Cell Tiss. Res. 179, 177-196 (1977)
Cell and Tissue Research 9 by Springer-Veflag 1977
The Lateral Hypothalamic Area An Ultrastructural Analysis* Jack C. Sipe** and Robert Y. Moore*** **Veterans Administration Hospital, San Diego, California, and **,***Department of Neurosciences, University of California at San Diego, La Jolla, California
Summary. An ultrastructural analysis of the rat lateral hypothalamic area (LHA) was undertaken in order to provide an initial step in the characterization of this complex area which appears to participate in a number of important neural functions. The organization of the normal tuberal LHA was compared to the area following acute and chronic denervating lesions. In the normal animal, the principal features of the LHA are the presence of lateral hypothalamic neurons, a major sagittal pathway (the medial forebrain bundle, MFB) and the interposed neuropil richly populated by a variety of synaptic terminal types. Alterations in the synaptic organization of the LHA following rostral and caudal MFB lesions were most pronounced in animals with acute and chronic caudal lesions. A 10% reduction of synaptic terminals containing 800-1000 A diameter dense core vesicles and a 10% increase in terminals containing lucent core vesicles was observed in animals with caudal lesions while no significant redistribution of synaptic terminal types occurred with rostral lesions. The preliminary degeneration experiments indicate that identification of the numerous and diverse afferents to the LHA neuropil may be aided by this method but that a detailed and systematic ultrastructural analysis will be required to identify sources of input with certainty.
Key words: Lateral hypothalamic area - Synaptic organization - Rat Ultrastructure.
Introduction The lateral hypothalamic area (LHA) contains the axons of the medial forebrain bundle (MFB) and a prominent nucleus, the lateral hypothalamic nucleus (LHN) Send offprint requests to: Jack C. Sipe, M.D., Department of Neurology, Veterans Administration Hospital, San Diego, Ca 92161,USA
* Presented in part at the 27th Annual Meeting of the American Academy of Neurology, Bal Harbour, FLA, 1975 ** Recipient of Research Associate Award, Veterans Administration **,*** Supported by the Veterans Administration and by NIH Grants NS 12080. Skilled technical assistance was provided by Robin Isaacs, Marilyn Woodward and Sharon Keigher
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(cf. G u r d j i a n , 1927; Bleier, 1963; Christ, 1969). The n e u r o n s of this nucleus give rise to a s c e n d i n g a n d descending projections into the adjacent medial h y p o t h a l a m i c zone (Guillery, 1957; Szentfigothai et al., 1968; Millhouse, 1969; R a i s m a n , 1971), a n d they receive i n p u t f r o m the b r a i n s t e m reticular f o r m a t i o n , medial h y p o t h a l a mus, other lateral h y p o t h a l a m i c n e u r o n s a n d basal f o r e b r a i n (Guillery, 1957; Szent/tgothai et al., 1968; Millhouse, 1969; N a u t a a n d H a y m a k e r , 1969; R a i s m a n , 1971). Since the m e d i a l h y p o t h a l a m i c nuclei receive relatively sparse projections f r o m e x t r a - h y p o t h a l a m i c sources ( N a u t a a n d H a y m a k e r , 1969; R a i s m a n , 1971), it is evident that the lateral h y p o t h a l a m i c nucleus is a n a t o m i c a l l y situated to represent a m a j o r i n t e g r a t i n g center between the b r a i n s t e m reticular f o r m a t i o n , basal f o r e b r a i n a n d m e d i a l h y p o t h a l a m u s . This is c o n f i r m e d by a n u m b e r of a b l a t i o n a n d s t i m u l a t i o n studies which have d e m o n s t r a t e d that the L H A plays a n i m p o r t a n t role in m a n y f u n c t i o n s (cf. Olds, 1962; Epstein, 1971; Rolls, 1975; Stricker a n d Z i g m o n d , 1976 for reviews) b u t these have generally been a t t r i b u t e d to the c o m p o n e n t s of the medial f o r e b r a i n b u n d l e or related p a t h w a y s traversing the L H A a n d adjacent areas. There has b e e n little a t t e n t i o n directed to the L H N even t h o u g h its n e u r o n s appear to participate i m p o r t a n t l y in f u n c t i o n s such as reward (Olds, 1973, 1974; Rolls, 1975). Because o f its evident f u n c t i o n a l significance, the present u l t r a s t r u c t u r a l study was u n d e r t a k e n in order to provide further i n f o r m a t i o n o n the o r g a n i z a t i o n of the lateral h y p o t h a l a m i c area.
Material and Methods The animals used in this study were adult female albino rats of the Sprague-Dawleystrain, 150-200gm. They were anesthetized with pentobarbital (40 mg/kg) and perfused with glutaraldehyde-parafomaldehyde solutions described perviously (Sipe et al., 1973). The LHA from the tuberal hypothalamus, between the optic chiasm and mamillary nuclei at approximately the level of the ventromedial nucleus, was dissected in a block approximately 1.5 • 1 mm, from 10 normal adult rats. The tissue blocks were post-fixed in cacodylate-buffered osmium tetroxide and aqueous uranyl acetate before dehydration and embedding in Epon. Thick sections were stained with methylene blue and used for further identification of areas to be examined by electron microscopy. Thin sections were mounted on uncoated grids, stained with both warm uranyl acetate in 50~ methanol and lead citrate and examined in the coronal, sagittal and horizontal planes. A large number of photographs of randomly selected areas through the LHA was made at medium and high magnification for categorization and counting of synaptic terminal types and other structures. In addition, cresyl violet-stained celloidin sections in the coronal and horizontal planes were studied by light microscopy. Two groups of animal were prepared with lesions. Each consisted of 6 animals with unilateral lesions either rostral to or caudal to the tuberal portion of the hypothalamus. The lesions were made as follows. With the animals under deep anesthesia, the head was placed in a small animal stereotaxic apparatus (Kopf Instruments, Tujunga, Ca) with the incisor bar 2 mm above the tooth bar. A burr hole was placed in the skull and a guillotine-type knife, 1.5 mm in diameter, was passed through the brain in the coronal plane until it reached the skull base. Both the rostral and caudal lesions were made with the center of the knife, 1.7 mm lateral to the midline. The rostral lesion was made at the level of the bregma and the caudal lesion 4.5 mm caudal to the bregma. Two animals at each time point from each group were sacrificed at 2 days, 4 days and at 4 and 8 months after the lesion was made. The method of sacrifice and preparation of tissue was as described above. The acute postoperative material, 2 and 4 day survival, was examined for evidence of axon terminal degeneration. The chronic brains were sectioned as described above and the terminals were categorized and counted from photographs .of each lesion type.
