamic neurons. These results support the general “regulation of food intake” thesis that there is reciprocal glucose action between the ventromedial nucleus and lateral hypothalamus. Glucoreceptor mechanisms in the liver can respond to changes in portal blood glucose concentrations (296) with the potential for modifying hypothain lamic neuron .a1 activity. When Schmidt (362) exam .ined this possibility urethan-anesthetized rats, she found that portal vein-infused glucose and hypertonic NaCl produced facilitatory and inhibitory responses from lateral nerve and T, cord section blocked these hypoth .alamic neuron 5. Splanchnic lateral hypoth .alamic ne uronal effec ts, whereas bilateral vagotomy enhanced


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them. No effects of intraportal infusion of glucose or hypertonic NaCl were found in the ventromedial nucleus. These lateral hypothalamic neurons did reveal a circadian rhythm: at 12 noon spontaneous firing occurred at slow rates of 0-0.5/s; at 5-9 P.M. optimal and consistent responses to intraportal hypertonic glucose or hypertonic NaCl occurred at higher rates of 30-40/s (363). These data suggest that neural input from liver receptors travels over splanchnic nerves and spinal cord pathways. The role these hepatic glucoreceptors play in regulation of food intake remains poorly defined. 3. Lipostatic

theory

Food intake and body weight in mammals correlate closely with a decrease (lipogenesis) and increase (lipolysis) in blood free fatty acids. Mogenson (268) proposed that by preventing the animal from adding weight the ventromedial nucleus acts as a brake, while the lateral hypothalamic area prevents the animal from losing fatty tissue. Feeding causes lipogenesis and an increase in blood insulin and glucose; fasting causes lipolysis, with its respective decrease in blood insulin and glucose. The increased free fatty acid concentration during hunger, however, can be assigned partially to the increased release of both growth hormone and epinephrine. Because of correlated changes in free fatty acid, glucose, and insulin, the glucose-sensitive neurons of the lateral hypothalamic area and the glucoreceptor neurons of the ventromedial nucleus may contain complex chemosensitive properties that respond to glucose levels as well as to free fatty acid and other metabolic products. When Oomura et al. (316) tested the free fatty acid effects on glucose-sensitive lateral hypothalamic neurons that were inhibited by glucose microiontophoresis, they found that free fatty acid facilitated these lateral hypothalamic glucosensitive neurons. Lateral hypothalamic neurons that were insensitive to glucose were also unresponsive to free fatty acid. In studies where free fatty acids were applied electro-osmotically to lateral hypothalamic glucosensitive and to ventromedial glucoreceptor neurons in the rat, the former facilitated and the latter inhibited (313). Approximately 50% of the glucose-sensitive neurons were responsive. These results suggest free fatty acids depolarize lateral hypothalamic neurons and hyperpolarize ventromedial nucleus neurons. In addition, chemosensitive neuronal monitoring of any increased blood free fatty acid would lead to ventromedial nucleus suppression, in turn causing simultaneous direct lateral hypothalamic activation and disinhibition: the now strongly motivated animal eats (313).

4.

Thermostatic

theory

The thermostatic theory of food intake (268) was not supported by the studies Anand et al. (3) performed in anesthetized cats. Local heating of the hypothalamus revealed that nonantidromically identified single ventrome-


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dial and lateral hypothal .amic cells were unresponsive; hypothalamic cells were thermosensitive. 5. Visceral-limbic

input

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NEURONS

only preoptic-

anterior

and behavior

a) Vagal input. In the fasted, anesthetized cat, Anand and Pillari (5) found single ventromedial nucleus neurons firing more slowly than single lateral hypothalamic neurons. Distension of the stomach and stimulation of the gastric vagal branches increased the spike frequency of the ventromedial nucleus neurons and decreased discharges of lateral hypothalamic neurons. The firing frequency of adjacent hypothalamic neurons was unchanged. When gastric nerves were sectioned, these responses disappeared. These data indicate a role in food intake regulation for mechanoreceptors in the stomach transmitted via the vagus nerves. b) OZfactory-gustatory input. The smell and taste of food provide important cues for food intake (260). When Scott and Pfaffman (367) recorded single- and multiple-unit activity from lateral hypothalamic neurons in the urethan-anesthetized rat, they found that by electrically stimulating the olfactory bulb or the lateral olfactory tract in the ventrolateral portion of the medial forebrain bundle they caused spike responses of variable latency (4-25 ms). Unit responses in this area were driven by odors in animals that were paralyzed to prevent breathing artifacts. Tail pinch failed to activate these olfaction-driven lateral hypothalamic units. Single nonantidromically identified cells in the medial forebrain bundle in the urethan-anesthetized rat (230, 329) responded to multiple odors. These lateral hypothalamic neurons showed some discrimination between natural and artificial odors. Olfactory input involving lateral hypothala .mic neurons may stimulate the same cell with food-related and sex-related stimuli (329). , Using the sam .e preparation, Scott and Pfaffman (368) studied the effect on lateral hypothalamic neurons of single shocks to the ipsilateral olfactory bulb and to airborne odors. Shortlatency units driven from olfactory bulb shock were more responsive to odors (368); lateral hypothalamic units exposed to a graded intensity series of different odors did not show as fine a degree of discrimination as olfactory bulb units (362). Inhibitory responses to olfactory stimuli were commonly seen in olfactory bulb units, but were uncommon in lateral hypothalamic neurons. Olfactory input received in the lateral hypothalamus probably modulates cell activity in the regulation of food intake. Using tritiated proline autoradiography and electrophysiological studies, Norgren (300) traced the third-order gustatory relay pathway from the dorsal pons to the dorsomedial corner of the internal capsule to the far-lateral hypothalamus, ending in the central nucleus of the amygdala. With its widespread preoptic-anterior hypothalamic and other hypothalamic connections, the amygdala provides an excellent site for modulating autonomic, endocrine, and consumatory functions (268; see Fig. 1). Lesioning the basolatera1 nucleus of the amygdala results in hyperphagia; when stimulated, it electrically arrests feeding behavior (268).