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Results
Light Microscopic Observations The part of the lateral hypothalamic area analyzed in the present study is the tuberal region lying between the optic chiasm rostrally and the mamillary region caudally (Fig. 1a). In this region the LHA is bordered by the zona incerta dorsally, the internal capsule laterally, the optic tract and pial surface ventrally and the medial hypothalamus medially. In sections stained by the Nissl method the LHA is characterized by the presence of the scattered polymorphic neurons of the lateral hypothalamic nucleus. These neurons are quite numerous throughout the LHA. At the medial border of the LHA they merge without a distinct demarcation with the cells of the ventral tuberal field of the medial hypothalamus. There is a wide range of neuron size in the lateral hypothalamic nucleus. The most prevalent cells are large (approximately 35-45 p~m in diameter) with prominent, darkly-staining Nissl substance and a centrally situated nucleus (Fig. 1 d). They vary in shape from fusiform to multipolar and in coronal sections appear scattered throughout the nucleus without any evident arrangement into groups. In horizontal sections, however, the neurons appear to be arranged, at least in part, into irregular rows which parallel the trajectory of medial forebrain bundle fibers (Fig. 1b). The remaining neurons in the LHA fall into two groups on the basis of size. One is composed of small neurons, usually 12-15 ~tm in diameter, with scanty cytoplasm and faint Nissl substance and the other is an intermediate group with most cells ranging from 18-25 ~tm in diameter (Fig. 1c). In sections stained for myelin, numerous small myelinated fibers are evident scattered throughout the LHA.
Electron Microscopic Observations The distinctive ultrastructure of the LHA is characterized by the presence of lateral hypothalamic neurons whose dendrites, radially arranged in the coronal plane, are intermingled with rostro-caudally oriented axons in thick bundles. This appearance is due to the presence of the axon bundles of the major sagittal pathway in the LHA, the medial forebrain bundle, and the neurons of the LHN. The neuropil interposed between these two major components is richly populated with a variety of synaptic terminals. Ultrastructural analysis of the LHA demonstrates three major components; the neuronal perikarya of the lateral hypothalamic nucleus, the axons of the medial forebrain bundle, and a complex neuropil composed chiefly of a varied array of synaptic terminals. The following description of the ultrastructural organization will emphasize those electron microscopic features that are distinctive to the normal LHA, since the subcellular structure of the area is similar in most respects to other regions of mammalian brain (Peters, Palay and Webster, 1970). This is followed by an analysis of the effects of rostral and caudal MFB lesions on the organization of the neuropil of the tuberal LHA.
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Lateral Hypothalamic Nucleus The cell bodies of the lateral hypothalamic neurons and their processes f o r m an almost continuous spectrum of cell morphology from the large neurons (35-50 Ixm in diameter) that contain abundant Nissl substance to small neurons (12-15 I~m in diameter) with extremely sparse Nissl substance (Fig. 2). In general, the cell bodies o f these neurons tend to be rounded or fusiform with the perikaryon occupied by a large nucleus which exhibits a rounded or slightly oval contour. The nuclear envelope has a typical, double-layered structure with periodic nuclear pores and shows a remarkable tendency toward invagination (Fig. 2) similar to that seen in other hypothalamic neurons (Clementi and Ceccarelli, 1970). A conspicuous, dense spherioidal nucleolus is surrounded by evenly dispersed delicate nuclear chromatin. Cytoplasmic organelles are identical to those in all neurons save for their distribution. The pattern of Nissl substance in the small and medium size neurons is characterized by only a few slender cisternae of granular endoplasmic reticulum which become less conspicuous in the smaller neurons (Fig. 4). Large neurons, on the other hand, display large rhomboid stacks of granular endoplasmic reticulum (Fig. 3) that are often disposed in a crescent shape about the nuclear envelope. Nissl substance is not confined to the perikaryon and is often observed extending into the main dendritic trunks. N u m e r o u s ribosomes and polyribosomal aggregates are particularly abundant in the smaller neurons where they lie free in the cytoplasm and are not closely associated with the surface o f the endoplasmic reticulum (Fig. 4). The Golgi apparatus, however, is very prominent in all neurons, but in the smaller neurons these stacks of broad, flattened cisternae are often more numerous than the Nissl bodies and are regularly present in the proximal dendrites (Fig. 2). M a n y small vesicles adjacent to the Golgi apparatus also extend f r o m the perikaryon into the proximal dendrites. Nearly all L H A neurons contain 1200-1500 diameter, smooth-walled vesicles (Fig. 3) with dense granules that are noted in the dendritic cytoplasm as well. Multivesicular bodies are occasionally situated in the Golgi region or lying free within the dendrites. In m a n y cells there are 600-700 A diameter coated or alveolate vesicles distinguished by radially arranged bristles projecting f r o m their surface. Although lipofuscin granules are inconspicuous, numerous lysosomes are contained within the neuronal cytoplasm. Subsurface cisternae with narrow channels closely applied to the cytoplasmic surface are evident in a few neurons where they are typically located adjacent to areas of p l a s m a l e m m a free o f axosomatic synaptic terminals (Fig. 2).
Fig. 1 a--d. Photomicrographs of the lateral hypothalamic area. a Coronal section of the tuberal hypo-
thalamus at the level analyzed in this study. Abbreviations: A, arcuate nucleus; F, fornix; IC, internal capsule; LH, lateral hypothalamic area; ME, median eminence; OT, optic tract; VM, ventromedial nucleus; VTA, ventral tuberal area. Cresyl violet stain, marker bar = 750 pro. b Horizontal section through lateral hypothalamic area showing rows of interfascicular oligodendroglia with interspersed neurons of the lateral hypothalamic nucleus. Cresyl violet stain, marker bar = 25 ~tm. c High power photomicrograph to illustrate small neurons of the area. Cresyl violet stain, marker bar = 8 rim. d Large neuron of the lateral hypothalamic area. Cresyl violet stain, marker bar = 8 ~tm
Fig. 2. Normal tuberal LHA, coronal plane. Typical small neuron contains sparse Nissl substance, abundant Golgi apparatus, subsurface cisternae (thin arrows) and a nucleolus-like body (boldarrow) in the cytoplasm. No axosomatic synaptic terminals are evident. Surrounding neuropil composed of myelinated and unmyelinated MFB axons, dendrites and synaptic terminals, x 10,250 Inset: Higher magnification of a cytoplasmic nucleolus-like body demonstrating a fenestrated appearance and the predominantly granular component of the matrix, x 22,000
Fig. 3. Cytoplasm of large LHA neuron illustrating large Nissl bodies (ER), Golgi apparatus (G), and rare dense granular vesicles (thin arrows). • 4,800 Fig. 4, LHA coronal plane. Bundle of unmyelinated MFB axons (center) is tightly compacted in the intercellular space between two small neurons. Neuronal cytoplasmic contents similar to Figure 2. N, nucleus; G, Golgi apparatus; MVB, muilivesicular body. x 26,000
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A conspicuous feature of some LHA neurons is the presence of a distinctive cytoplasmic inclusion body. These nucleolus-like bodies (NLB) measure up to 2 lxm in diameter and are exclusively found in the neuronal cytoplasm (Fig. 2). At higher magnification the NLB are composed of two principal components; moderately dense 150-200 A diameter granules and occasional filaments approximately 70 A in diameter (Fig. 2, inset). Frequently NLB's exhibit electron lucent clearing in the matrix of the dense granules producing a fenestrated appearance (Fig. 2, inset). Ribosomes and mitochondria may be seen adhering to the outer surface of many NLB's.
Medial Forebrain Bundle The second major division of the LHA is the medial forebrain bundle which contributes large groups of axons to the neuropil that are principally fibers of passage (Millhouse, 1969). Two populations of axons can be recognized in the medial forebrain bundle. Small, unmyelinated axons are grouped in fascicles which are intermingled with individual myelinated axons (Fig. 5). The strikingly homogeneous group of small unmyelinated axons traverses the MFB in parallel bundles that run in a rostro-caudal plane. In the coronal sections these axons measure 100-400 nm in diameter and exhibit the full range of size variability within any one bundle (Fig. 5). Neurofilaments, microtubules, smooth endoplasmic reticulum, and occasional mitochondria are the most common axoplasmic contents. Frequently, 800-1000 A diameter, dense granular vesicles may be found within the axoplasm or collected near terminal enlargements of synaptic endings (Fig. 5, inset). In sagittal sections, the individual axons are tightly grouped and and have extensive, regular linear contours without branching points. It is only on rare occasions that these small axons are observed to expand into synaptic terminal enlargements within the neuropil. A second prominent component of the MFB is the presence of individual myelinated axons 0.5 to 2 Ixm in diameter (Fig. 5). These axons, unlike the unmyelinated fibers, are present individually scattered among the other axons, often in company with oligodendrocytes. A thin to moderately thick myelin sheath surrounds the axolemma and axoplasmic contents, the latter mainly being formed by numerous neurofilaments and microtubules together with variable smooth endoplasmic reticulum and mitochondria. In contrast to the small unmyelinated axons, dense granular vesicles or other neurosecretory vesicles are not a conspicuous feature of individual myelinated axons. On rare occasions, single 1000 A diameter dense granular vesicles may be found within the axoplasm of these large axons.
The LHA Neuropil The neuropil contains a substantial dendritic population which takes the form of large dendritic trunks occasionally observed to be in continuity with neuronal perikarya (Fig. 2). In addition, the neuropil is densely populated with the more numerous secondary and tertiary dendritic branches that contain projections
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Fig. 5. LHA neuropil, coronal plane. Prominent components include myelinated and unmyelinated axons of MFB, some of which contain 800-100 A diameter dense granular vesicles (arrows), synaptic terminals, and dendrites (D). Cytoplasm of two small neurons appears below and at lower left. x 26,400 Inset: Higher magnification view of synaptic terminal that contains abundant 800-1000 A diameter dense core vesicles mixed with lucent core vesicles. • 45,200
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Fig. 6. Representative axosomatic and axodendritic synaptic terminal types encountered in the analysis of LHA neuropil. Axosomatic dense core vesicle-containing terminals (DCIO are mingled with terminal (center) containing pleomorphic spherical or elliptical lucent core vesicles (L C V). Axodendritic terminal (upper left) contains only lucent spherical vesicles, x 45,000 Fig. 7. This axodendritic terminalillustrates the distinctive crystalline-like array of compactly organized lucent vesicles, x 37,000
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and spines. Large dendrites are frequently seen lying at right angles to the longitudinally oriented axons of the medial forebrain bundle. That an extensive dendritic arborization is present is evident because of the presence of numerous, slender dendritic branches measuring 0.4 to 1.8 gm in diameter. Abundant synaptic terminals are present in the neuropil and these make contact principally with dendritic branches (Figs. 2 and 5). Compared with the visual cortex (Valverde, t967) and other areas of cortex (Peters, Palay and Webster, 1970), dendritic spines are relatively sparse in their distribution, even on the most terminal dendritic branches. Spines measuring 0.4 to 1.4 gm in greatest dimension are distinguished from dendritic branches by their lack of cytoplasmic organelles, especially microtubules, and the presence of fine, fibrillar material (Fig. 8 a) characteristic of dendritic spines even in the absence of a spine apparatus (Peter, Palay and Webster, 1970). Another component of the neuropil is the rich network of synaptic terminals. Two populations of synaptic endings are recognized in the LHA; axosomatic synapses terminating upon cell bodies and axodendritic synaptic terminals present in the dendritic field (Fig. 6). Axosomatic synapses are, in general, infrequent in the LHA. Less than 10~ of the cell body surface appears to be occupied by such terminals since individual neurons rarely show more than four cell surface terminals in a coronal section and it is more common that no axosomatic ending are evident in a single section (Fig. 2). Two types of axosomatic synaptic terminals are encountered and, in a count of 100 of these terminals, the two groups appear to occur with equal frequency. The first type is terminals containing 800-1000 A diameter, dense-core vesicles among greater numbers of 400-600 A lucent spherical vesicles (Fig. 6). A second type contains only lucent core spherical vesicles or pleomorphic spherical and slightly elliptical vesicles (Fig. 6). The pre- and postsynaptic specializations and cell organelles in the subjacent neuronal cytoplasm do not differ structurally from chemical axosomatic synapses described in other regions of mammalian brain (Peters, Palay and Webster, 1970). Axodendritic synaptic terminals comprise the majority of terminals in the neuropil and are represented by three common types (Table 1). Approximately 50~o of 1000 axodendritic synaptic terminals counted in the LHA, contain one or more 800-1000 A diameter dense granular vesicles amid a more numerous population of 400-600 A diameter, lucent spherical vesicles (Fig. 8 a). A second type of terminal contains a compactly organized array of slightly smaller 300-500 A diameter, spherical vesicles that have the appearance of crystalline-like structure (Figs. 7 and 8 b). This terminal type is infrequent and contributes less than 3~o to the total population of axodendritic terminals. The remainder of the terminals (47~) belong to the third type which contains only spherical or pleomorphic spherical and slightly elliptical vesicles distributed throughout the terminal (Figs. 6 and 8 a). Like many other sites of chemical synapse in the mammalian brain, the pre- and post-synaptic membrane specializations can usually be separated into asymmetrical synapses of the Gray type I and symmetrical synapses of the Gray type II. In the tuberal LHA, the Gray type II terminals comprise about 7 0 ~ of the synaptic specializations primarily at axosomatic and axodendritic contacts (Fig. 6). This is especially evident on the larger dendritic branches although type II specializations were occasionally encountered on smaller dendritic branches.