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c) Limbic-midbrain-basal ganglia. Single nonantidromically identified units in the ventromedial nucleus respond to electrical stimulation of the septal region and amygdala but not to excitation of the midbrain tegmentum (281, 282). Amygdaloid influences on ventromedial neurons arise from the corticomedial and basolateral nuclei (281, 282). Basolateral-induced activity travels along the ventral amygdalofugal pathway, producing an initial shortlatency ventromedial activation, followed by inhibition (281,282). Corticomedial-induced activity travels via the stria terminalis to inhibit ventromedial cells (86, 87, 281, 282). This inhibition may be mediated by a long, slowconducting axon without interposition of an inhibitory interneuron (86, 87, 281, 282). Dreifuss et al. (86, 87) demonstrated convergence and interaction of stria terminalis and ventral amygdalofugal impulses on single ventromedial nucleus neurons. Morphological and electrophysiological studies suggested two types of neurons in the ventromedial nucleus responsive to electrical stimulation of the amygdala nuand the lateral edge of the ventromedial cleus. Stimulation of this lateral edge of the ventromedial nucleus activated large-amplitude spikes associated with a negative slow potential that probably originates from interneurons (283). Stimulation of the two amygdaloid efferent pathways, the stria terminalis and the ventral amygdalofugal, activated these ventromedial interneurons, leading to inhibition of the larger multipolar ventromedial nucleus neurons (283). Miller and Mogenson (265) found facilitatory or inhibitory effects on lateral hypothalamic nonantidromically identified single units after septal areas in the urethan-anesthetized rat were stimulated electrically. The modulatory effect from this stimulation of lateral hypothalamic neuron firing patterns was dependent on the resting firing frequency at the time. A slowly firing cell was accelerated and a faster firing, one was inhibited for a relatively constant level of neural activity (265). Although a wide region of the limbic system activated lateral hypothalamic units, the ventromedial nucleus and preoptic-anterior hypothalamic area yielded more responsive cells than did the basomedial or basolateral amygdala, stria terminalis, septum and ventral hippocampus. This study indicated that afferent projections converge extensively on lateral hypothalamic neurons (408). By recording extracellularly and intracellularly in the ether-anesthetized rat, Oomura et al. (313) found amygdala-induced excitation and inhibition of lateral hypothalamic neurons with excitatory-inhibitory postsynaptic potential sequences and powerful, long-lasting inhibitory postsynaptic potential effects. These results indicate that the inhibitory effect of the amygdala on lateral hypothalamic neurons is the result of long-lasting inhibitory postsynaptic potentials exerted through the stria terminalis (317). Whether these neuronal effects relate directly or indirect1 .Y to feeding behavior requires more direct studies in the behaving animal. 0 ther work by Oomura et al. (315) showed excitatory-inhibitory single-unit activity responses in the lateral hypothalamus after electrical stimulus of the globus pallidus and substantia nigra in the rat (319, and Tsubokowa and Sutin (403) found midbrain-induced excitation and inhibition of ventromedial nucleus neurons.


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Behavior. By auto- and cross-correlated single-unit recordings in the acute, anesthetized cat, Oomura et al. (319) fou nd the functions for simultaneously discharged ventromedial and lateral hypothalamic neurons. Ether anesthesia and electrically stimulated preparations confirmed these observations. In chronic units in the unanesthetized cat, Oomura’s group (314) found a reciprocal relationship between cell discharges in the ventromedial nucleus and lateral hypothalamus during natural sleep, in the alert state, or during searching for food. During paradoxical sleep (REM, fast wave), the singleunit discharge frequencies in lateral hypothalamus and ventromedial nucleus were of the same order of magnitude as those observed for the alert state. Neurons classified as specific or nonspecific according to their pattern of response, were intermingled (314). In the waking, freely moving, eating rat, Hamburg (148) found consi .stently decreased la teral hypothalamic singleand multiple-unit activity that returned to control levels of firing during food removal even though chewing and swallowing continued. No consistent changes in single- and multi ple-unit activity were found elsewhere in the hypothalamus duri ng eating (148). These results indicate that the specific neuronal inhibition during eating appears to reflect the motivational state rather than the motor action of chewing and swallowing alone. In order to ascertain the temporal and functional relationships between lateral hypothalamic neuronal response and hunger-induced behavior, like bar pressing for food, Ono et al. (312) studied chronic monkeys. Recordings from lateral hypothalamic single cells in monkeys during food intake and during stimulation of basolateral amygdala and fronto-orbital cortex showed two separate periods of excitation: prior to bar press and after bar press. Electrical stimulation of the amygdala and fronto-orbital cortex produced prolonged neuronal and feeding behavior inhibition. Apparently, after information from intrinsic and extrinsic factors and from central structures, like the limbic system and the frontal cortex, is received and processed in the lateral hypothalamus, hunger-alleviating behavior (feeding) begins. These somewhat fragmentary data do not, however, allow any final conclusions regarding lateral hypothalamic or ventromedial nucleus neurons and feeding behavior to be drawn. Further studies are needed with more precise anatomical, chemical, and physiological definition of these neuronal pools and their connections. 6. Drug

effects

a) Biogenic amines. In order to determine the chemical sensitivity of hypothalamic cells involved in the reciprocal regulation of food intake, Oomura et al. (322) applied putative transmitters and other drugs microiontophoretically into the ether-anesthetized rat’s ventromedial and lateral hypothalamic area. Acetylcholine and norepinephrine excited nonantidromically identified, lateral hypothalamic single neurons, whereas acetylcholine decreased and norepinephrine increased as often as it decreased ventromedial neuronal activity. Atropine blocked both ventromedial and lateral hypothalamic neurons. Serotonin decreased ventromedial neuronal firing equally.


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Gamma-aminobutyric acid plus glycine was found to be a general depressant, glutamate a general stimulant in both hypothalamic areas (317). Strychnine blocked the action of glycine; bicuculline antagonized the reduction in cell firing when GABA was applied to ventromedial nucleus cells (85). Since the distribution of cells sensitive to acetylcholine and norepinephrine overlaps in the ventromedial and lateral hypothalamic areas, it is difficult to interpret the behavioral effects of rat intrahypothalamic microi .njections. These apparently show acetylcholine producing drinking and norepinephrine producing eating. When microinjected into the lateral hypothalamus, angiotensin produces drinking (428, 429). When fenfluramine was administered intravenously in the ether-anesthetized cat, this amphetamine derivative with strong appetite-depressant effects, but witho ut sympathetic activity, caused ventromedial sing1 .e-cell activi .ty to increase. Simultaneously, lateral hypothalamic neuronal discharges were depressed (219). Unraveling the role of biogenic amines in the regulation of food intake requires further effort and precision in experimental design. b) AZcohoZ and morphine. In a study of the effects of alcohol on lateral hypothalamic neurons, Wayner et al. (427, 429) found that 53% of the nonantidromically identified single neurons increased discharge rates, 32% decreased, and the remainder were unchanged. Eidelberg and Bond (110) found excitatory-inhibitory effects of morphine on hypothalamic single-unit activity in naive and addicted rats. Kerr et al. (216) studied the effects of morphine and its antagonists, naloxone and nalorphine, on simultaneously recorded single- and multiple-unit activity in the ventromedial and lateral hypothalamic areas of naive and morphi ne-dependent, urethan-anesthetized rats. Morphine had opposing effects on cell activity. It excited ventromedial and depressed lateral hypothalamic neuronal activity. The antagonists reversed these effects decisively. During withdrawal, addicted animals showed lateral hypothalamic firing rates greater than those recorded for naive. When morphine was withdrawn from the morphine-dependent rat, methadone excited both ventromedial and lateral hypothalamic neurons. Morphine follow-up reversed these effects. The reciprocal effects of these narcotics might explain, in part, the addictive properties of these narcotics on the hypothalamic level. How these results relate to the endogenous morphinomimetic brain peptides, the enkephalins and endorphins, remains unexplored at this time. 7. Summary Ventromedial and lateral hypothalamic neurons are associated with feeding behavior. Humoral and nonhumoral factors found to modify the firing patterns of nonantidromically identified “feeding” neurons include glucose, free fatty acids, vagal input, olfactory-gustatory input, motivational states, putative transmitters, alcohol, and morphine. The relationship of these neurons to feeding remains an area of controversy, but this is to be expected considering the complexities of the activity. As the chemical nature of hypothalamic neurons becomes known, the combination of immunohistochemical


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and anatomical techniques ther to our understanding

C. Neurons

Associated

with physiological studies should of the neural control of feeding.

with Cardiovascular

635

NEURONS

contribute

fur-

Function

I. Introduction Electrical stimulation of the hypothalamus can produce changes in arterial blood pressure, heart rate, and peripheral blood flow; hypothalamic lesions can alter cardiovascular reflexes (53, 233). In order to examine the cellular basis for some of these cardiovascular effects, workers recorded from single hypothalamic neurons during changes in arterial bood pressure, baroreceptor input, and atria1 pressure. The results support the concept that hypothalamic single cells are involved in mammalian cardiovascular function (233; see Figs. 1 and 6). 2. Pressosensitive

neurons

In the pentobarbital-anesthetized cat, Baust and Katz (20) showed that in a discrete region of the posterior hypothalamus 90% of the single neurons studied were excited during epinephrine-induced blood pressure elevation, the firing rates directly proportional to pressure changes. Posterior hypothalamic responses to intravenous epinephrine were abolished with blood pressure clamping but not with denervating the sinoaortic baroreceptors. With independent manipulation of blood pressure and hypothalamic blood flow, with operative isolation of the posterior hypothalamus, these cell responses followed the changes in blood pressure but not blood flow (21). In contrast to these excitatory effects in the posterior hypothalamus, 80% of the midbrain reticular neurons were inhibited by a rise in blood pressure (22). In similar studies in anesthetized cats, Frazier et al. (132) found evidence for two populations of pressure-sensitive cells in the posterior hypothalamus: 1) cells that responded with increased firing rates (55%) to epinephrine-induced arterial blood pressure rise and 2) cells that responded with decreased firing rates (75%) to aortic-occlusion-induced blood pressure rise. The role these apparent pressure-sensitive neurons play in the regulation of cardiovascular reflexes is still unclear. 3 . Baroreceptor

neurons

In order to determine if discrete regions of the anterior hypothalamic depressor area were involved in the integration of the baroreceptor reflex, Spyer (380) recorded from single units in chloralose-anesthetized rats. Three groups of neurons were found: cells markedly excited by increased intrasinusal pressure, cells with a “burst-on” discharge, and cells inhibited by blood