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Table 1. Relative distribution of synaptic terminal types
Normal LHA DCV terminals LCV terminals Compact V terminals
50~ 47~ 3~
Following rostral lesions
DCV terminals LCV terminals Compact V terminals
Acute
Chronic
48% 49% 3%
45-50~o 45-50% 1- 2%
Acute
Chronic
Following caudal lesions
DCV terminals LCV terminals Compact V terminals
37~ 61 ~ 2~/o
40~ 58~ 2~
Abbreviations: DCV = Dense Core Vesicle; LCV = Lucent Core Vesicle; V = Vesicle. See text for definitions of lesions and terminal types
The Gray type I synapses make up the remaining 30~ and are found mainly on dendritic spines and terminal dendritic branches (Fig. 8 a) but occasional exceptions to this rule are encountered. This ratio of Gray I to Gray II synaptic types is similar to that observed in the rat suprachiasmatic nucleus (Giildner, 1976). Dendrodendritic, dendrosomatic and axoaxonal synapses were not observed in the normal LHA although the former two types of synapses have been described in the suprachiasmatic nucleus (Giildner, 1976).
Neuroglia Nearly the whole of the neuroglial population is composed of oligodendrocytes while the remainder is represented by widely spaced protoplasmic astrocytes. Oligodendrocytes are the most numerous among the myelinated fibers of the medial forebrain bundle but they are also distributed as perineuronal satellite cells. A few protoplasmic astrocytes occur in the LHA and are easily distinguished by their intracytoplasmic fascicles of 80-90 A diameter glial filaments. Although astrocytic cell bodies appear to be infrequent, abundant small astrocytic processes are distributed throughout the neuropil in such a manner as to isolate or surround the receptive surfaces of neurons and synaptic terminals. It is within the neuropil that these slender isolating glial processes are connected by a system of membrane specializations that have the features of gap or nexus junctions (Fig. 8 e) (Brightman and Reese, 1969; Sotelo and Llin~ts, 1972; Weinstein and McNutt, 1972).
Fig. 8. a Representative Gray I synapse (lower left) upon dendritic spine with terminal that contains several 800-I000 ~, diameter DCV mixed with more numerous spherical LCV. Terminal (upper right) contains pleomorphic spherical and slightly elliptical LCV. • 25,000. b Terminal containing compactly organized array of slightly smaller 400-500A diameter lucent spherical vesicles. • 35,000. c Degenerating Gray I axodendritic synaptic terminal 4 days following a caudal MFB lesion (midbrain hemisection). • 46,000. d Degenerating synaptic terminal following acute caudal MFB lesion. Terminal contains at least two dense core vesicles (arrows). x 58,700. e Gap junction (center) joins two slender isolating astrocytic processes in the neuropil. S, synaptic terminal, x 183,700
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These junctions are formed by the outer leaflets of the glial membrane converging to produce a cleft or gap 20-30 A wide between the apposed membranes. In material stained "en bloc" with aqueous uranyl acetate, a lattice-like structure with transverse dense lines repeating every 80-90 A is evident in the junction (Fig. 8e). The overall width of the septalaminar junction is 160 A. In tangential sections the gap junctions appear to be composed of polygonal subunits with 90 center to center spacing.
Effects of Lesions on the Structural Organization of the LHA Lesions placed either in the rostral LHA in the preoptic area or caudally at the junction between the midbrain and diencephalon, allowed analysis of both acute and chronic effects of denervation on the structure and synaptic organization of the LHA (Table 1).
Acute Caudal Lesions Electron microscopic study of the LHA 2 and 4 days following midbrain hemisection reveals numerous degenerating synaptic terminals and axons (Fig. 8c and d). A count of degenerating axons indicates that 7 5 ~ are myelinated and 25~o unmyelinated. Of the degenerating synaptic terminals that could be classified according to their contents, 6 0 ~ contain at least one dense core vesicle (Figs. 8 c and d), 5 ~ contain lucent core vesicles and the remaining 3 5 ~ cannot be classified with certainty due to the marked electron density of the degenerating terminal matrix. While numerous degenerating axons and terminals are evident at both 2 and 4 days after interruption of all ascending input into the LHN, it should be emphasized that these represent only a relatively small proportion of the total axons and terminals in the LHN.
Chronic Caudal Lesions Two animals with caudal lesions were allowed to survive 4 months before examination of the synaptic organization in the LHA. In these animals there appears to be significant rearrangement of the synaptology resulting in an alteration in the distribution of terminal types. Terminals with one or more dense granular vesicles comprised only 40~o in animals with chronic caudal lesions compared to 50~o in the normal LHA. The contribution of terminals with lucent core vesicles of either the spherical or elliptical types increased to 5 8 ~ in the LHA of animals with chronic caudal lesions in contrast to the normal distribution of47~o. The presence of terminals with compact lucent vesicles remained unchanged at 2 ~ in these lesioned animals. In the chronically denervated LHA, no change in LHA is evident other than the change in frequency of terminal types. In particular, no increase in vacant dendritic space or other aspects of LHA organization is apparent.
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Acute Rostral Lesions
Animals subjected to rostral transection of the LHA in the preoptic area and sacrificed after 2 to 4 days exhibit relatively few degenerating terminals or axons in contrast to the prominent short term degeneration observed in animals with caudal transection. Chronic Rostral Lesions
Two animals surviving 8 months with a rostral lesion were studied. These showed no evident alteration of the terminal distribution pattern as observed in animals with chronic caudal lesion. In the animals with a rostral lesion, approximately 50% of the terminals contained at least one dense core vesicle, 49% contained only lucent core vesicles and approximately 1~ were terminals with compactlyorganized vesicles in a crystalline-like fashion. This distribution of terminal type conforms closely to that described for normal control animals above in which 50% of the terminals counted contained large, dense-core vesicles, 47% contained spherical or pleomorphic vesicles and 3 ~ the compactly-organized vesicles.