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NEURONS ASSOCIATED WITH CARDIOVASCULAR FUNCTJON BARORECEPTOR CHEMORECEPTOR

& H VASODEPRESSOR l VASOPRESSOR A PRESSOSENSITIVE

FIG. 6. Hypothalamic neurons associated with cardiovascular function. An elevated hypothalamic arterial blood pressure is detected by the posterior hypothalamic pressure-detector neurons, which activate the posterior hypothalamic vasodepressor interneurons and inhibit the posterior hypothalamic vasopressor interneurons. Simultaneously, afferent input from the carotid and aortic baroreceptors, and at other times from chemoreceptors and atria1 volume receptors, excites the anterior hypothalamic vasodepressor interneurons and inhibits the anterior hypothalamic vasopressor interneurons. These anterior hypothalamic cardiovascular interneurons transmit impulses to posterior hypothalamic vasodepressor and vasopressor interneurons. The posterior hypothalamic vasodepressor interneurons send impulses to the lower brainstem and spinal cord for inhibition of sympathetic vasoconstrictor tone, reduction of arterial blood pressure, and alteration of regional peripheral blood flow. Reciprocal inhibition of anterior and posterior hypothalamic vasodepressor interneurons further insures a reduction in blood pressure. During a reduction of posterior hypothalamic arterial blood pressure and systemic arterial blood pressure the vasopressor interneurons are activated. Input from limbicmidbrain, neocortical, and chemically specific pathways modulates the activities of these vasodepressor and vasopressor interneurons (not shown). For further discussion see text. Abbreviations: AC, anterior commissure; AP, anterior pituitary gland; OC, optic chiasm; PI’, posterior pituitary gland; n VASODEPRESSOR, vasodepressor interneurons; 0 VASOPRESSOR, vasopressor interneurons; A PRESSOSENSITIVE, posterior hypothalamic pressure detector. (Based on references cited in sect. 1vC.1

pressure that was elevated within a vascularly isolated carotid sinus. These data suggest that the anterior hypothalamic depressor area may represent the rostra1 extension of the integrative center for the carotid sinus baroreceptor reflex.


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In a medial hypothalamic pressor area Thomas and Calresu (401) recorded single units in the chloralose-anesthetized cat that were inhibited (30%) or excited (14%) by electrical stimulation of the carotid sinus nerve. Electrically inhibited units were also inhibited by selectively activated baroreceptors and by norepinephrine-induced blood pressure rise, but not by sodium cyanide. Neurons excited when the carotid sinus nerve was electrically stimulated were also excited by the close intra-arterial injection of sodium cyanide that selectively activated the carotid body chemoreceptors. These results indicate that the medial hypothalamic pressor area receives baroreceptor and chemoreceptor inputs and perhaps influences cardiovascular reflex activity. Units recorded in the supraoptic and paraventricular areas, anterior, lateral, and medial dorsal hypothalamus (141) were found responsive to left (26) and right (141) atria1 stretch, respectively. In Dial-urethan-anesthetized cats, Menninger and Frazier (262) found that 15% of the hypothalamic neurons tested were responsive to NaCl injected in both the left atria1 and intracarotid areas. In chloralose-anesthetized cats, Grizzle et al. (141) found 64% of the medial-dorsal hypothalamic units were responsive to right atria1 stretch. These results suggest that atria1 volume-receptor activity travels along vagus afferents to influence hypothalamic neurons that are involved in regulating the anterior [ adrenocorticotrophin (l41)] and posterior [ vasopressin (262)] pituitary and cardiovascular defense against hemorrhage.

4. Summary Hypothalamic neurons are involved in the neural regulation of the cardiovascular system. Pressosensitive neurons in the posterior hypothalamus respond directly to a rise in arterial blood pressure with increased or decreased firing rates. Other neurons in the lateral, anterior median, and medial dorsal hypothalamus respond to baroreceptor input from carotid sinus and atria1 receptors. Futher studies are needed to indicate the role of these hypothalamic neurons in cardiovascular function. D. Behavior

and Hypothalamic

Unit Activity

1. Introduction The major ascending and descending pathways connecting the lower brainstem with the limbic-neocortical regions provide input to the hypothalamic neurons that are thought to be involved in feeding, drinking, sexual, and thermoregulatory behavior. The major sensory-motor systems must of necessity be considered for their role in activating a particular function in the hypothalamic neuroendocrine and nonendocrine neurons. The fact that separating the humoral from the synaptic influences on these units is difficult has not deterred researchers (see Figs. 1 and 7).