Discussion
Investigations of the ultrastructure of the hypothalamus have concentrated on the medial hypothalamic nuclei and, particularly, those concerned with hypothalamic-pituitary relations (cf. Clementi and Ceccarelli, 1970; Knigge and Silverman, 1974, for reviews). It has been evident for some time, however, that the LHA serves not only as a conduit for the fibers of the MFB but contains a prominent nucleus with numerous connections and a complex synaptic architecture. In addition, there is substantial evidence (cf. Szent~igothai et al., 1968; Millhouse, 1969; Nauta and Haymaker, 1969; Raisman, 1971, for reviews) that the lateral hypothalamic nucleus is uniquely situated in that it receives input both from ascending and descending components of the MFB and the medial hypothalamic nuclei, and projects upon the medial hypothalamic nuclei, as well as into the ascending and descending MFB. On this background it appeared important to undertake an analysis of the LHA ultrastructure as a basis for understanding its functional organization. The present study represents an initial step in this analysis. Its intent was to provide a description of the LHA at the tuberal level. This level was chosen because of its morphological relationship to the medial hypothalamic areas which constitute the "hypophysiotrophic" area of the hypothalamus (Szent~igothai et al., 1968). The description has included the neurons of the LHA, the neuropil, the glia and the effects of acute and chronic denervation on the synaptic organization of the LHA. L H A Neurons
The ultrastructural features of the lateral hypothalamic area are similar in many respects to other brain areas and, particularly, to other hypothalamic nuclei,
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Neurons of the LHA have the characteristics of non-neurosecretory hypothalamic cells as detailed by Clementi and Ceccarelli (1970) in that they lack the large 1800-2400 A diameter cytoplasmic neurosecretory granules that are prevalent in the supraoptic-paraventricular complex and arcuate nucleus (Subro and Pellegrino de Iraldi, 1969; Clementi and Ceccarelli, 1970; Bandaranayake, 1971; Kalimo, 1971; Adamo, 1972). The presence of well-developed Golgi apparatus and only a few cisternae of granular endoplasmic reticulum together with scattered 8001200/~ diameter dense granular cytoplasmic vesicles are features that have been described in neurons of the anterior and suprachiasmatic nuclei (Clementi and Ceccarelli, 1970). Many large, amorphous, dense membrane bound bodies are present in the neuronal cytoplasm and are similar in appearance to lysosomes described in other hypothalamic neurons (Clementi and Ceccarelli, 1970). Subsurface cisternae that appear unrelated to axosomatic terminals are comparable to those extensions of endoplasmic reticulum seen in cortical neurons and in Purkinje cells (Peters, Palay and Webster, 1970). The distinctive, nucleolus-like bodies (NLB) observed in the present study are similar in every respect to the NLB's reported in neurons of several mammals and lower vertebrates (Santolaya, 1973). Since the first reports of NLB in neurons of the rat hypothalamus (Shimizu and Ishii, 1965), nearly all hypothalamic nuclei have been found to contain these bodies (Adamo, 1972; Santolaya, 1973; Anzil et al., 1973) and, as shown in this study, the LHA is no exception.
LHA Neuropil Ultrastructurally, the organization of the LHA differs significantly from other hypothalamic regions in the following respects. First, as noted above, the neurons of the lateral hypothalamic nucleus do not contain the characteristic organelles of secretory neurons (Clementi and Ceccarelli, 1970). Secretory granules are only infrequently encountered in the neuronal cytoplasm and are not a constant feature of either the small or large neurons. Second, a distinctive feature of the LHA is the compact arrangement of the rostro-caudally oriented MFB fibers among the neuronal cell bodies. Although small, unmyelinated fibers are a very common finding in many hypothalamic nuclei, their arrangement into compactly organized parallel bundles mingled with individual myelinated axons is characteristic of the large MFB pathway and is similar to the arrangement illustrated by Adamo (1972) in the lateral preoptic area (LPA) and described in the medial preoptic area (MPA) by Prince and Jones-Witters (1974). This is in accord with the view that the LPA is a rostral continuation of the LHA (Bleier, 1963; Crosby and Showers, 1969) and, thus, exhibits a similar structure. In contrast to other major hypothalamic regions, the LHA neuropil has the appearance of a more regularly organized structure due to the presence of MFB axons arranged in bundles. Finally, the dendritic arborization of the lateral hypothalamic path neurons is generally oriented perpendicular to the axon bundles of the MFB (Millhouse, 1969). These differences from other hypothalamic nuclei are important factors that contribute to the distinctive fine structural organization of the LHA. In the present study, analysis of the synaptic types in the LHA has shown three main variations. The synaptic types are not unique to the LHA, for similar terminals have been illustrated in many areas of the CNS including other hypothalamic
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nuclei (Clementi and Ceccarelli, 1970; Ifft and McCarthy, 1974). We recognize, as do others, that division of synaptic types on the basis of morphology alone must he undertaken with caution. However, the striking differences between synaptic terminals in the LHA suggests that a functional differentiation may correlate with their morphology. The most unusual synaptic type is so infrequent that it comprises less than 3 ~ of the available axodendritic synaptic terminals. In this type, numerous lucent spherical vesicles are compacted tightly together, usually in the central portion of the terminal, to form the appearance of a crystalline-like array. Although the morphology of the lucent vesicles is not usual, they are of uniformly smaller size than lucent vesicles noted in other types of terminals. Synaptic endings with a nearly identical vesicle population have been illustrated by Clementi and Ceccarelli (1970) in the anterior hypothalamic nucleus, by Ifft and McCarthy (1974) in the supraoptic nucleus and by Gfildner (1976) in the suprachiasmatic nucleus. The presence of this terminal type in more than one hypothalamic nucleus suggests that the synaptic endings may have a uniform function in the areas in which they are present but the details of functional aspects of this type of terminal remain to be elucidated. A similar, tightly-compacted synaptic terminal containing a population of dense granular vesicles and lucent spherical vesicles has recently been described in the superior cervical ganglion of cats (Birks, 1974). Unlike the compactly organized terminals in the LHA, many superior cervical ganglion terminals, presumed to contain acetylcholine, also contain dense granular vesicles. The identification of this type of terminal in the superior cervical ganglion and the changes occurring with stimulation (Birks, 1974) suggests that it may represent a functional variant of a more typical lucent core vesicle terminal. Our observations indicate that axodendritic terminals with 800-1000 A diameter dense granular vesicles occur with a frequency of 50~. Approximately 4 7 ~ of the axodendritic terminals contain lucent vesicles of variable shape. Since histochemical studies have shown that the MFB is a major ascending pathway of monoamine neuron systems (cf. Anden et al., 1966; Ungerstedt, 1971, for reviews) and these pathways appear to give off terminals in the LHA (Ungerstedt, 1971; Lindvall and Bj6rklund, 1974), the question arises as to whether some of these dense core-vesicle-containing terminals arise from monoamine neurons. This cannot be stated with certainty from the present material but it is likely that some do fall into this class. Bloom (1972) has emphasized the difficulty of correlating vesicle morphology with neurotransmitter content. Nevertheless, large numbers of monoamine axons are traversing the area studied and Descarries and his associates (Descarries et al., 1975, 1977) have demonstrated that large dense core vesicles are a uniform feature of the terminals of serotonin and noradrenaline neurons in the central nervous system. Thus, both the lucent core and large dense core containing terminals undoubtedly represent axons arising from a wide variety of sources and employing a number of substances as neurotransmitters.