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NEURONS ASSOCIATED WITH BEHAVIOR LIMBIC -MIDBRAIN

n

GOLGI II MH . GOLGI I LH A RECEPTORS

FIG. 7. Hypothalamic neurons associated with behavior. During an altered behavioral state such as the defense reaction or self-stimulation, the hypothalamic detector neurons detect various alterations in the internal milieu (glucose, nonesterified fatty acids, blood pressure, epinephrine, temperature, osmotic pressure, etc.) and signal these changes in the arterial blood to interneurons in the medial hypothalamus and to interneurons in the lateral hypothalamus. Afferent input received from neocortical, olfa&ry, and visceral pathways via the limbic-midbrain pathways activates chemically specific pathways with resultant vegetativesomnolent behavior (medial hypothalamus) or defense-aggressive behavior (lateral hypothalamus). For further discussion see text. Abbreviations:. AC, anterior commissure; AP, anterior pituitary gland; OC, optic chiasm; PP, posterior pituitary gland; n GOLGI II MH, medial hypothalamic short-axoned interneurons; 0 GOLGI I LH, lateral hypothalamic long-axoned interneurons; A RECEPTORS, receptors of changes in the internal milieu. (Based on references cited in sect. IvD.)

2. Hypothalamic

deafferentation

In order to study separately the effects of humoral and neural input to hypothalamic neurons, Cross and Kitay (68) used a cylindrical cutting device to isolate the hypothalamus of the rat from the surrounding diencephalon. In these hypothalamic islands, cells fired at rates faster than in intact preparations. Sensory stimuli failed to change hypothalamic discharge rates. Subsequently, Cross and Dyer (64) studied the effects of anesthesia on rat hypothalamic island units, finding no change in firing rates before or after urethan.


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After intravenous subanesthetic doses of sodium methohexital, however, they found unit activity depressed. They also examined the effects of hormone on hypothalamic island cells, as discussed earlier (63; see sect. III, Cl ). Anesthesia activated posterolateral hypothalamic neurons (264; see Fig. 7). 3. Sleep-waking

activity

In early studies the effects that hormones and afferent stimuli have on hypothalamic neurons (17) and the role of sleep-waking activity were not clearly recognized (28). Sawyer and co-workers (27, 231, 339) found a close correlation among single-unit activity, integrated multiple-unit activity, and the effects of vaginal and other sensory stimuli and hormones on EEG activity. These workers concluded that it was necessary to monitor cortical EEG simultaneously with hypothalamic units in the urethan-anesthetized rat in order to determine whether a particular sensory stimulus or hormonal action was specific or nonspecific. Lincoln (244) defined three EEG patterns associated with the sleep cycle in the urethan-anesthetized rat, showing that each pattern had different effects on hypothalamic cell firing. In the unanesthetized rabbit, Findlay and Hayward (129) found a high correlation of cell discharge with the behavioral state. The highest discharge rates occurred with fast-wave sleep, the lowest during slow-wave sleep. The resulting interspike-interval histograms showed marked similarity. The complexity of cell activity alterations during different states of sleep and waking indicates that the hypothalamus possesses a cell population less homogeneous than most of the other brain areas studied. Others found similar results with integrated multiple-unit activity in sleep (195). In view of the lack of unit activity change in the rat hypothalamic islands studied by Cross and his group (64, 68), it seems probable that caudal brainstem mechanisms involved in sleep-waking activity are responsible for these behavioral changes in hypothalamic unit activity. In their analysis of preoptic neurons responding to a change in EEG activity, Pfaff and Gregory (330) noted that a higher proportion of cells accelerated during EEG desynchrony in normal, urethan-anesthetized male rats than in castrated. Preoptic units that increased firing rates during EEG activation tended to be the same units that showed excitatory responses to odors. Preoptic units correlated with EEG tended to be among units that showed no correlation, rather than being restricted to a clearly defined preoptic subregion. Whether the presence of male hormone in the rat enhances reticular-preoptic, preoptic-reticular, or olfactory-preoptic circuits requires further study. The difficulty in separating these diverse EEG activities makes study of sleep-waking effects of hypothalamic units limited in value. 4. Conditioned Exteroceptive tors, and hormone

responses and enteroceptive inputs, glucoreceptors, detectors that relay signals from the internal

somatorecepenvironment


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converge on hypothalamic neurons to provide a neuronal network ideally suited for associative functions in learning. Motivational and emotional states important to the learning of new associations have been related to hypothalamic neurons, but whether such neurons have a direct relationship to learning is unclear. Using adversive Pavlovian conditioning in the cat, Umemoto et al. (405) found four patterns of hypothalamic single cell discharges. Type I showed facilitation (ZOO-Hz tone) during conditioned stimulus; type II showed inhibition. Type III showed facilitation after conditioned stimulus; type IV showed inhibition. Neurons with type I and II patterns may be responding to the appearance of a warning signal, that is, anticipating a noxious stimulus. Type III and IV neurons seem to respond to the disappearance of a warning, that is, experiencing relief after danger is gone (405). During appetitive Pavlovian conditioning in the rat, Olds (308) recorded single- and multiple-unit activity in the medial forebrain bundle, finding two kinds of response: 1) innate, occurring less than 80 ms after auditory conditioned stimulus; 2) learned, occurring 40-80 ms after stimulus but before behaviorally learned (latency, 80-120 ms) and after many of the innate responses. On the basis of these data, Olds concluded that hypothalamic neurons participate in the cognitive process as regional areas for the positive reinforcement of converging input-output sequences, but not as primary information processors. These hypothalamic neurons may function as the common pathway where information, reinforcement, and initiation of behavior are integrated to produce the final motor output (see Fig. 7). In further studies of appetitive Pavlovian conditioning in the rat, Linseman and Olds (252) recorded from single- and multiple-unit activity in the preoptic area. The latency of the conditioned brain response in eight cases was 20 ms or less, prior to any behavioral sign and possibly prior to learned responses in any system afferent to the recording site. Hypothalamic, preoptic, and other basal forebrain cells showed brief inhibitory response to conditioned stimulus. Whether such inhibitory responses are nonspecific correlates of arousal associated with conditioning or more specifically related to positive reinforcement (food reward) required further study. Linseman (251) then examined preoptic-hypothalamic cells during both appetitive and adversive Pavlovian conditioning, finding inhibitory responses afterward in both situations. She concluded that preoptic-anterior hypothalamic units inhibited after appetitive or adversive conditioning may represent the effects of transient arousal rather than specific responses to unconditioned stimulus. 5. Self-stimulation