L H A Glia
Neuroglial elements in the LHA differ substantively from those of other hypothalamic nuclei in one important respect. This is the finding of numerous slender
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glial processes connected by gap junctions and interposed between the active surfaces of neurons, dendrites and synaptic terminals. Such a distribution of asstrocytic processes is well known in different regions of the nervous system (Peters and Palay, 1965; Peters, Palay and Webster, 1970). Throughout the LHA these glial processes are interconnected by gap or nexus junctions of the type encountered in the central nervous system but only recently found to be a feature of isolating astrocytic processes (Morales and Duncan, 1975; Sipe and Moore, 1976).
Degeneration Studies Degeneration studies offer the opportunity to study not only alterations in the distribution of synaptic terminals in the area but also to identify the sources of afferent and efferent projections. Because the number of afferents to the LHA is so great and their sources so diverse, it was beyond the scope of this introductory study to attempt to identify sources of input to the LHA and the location and types of their terminals on LHA neurons. This promises to be a difficult task for the future because of the limited number of synaptic terminal types evident in routine electron microscopic study and the known variety in their sources of origin. The preliminary degeneration experiments carried out in the present study, however, have indicated that this method can contribute to our understanding of the organization of the LHA neuropil. Studies employing rostral and caudal lesions have resulted in two different patterns of terminal degeneration in the acute phase. Rostral preoptic lesions produced little degeneration of synaptic terminals in the tuberal LHA. This failure to produce more evidence of degenerating terminals and axons may reflect several factors. First, many of the descending projections in the MFB terminate quite rostrally (Raisman, 1971). Second, the postoperative survival periods selected may not be optimal for the descending systems. And, although more degenerating terminals are noted within the caudal lesions (see below), this may represent a general problem with the degeneration studies. The medial forebrain bundle is made up almost entirely ofunmyelinated axons and such axons have been shown at least in the periphery, to undergo a very rapid degeneration which is difficult to visualize (Roth and Richardson, 1969). With acute midbrain hemisection, however, numerous degenerating axons and synaptic terminals were observed in the tuberal LHA. Up to 60~o of these degenerating synaptic terminals contained at least one large dense core vesicle. As discussed above, it would appear likely that at least some of the degenerating terminals with large dense core vesicles in animals with acute caudal lesions represent ascending brainstem monoamine neuron axons in the MFB terminating on neurons of the LHA. Animals allowed to survive 4 to 8 months following both rostral and caudal lesions showed alterations of the synaptic terminal population when compared to normal controls. In animals with long-standing caudal hemisection, the relative number of synaptic terminals containing at least one dense core vesicle dropped to 40~o compared to 50~o in normal animals while the relative number of lucent vesicle terminals rose to 57~o compared to 47~o in normal controls. However, in animals with chronic rostral lesions, the quantitative distribution of terminal types was essentially unchanged from normal controls. The problem of determining whether or not an actual reorganization of syn-
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aptic terminal space occurs in animals with chronic caudal lesions cannot be resolved at this time since the quantitative differences in terminal types may only reflect terminal loss without reorganization. For example, analyses of vacant terminal space in the dendritic arborization of normal LHA neurons, as well as lesioned animals would need to be undertaken in order to determine if changes in synaptic organization could be related to significant changes in available synaptic terminal space. Nevertheless, the chronic alteration in the proportion of terminals with large dense core vesicles compared to those with lucent core vesicles following caudal lesions indicates that a significant proportion of the dense core vesicle terminals arise from brainstem neurons. The present study was devoted to characterizing the ultrastructure of the LHA in the normal animal and in the animal with acute and chronic denervation of the area. It is meant as a preliminary investigation to provide a background for subsequent studies into the organization of this complex area. The morphological analysis of the LHA appears to be a worthwhile undertaking in that it forms a background for understanding the participation of this region in such important neural functions as the mechanisms of reward (Olds, 1972; Olds, 1974, 1975; Rolls, 1975). References Adamo, N.J.: Ultrastructural features of the lateral preoptic area, median eminence, and arcuate nucleus of the rat. Z. Zellforsch. 127, 483491 (1972) Anden, N.-E., Dahlstr/Sm, A., Fuxe, K., Larsson, K., Olson, L., Ungerstedt, U.: Ascending monoamine neurons to the telencephalon and diencephalon. Acta physiol. Scand. 67, 31 3.326 (1966) Anzil, A.P., Herrlinger, H., Blinzinger, K. : Nucleolus-like inclusions in neuronal perikarya and processes: phase and electron microscopic observations on the hypothalamus of the mouse. Z. Zellforsch. 146, 329-337 (1973) Bandaranayake, R.C.: Morphology of the accessory neurosecretory nuclei and of the retrochiasmic part of the supraoptic nucleus of the rat. Acta anat. (Basel) 80, 14-22 (1971) Birks, R.I.: The relationship of transmitter release and storage to fine structure in a sympathetic ganglion. J. Neurocytol. 3, 133-160 (1974) Bleier, R.H.: The hypothalamus of the cat. Baltimore: John Hopkins Press 1961 Bloom, F.: Localization of neurotransmitters by electron microscopy. Res. Publ. Ass. nerv. ment. Dis. 50, 25-57 (1972) Brightman, M.W., Reese, T.S. : Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 40, 648-677 (1969) Christ, J.E. : Derivation and boundaries of the hypothalamus with atlas of hypothalamic grisea. In: W. Haymaker, E. Anderson, W.J.H. Nauta (eds.), The hypothalamus, pp. 13.60. Springfield, Illinois: C.C. Thomas 1969 Clementi, F., Ceccarelli, B.: Fine structure of rat hypothalamic nuclei. In: L. Martini, M. Motta, F, Fraschini (eds.), The hypothalamus, pp. 1743. New York: Academic Press 1970 Crosby, E.C., Showers, M.J.C.: Comparative anatomy of the preoptic and hypothalamic areas. In: W. Haymaker, E. Anderson, W.J.H. Nauta (eds.), The hypothalamus, pp. 61-135. Springfield, Illinois: C.C.Thomas 1969 Descarries, L., Raudet, A., Watkins, K.C.: Serotonin nerve terminals in adult rat neocortex. Brain Res. 100, 563.588 (1975) Descarries, L., Watkins, K.C., Lapierre, Y.: Noradrenergic axon terminals in the cerebral cortex of the rat. III. Topometric ultrastructural analysis. Brain Res. (In press, 1977) Epstein, A.N.: The lateral hypothalamic syndrome: its implications for the physiological psychology of hunger and thirst. Progr, physiol. Psychol. 4, 263.317 (1971) Gtildner, F.-H.: Synaptology of the rat suprachiasmatic nucleus. Cell Tiss. Res. 165, 509-544 (1976) Guillery, R.W. : Degeneration in the hypothalamic connections of the albino rat. J. Anat. (Lond.) 91, 91-115 (1957)