behavior

The anatomical region most closely associated with self-stimulation behavior is the medial forebrain bundle, which spreads in both directions from the paleocortex through the lateral hypothalamus to the midbrain. In a study of single- and multiple-unit activity during and immediately after self-stimulation behavior in the rat, Ito and Olds (193) found a high correlation between


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the stimulation and the driven anterior cingulate, on the one hand, and inhibited hippocampal neurons, on the other. During self-stimulation, hypothalamic units showed either facilitation or inhibition. The authors argue that medial forebrain bundle fibers rather than “path” neurons represent the key elements involved in the neural substrate for self-stimulation (193). Later, Ito (191) reexamined the effects of medial forebrain bundle self-stimulation on the ra t hY pothalam .ic neurons. Although there were inhibitory effects in middle and anterior lateral hypothalamic ” path” neurons, lateral preoptic neurons were excited. These data were interpreted to indicate that intrahypothalamic medial forebrain bundle fibers mediated the inhibitory effects, while extrahypothalamic fibers mediated the excitatory effects. Recently, in an analysis of lateral hypothalamic Ipath” neurons in the pentobarbital-anesthetized rat, Ito (192) found that orthodromic facili tation and inhibition and antidromic activation followed electrical stimulation of the medial forebrain bundle at the level of the mesodiencephalic junction. He therefore speculated (192) that two types of relay neurons were present in the lateral hypothalamus: type A, part of an ascendi ng pathway with an axon projecting to the midbrain; and type B, part of an ascending pathway. Intracellularly recorded spike potentials of these neurons deteriorated rapidly. Only the hyperpolarization that corresponded in its time course to the suppression of intracellular spike discharges remained after medial forebrain bundle stimulation. It was suggested that the inhibition was elicited via recurrent routes localized within the hypothalamus. 6. Defense

behavior

In the cat, affective defense behavior (hissing or striking) evoked by a second attacking cat inhibited, facilitated, or did not change the firing rates of hypothalamic single cells in a manner similar to other types of control manipulations (1). Four cells were found in the dorsal midbrain central grey matter that were driven specifically during defense behavior. Defense behavior induced by hypothalamic electrical stimulation markedly accelerated lateral hypothalamic neurons close to (0.7-1.9 mm) and at a distance from (2.0-5.2 mm) the stimulating electrode (186). Such electrical stimulation in the lateral hypothalamus of the cat apparently differs from the self-stimul .ation studies in the rat by the intensity of the stimulation and the location of the electrode. The excitatory pathway described by Ito (192) may well correspond to the loci eliciting attack behavior and medial forebrain bundle neuronal excitation described by Huang and Flynn (186).

7. Peripheral

and central

input

mechanisms

In the acutely prepared, paralyzed, locally anesthetized cat, Stuart et al. (385, 386) found that single units in the posterior hypothalamus responded to bladder distension (30%) and to sciatic nerve stimulation (35%) with excit-


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atory or inhibitory responses. Either more than 80% of these bladder distension units responded to sciatic stimulation or simultaneous sciatic stimulation modified the firing rate (385, 386). Shigenaga et al. (374) and Takaori et al. (396) discovered excitatory-inhibitory nonantidromically identified singleunit responses in the posterior hypothalamus after electrical stimulation of tooth pulp and inferior alveolar nerve and after thermal stimuli applied to the stomach of the rat. Anterior and ventral posterior hypothalamic units were not as responsive to visceral and somatic stimuli as the dorsal hypothalamic units were. Units in the preoptic-anterior hypothalamus (67) and lateral hypothalamus (353) were found to respond to clicks, light flashes, and cutaneous stimuli in the urethan-anesthetized rabbit (67) and the unanesthetized, immobilized cat (353). Dafny and Feldman (76) found 50% of the anterior and posterior hypothalamic nonantidromically identified single-unit activity responsive to photic, acoustic, and sciatic nerve stimulation with convergence of sensory stimuli. There was a high correlation among the responsiveness of the three sensory modalities on posterior hypothalamic cells. Mandelbrod and Feldman (257) found that single cells generally showed sensory convergence in the median eminence of the urethan-anesthetized rat. Posterior hypothalamic neurons in the acutely prepared, paralyzed, locally anesthetized cat tended to show a similar direction of effect (augmentation or reduction of firing rates) from the central (limbic and striatal) stimulation, when these central stimuli were paired with visceral (bladder distension) or somatic (sciatic stimulation) stimuli (385, 386). Anterior hypothalamic nonantidromically identified single-unit activity in acute, pentobarbital-anesthetized cats accelerated or decelerated to sciatic nerve, photic, acoustic stimulation, but were inhibited by caudate stimulation and by caudate lesions (74). There may be a topographic relationship between the amygdala and the hypothalamus. Electrical stimulation of the medial part of the basal nucleus of the amygdala in the acute, paralyzed cat drives hypothalamic nonantidromically identified single-unit activity in the medial hypothalamus. Stimulation of the lateral part of the basal nucleus of the amygdala drives hypothalamic single-unit activity in the lateral hypothalamus (108,109). In the acute, paralyzed cat, electrical stimulation of the midbrain reticular formation excited or inhibited posterior hypothalamic single-unit activity (75). Stimulation in the septum or hippocampus inhibited posterior hypothalamic singleunit activity uniformly (75). In the rat, the dorsal hippocampus produced a predominantly inhibitory effect on medial hypothalamic units. In contrast to these studies of inhibition in the acutely prepared cat and rat (73, 75, 385, 386), Poletti et al. (332, 333) suggested that single pulse and volleys to the hippocampus of the chronic squirrel monkey caused initial acceleration of the preoptic-anterior hypothalamic units (83%). Cerebellar stimulation excites and inhibits hypothalamic units (8). Siegel and Wang (376) found excitatory and inhibitory nonantidromically identified single-unit responses in the rostral hypothalamus after electrical stimulation of the basal forebrain structures in the cat. As shown in Figures l-7, the hypothalamus is the crossroads


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for a multiplicity of peripheral and central input pathways that act on neurons related to behavior. Further progress in the identification of neuronal pools important for these behavioral mechanisms depends on the correlation of the anatomical and chemical cell types with those physiologically defined functional cell types. 8. Pharmacological

effects

After systemic injections of methscopolamine, physostigmine, and neostigmine designed to bring out the neuronal mechanisms that underlie the cholinergic arousal reaction and the cause of the stimulated arousal reaction, Olds and Eibergen (309) studied single- and multiple-unit activity in the rat hypothalamus, midbrain, and hippocampus. All three drugs caused increased movement that became rhythmic tremors. Methscopolamine, a peripheral anticholinergic, caused the discharge rate of hypothalamic neurons to increase, but the discharge rate of midbrain and hippocampus single and multiple units remained the same. Neostigmine, an anticholinesterase agent that does not cross the blood-brain barrier, caused the animal’s rhythmic tremor to increase without any increase in cell discharge. Physostigmine, an anticholinesterase agent that crosses the blood-brain barrier, caused the activity of hypothalamic neurons to remain the same or increase slightly, while hippocampal and midbrain neurons decreased in activity. The authors concluded that the hypothalamus contained neurons responsive to cholinergic agents acting at the periphery. As measured by the movement detector, drugs acting centrally at the doses used produced minor peripheral effects, while drugs acting peripherally at these doses produced large peripheral effects. Olds and Ito (310) examined the effects of adrenergic and cholinergic action on behavior and single- and multiple-unit activity in the self-stimulating rat. Tetrabenzamine, a catecholamineand indoleamine-depleting agent, suppressed self-stimulating behavior and reduced the suppressant effect that self-stimulation in the posterior hypothalamus had on neuronal activity in the medial forebrain bundle. Amphetamine partially reinstated the suppressan t action of stimulation on neuronal activity recorded in the medial forebrain bundle. These results suggest that catecholamine modulates self-stimulating behavior and also neuronal activity correlated with self-stimulation. Such modulation could be specific to the reward pathway. Physostigmine and scopolamine produced effects identical to tetrabenzamine and amphetamine on self-stimulation behavior, but were ineffective on neuronal activity. Neuronal responses did not correlate with the self-stimulated brain reward, suggesting that the cholinergic systems d.iffuse the brain-reward response. For research on the effects of drugs on conditioned emotional behavior and conditioned hypothalamic neuronal activity in the rat, Umemoto and Olds (406) studied single- and multiple-unit activity. The benzodiazepines, chlordiazepoxide and diazepam, disinhibited the lever-pressing responses for food in half the subjects studied. The benzodiazepines simultaneously reduced the background rate of neuronal activity and the conditioned stimulus-


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correlated rate of discharge. The peak effect of the drugs on behavior correlated well with their peak effect on the conditioned stimulus-correlated neuronal activity and on the background-period activity. 9. Summary .

As shown in Figures 1-7, the hypothalamus contains the neuronal circuitry essential to the several behavioral states on which the survival of self and preservation of the species rely. Single-cell activity in the hypothalamus can alter when variations in sleep-waking behavior cause generalized changes in EEG, and hypothalamic neurons have been shown to be related to motivational and emotional states important for learning new associations. During Pavlovian appetitive and adversive conditioning, hypothalamic single- and multiple-unit activity shows specific excitatory and inhibitory responses. Hypothalamic neurons in the medial forebrain bundle show facilitation or inhibition during self-stimulation. Peripheral somatosensory, visceral, and central limbic afferents converge on and activate single posterior hypothalamic neurons. In addition, these neurons of behavior are susceptible to a diversity of pharmacological agents. Future study of single cells in the unanesthetized mammal’s hypothalamus with precise chemical, anatomical, and physiological identification may clarify the role these cells play in feeding, drinking, motivational, sexual, and maternal behavior. V.