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Gurdjian, E.S. : The diencephalon of the albino rat. Studies on the brain of the rat, No. 2. J. comp. Neurol. 43, 1-114 (1927) Ifft, J.D., McCarthy, U : Somatic spines in the supraoptic nucleus of the rat hypothalamus. Cell Tiss. Res. 148, 203-211 (1974) Kalimo, H.: Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat. Z. Zellforsch. 122, 283-300 (1971) Knigge, K.M., Silverman, A.J.: Anatomy of the endocrine hypothalamus. In: E. Knobil, W.H. Sawyer (eds.), The pituitary gland and its neuroendocrine control, Part 1, pp. 1-32. Washington: American Physiological Society 1974 Lindvall, O., Bj6rklund, A.: The organization of the ascending catecholamine neuron system in the rat brain. Acta physiol, scand., Suppl. 412, 1-48 (1974) Millhouse, O.E.: A Golgi study of the descending medial forebrain bundle. Brain Res. 15, 341-363 (1969) Morales, R., Duncan, D. : Spezialized contacts of astrocytes with astrocytes and with other cell types in the spinal cord of the cat. Anat. Rec. 182, 255-266 (1975) Nauta, W.J.H., Haymaker, W.: Hypothalamic nuclei and fiber connections. In: W. Haymaker, E. Anderson, W.J.H. Nauta (eds.), The hypothalamus, pp. 136-209. Springfield, Illinois: C.C. Thomas 1969 Olds, J.: Hypothalamic substrates of reward. Physiol. Rev. 42, 554-604 (1962) Olds, M.E.: Unit responses in the medial forebrain bundle to rewarding stimulation in the hypothalamus. Brain Res. 80, 479-495 (1974) Olds, M.E. : Short-term changes in the firing pattern of hypothalamic neurons during Pavlovian conditioning. Brain Res. 58, 95-116 (1973) Peters, A., Palay, S.L.: An electron microscope study of the distribution and patterns of astroglial processes in the central nervous system. J. Anat. (Lond.) 99, 419 (1965) Peters, A., Palay, S.L., Webster, H.D.: The fine structure of the nervous system. New York: Harper and Row, Publishers, Inc., 1970 Prince, F.P., Jones-Witters, P.H.: The ultrastructure of the medial preoptic area of the rat. Cell Tiss. Res. 153, 517-530 (1974) Raisman, G.: Some aspects of the neural connections of the hypothalamus. In: L. Martini, M. Motta, F. Fraschini (eds.), The hypothalamus. New Yorl~: Academic Press 1970 Rolls, E.: The brain and reward. New York: Pergamon Press 1975 Roth, C.D., Richardson, K.C.: Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy. Amer. J. Anat. 124, 341-360 (1969) Santolaya, R.C.: Nucleolus-like bodies in the neuronal cytoplasm of the mouse arcuate nucleus. Z. Zellforsch. 146, 319-328 (1973) Shimizu, N., Ishii, S.: Electron-microscopic observations on nucleolar extrusion in nerve cells of the rat hypothalamus. Z. Zellforsch. 67, 367-372 (1965) Sipe, J.C., Moore, R.Y.: Astrocytic gap junctions in the rat lateral hypothalamic area. Anat. Rec. 185, 247-252 (1976) Sipe, J.C., Vick, N.A., Schulman, S., Fernandez, C. : Plasmocid encephalopathy in the Rhesus monkey: a study of selective vulnerability. J. Neuropath. exp. Neurol. 32, 446-457 (1973) Sotelo, C., Llin~ts, R.: Specialized membrane junctions between neurons in the vertebrate cerebellar cortex. J. Cell Biol. 53, 271-289 (1972) Stricker, E.M., Zigmond, M.J.: Recovery of function after damage to central catecholamine-containing neurons: a neurochemical model for the lateral hypothalamic syndrome. Progr. Psychobiol. Physiol. Psych. 6, 121 188 (1976) Suburo, A.M., Pellegrino de Iraldi, A.: An ultrastructural study of the rat's suprachiasmatic nucleus. J. Anat. (Lond.) 105, 439-446 (1969) Szentfigothai, J., Flerk6, B., Mess, B., Halfisz, B.: Hypothalamic control of the anterior pituitary. Akadrmiai Kiad6 (Budapest) 1968 Ungerstedt, U.: Stereotaxic mapping of the monoamine pathways in the rat brain. Acta physiol. scand., Suppl. 367, 1-48 (1971) Valverde, F.: Apical dendritic spines of the visual cortex and light deprivation in the mouse. Exp. Brain Res. 3, 337-352 (1967) Weinstein, R.S., McNutt, N.S.: Cell junctions. New Engl. J. Med. 286, 521-524 (1972)
Accepted December 20, 1976
Cell and Tissue Research 9 by Springer-Veflag 1977
The Lateral Hypothalamic Area An Ultrastructural Analysis* Jack C. Sipe** and Robert Y. Moore*** **Veterans Administration Hospital, San Diego, California, and **,***Department of Neurosciences, University of California at San Diego, La Jolla, California
Summary. An ultrastructural analysis of the rat lateral hypothalamic area (LHA) was undertaken in order to provide an initial step in the characterization of this complex area which appears to participate in a number of important neural functions. The organization of the normal tuberal LHA was compared to the area following acute and chronic denervating lesions. In the normal animal, the principal features of the LHA are the presence of lateral hypothalamic neurons, a major sagittal pathway (the medial forebrain bundle, MFB) and the interposed neuropil richly populated by a variety of synaptic terminal types. Alterations in the synaptic organization of the LHA following rostral and caudal MFB lesions were most pronounced in animals with acute and chronic caudal lesions. A 10% reduction of synaptic terminals containing 800-1000 A diameter dense core vesicles and a 10% increase in terminals containing lucent core vesicles was observed in animals with caudal lesions while no significant redistribution of synaptic terminal types occurred with rostral lesions. The preliminary degeneration experiments indicate that identification of the numerous and diverse afferents to the LHA neuropil may be aided by this method but that a detailed and systematic ultrastructural analysis will be required to identify sources of input with certainty.
Key words: Lateral hypothalamic area - Synaptic organization - Rat Ultrastructure.