GENERAL

CONSIDERATIONS

A. Classification

of Hypothalamic

Neurons

At present, there is no good method for classification of hypothalamic neurons. In this review single cells studied by electrophysiological techniques were divided into endocrine and nonendocrine categories, yet to clearly label a particular cell under study as a member of one of these groups is not possible. Rather, investigators interested in one area of hypothalamic function qualify their results from a particular functional bias. The difficulty remains, clouded by the angle of visionis the hypothalamic neuron a detector-receptor element, an interneuron, or an effector cell? Because the hypothalamus is made up of a diffuse network of small fibers and cells with only poorly differentiated nuclei and tracts, it is not readily amenable to electrophysiological probing for systematic unit analysis. Furthermore, the small size of the hypothalamus and the many different types of functional states represented compound the problems confronting the dedicated researcher. The anatomical, chemical, and functional characteristics of the nonendocrine neurons involved in thermoregulation, feeding, cardiovascular, and behavioral activities are uncertain. The studies described under these categories in section IV provide only circumstantial evidence, at best, that the cells under study actually are involved in thermoregulation, feeding, cardio-


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vascular activity, and behavior. It is not possible at this time to study the same neuron from animal to animal, from species to species, because one neuron, uncharacterized, is one neuron, unidentifiable. In the study of the parvocellular neuroendocrine cells the recent chemical identification of the hypophyseotrophic hormones, luteinizing-hormone-releasing hormone, thyrotropin-releasing hormone, and somatostatin has allowed immunohistochemical identification of hypothalamic cell somata that contain LHRH and somatostatin. None of the studies of parvocellular neuroendocrine cells discussed in section III can claim to be recordings from chemically identified LHRH or somatostatin peptidergic neurons. Such studies remain to be performed. Of all the hypothalamic neurons, the magnocellular neuroendocrine cells are the most precisely characterized. These peptidergic neurons reside in the supraoptic and paraventricular nuclei and in the internuclear zone; they synthesize oxytocin, vasopressin, and neurophysin; and their vesicle-laden axons transport these peptides to the posterior pituitary gland neurohemal junction. As described in section II, these neurons can be antidromically identified and histologically localized within magnocellular nuclei. The recent immunohistochemical demonstration of a dual population of vasopressinergic and oxytocinergic neurons mixed in these magnocellular nuclei, however, complicates the problem of identification. It is not possible to know, on the basis of antidromic latency or cell-firing characteristics, which cell is oxytocinergic and which is vasopressinergic. None of the studies of magnocellular neuroendocrine cells quoted in section II can claim to be recordings from chemically identified vasopressinergic or oxytocinergic neurons. Such studies remain to be performed. B. Peptidergic

Hypothalamic

Neurons

The major flaw in previous studies of hypothalamic neurons has been the lack of chemical identification of the cells under study. Such specific chemical typing remained remote until the methods of immunohistochemistry were applied to endocrine neurons with consequent identification of vasopressin, oxytocin, LHRH, and somatostatin peptidergic neurons in the hypothalamus. The recent chemical identification of a variety of additional hypothalamic and extrahypothalamic peptides, such as substance P, neurotensin, angiotensin, enkephalins, endorphins, and others, indicates the promise of further cell localization of neuropeptides. These potent peptides can act on adjacent neurons as neurotransmitters an’d neuromodulators. Future electrophysiological and immunohistochemical study of peptidergic hypothalamic neurons promises to provide important specific information on the endocrine and nonendocrine functions of the hypothalamus. I thank Ms. S. Curtis for editorial assistance, Dr. T. A. Reaves for helpful criticism of the manuscript, and Ms. S. Void for assistance in typing the manuscript. The author’s research quoted in this review was supported in part by Research Grant NS13411 from the Public Health Service and a Grant from the Sloan Foundation to the Neurobiology Program.


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Myocardial bridges: morphological and functional aspects.

The vagina. Morphological, functional and ecological aspects.

Morphological and functional identifications of catfish retinal neurons. II. Morphological identification.

Intracellular Ca2+ stores in neurons. Identification and functional aspects.

Morphological and functional identifications of catfish retinal neurons. III. Functional identification.

Morphological characterization of immortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone.

Biochemical, morphological, and functional aspects of systemic and local vitamin A deficiency in the respiratory tract.

Morphological aspects of myocardial bridges.

[Morphological aspects of placental insufficiency].

In vitro evidence for modulation of morphological and functional development of hypothalamic immunoreactive atrial natriuretic peptide neurons by cyclic 3',5'-adenosine monophosphate.

[Morphological and genetic aspects of Spitz tumors].

Clinical and Morphological Aspects of Warthin's Tumor.

or castration.

Functional and morphological assessment of diaphragm innervation by phrenic motor neurons.

Neurovascular Interaction Promotes the Morphological and Functional Maturation of Cortical Neurons.

Morphological and functional identifications of catfish retinal neurons. I. Classical morphology.

Cell death. General features and morphological aspects.

Hypothalamic neurons secreting vasopressin and neurophysin.

[Quantitative morphological aspects of placental insufficiency].

Morphological aspects of the human vestibular nerve.

Genetic aspects of hypothalamic and pituitary gland development.

Network of hypothalamic neurons that control appetite.

Morphological analysis of the neurons in the area of the hypothalamic magnocellular dorsal nucleus of the guinea pig.

Mucopolysaccharides in hypothalamic neurons of the rat.