Introduction The lateral hypothalamic area (LHA) contains the axons of the medial forebrain bundle (MFB) and a prominent nucleus, the lateral hypothalamic nucleus (LHN) Send offprint requests to: Jack C. Sipe, M.D., Department of Neurology, Veterans Administration Hospital, San Diego, Ca 92161,USA
* Presented in part at the 27th Annual Meeting of the American Academy of Neurology, Bal Harbour, FLA, 1975 ** Recipient of Research Associate Award, Veterans Administration **,*** Supported by the Veterans Administration and by NIH Grants NS 12080. Skilled technical assistance was provided by Robin Isaacs, Marilyn Woodward and Sharon Keigher
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(cf. G u r d j i a n , 1927; Bleier, 1963; Christ, 1969). The n e u r o n s of this nucleus give rise to a s c e n d i n g a n d descending projections into the adjacent medial h y p o t h a l a m i c zone (Guillery, 1957; Szentfigothai et al., 1968; Millhouse, 1969; R a i s m a n , 1971), a n d they receive i n p u t f r o m the b r a i n s t e m reticular f o r m a t i o n , medial h y p o t h a l a mus, other lateral h y p o t h a l a m i c n e u r o n s a n d basal f o r e b r a i n (Guillery, 1957; Szent/tgothai et al., 1968; Millhouse, 1969; N a u t a a n d H a y m a k e r , 1969; R a i s m a n , 1971). Since the m e d i a l h y p o t h a l a m i c nuclei receive relatively sparse projections f r o m e x t r a - h y p o t h a l a m i c sources ( N a u t a a n d H a y m a k e r , 1969; R a i s m a n , 1971), it is evident that the lateral h y p o t h a l a m i c nucleus is a n a t o m i c a l l y situated to represent a m a j o r i n t e g r a t i n g center between the b r a i n s t e m reticular f o r m a t i o n , basal f o r e b r a i n a n d m e d i a l h y p o t h a l a m u s . This is c o n f i r m e d by a n u m b e r of a b l a t i o n a n d s t i m u l a t i o n studies which have d e m o n s t r a t e d that the L H A plays a n i m p o r t a n t role in m a n y f u n c t i o n s (cf. Olds, 1962; Epstein, 1971; Rolls, 1975; Stricker a n d Z i g m o n d , 1976 for reviews) b u t these have generally been a t t r i b u t e d to the c o m p o n e n t s of the medial f o r e b r a i n b u n d l e or related p a t h w a y s traversing the L H A a n d adjacent areas. There has b e e n little a t t e n t i o n directed to the L H N even t h o u g h its n e u r o n s appear to participate i m p o r t a n t l y in f u n c t i o n s such as reward (Olds, 1973, 1974; Rolls, 1975). Because o f its evident f u n c t i o n a l significance, the present u l t r a s t r u c t u r a l study was u n d e r t a k e n in order to provide further i n f o r m a t i o n o n the o r g a n i z a t i o n of the lateral h y p o t h a l a m i c area.
Material and Methods The animals used in this study were adult female albino rats of the Sprague-Dawleystrain, 150-200gm. They were anesthetized with pentobarbital (40 mg/kg) and perfused with glutaraldehyde-parafomaldehyde solutions described perviously (Sipe et al., 1973). The LHA from the tuberal hypothalamus, between the optic chiasm and mamillary nuclei at approximately the level of the ventromedial nucleus, was dissected in a block approximately 1.5 • 1 mm, from 10 normal adult rats. The tissue blocks were post-fixed in cacodylate-buffered osmium tetroxide and aqueous uranyl acetate before dehydration and embedding in Epon. Thick sections were stained with methylene blue and used for further identification of areas to be examined by electron microscopy. Thin sections were mounted on uncoated grids, stained with both warm uranyl acetate in 50~ methanol and lead citrate and examined in the coronal, sagittal and horizontal planes. A large number of photographs of randomly selected areas through the LHA was made at medium and high magnification for categorization and counting of synaptic terminal types and other structures. In addition, cresyl violet-stained celloidin sections in the coronal and horizontal planes were studied by light microscopy. Two groups of animal were prepared with lesions. Each consisted of 6 animals with unilateral lesions either rostral to or caudal to the tuberal portion of the hypothalamus. The lesions were made as follows. With the animals under deep anesthesia, the head was placed in a small animal stereotaxic apparatus (Kopf Instruments, Tujunga, Ca) with the incisor bar 2 mm above the tooth bar. A burr hole was placed in the skull and a guillotine-type knife, 1.5 mm in diameter, was passed through the brain in the coronal plane until it reached the skull base. Both the rostral and caudal lesions were made with the center of the knife, 1.7 mm lateral to the midline. The rostral lesion was made at the level of the bregma and the caudal lesion 4.5 mm caudal to the bregma. Two animals at each time point from each group were sacrificed at 2 days, 4 days and at 4 and 8 months after the lesion was made. The method of sacrifice and preparation of tissue was as described above. The acute postoperative material, 2 and 4 day survival, was examined for evidence of axon terminal degeneration. The chronic brains were sectioned as described above and the terminals were categorized and counted from photographs .of each lesion type.
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Results
Light Microscopic Observations The part of the lateral hypothalamic area analyzed in the present study is the tuberal region lying between the optic chiasm rostrally and the mamillary region caudally (Fig. 1a). In this region the LHA is bordered by the zona incerta dorsally, the internal capsule laterally, the optic tract and pial surface ventrally and the medial hypothalamus medially. In sections stained by the Nissl method the LHA is characterized by the presence of the scattered polymorphic neurons of the lateral hypothalamic nucleus. These neurons are quite numerous throughout the LHA. At the medial border of the LHA they merge without a distinct demarcation with the cells of the ventral tuberal field of the medial hypothalamus. There is a wide range of neuron size in the lateral hypothalamic nucleus. The most prevalent cells are large (approximately 35-45 p~m in diameter) with prominent, darkly-staining Nissl substance and a centrally situated nucleus (Fig. 1 d). They vary in shape from fusiform to multipolar and in coronal sections appear scattered throughout the nucleus without any evident arrangement into groups. In horizontal sections, however, the neurons appear to be arranged, at least in part, into irregular rows which parallel the trajectory of medial forebrain bundle fibers (Fig. 1b). The remaining neurons in the LHA fall into two groups on the basis of size. One is composed of small neurons, usually 12-15 ~tm in diameter, with scanty cytoplasm and faint Nissl substance and the other is an intermediate group with most cells ranging from 18-25 ~tm in diameter (Fig. 1c). In sections stained for myelin, numerous small myelinated fibers are evident scattered throughout the LHA.
Electron Microscopic Observations The distinctive ultrastructure of the LHA is characterized by the presence of lateral hypothalamic neurons whose dendrites, radially arranged in the coronal plane, are intermingled with rostro-caudally oriented axons in thick bundles. This appearance is due to the presence of the axon bundles of the major sagittal pathway in the LHA, the medial forebrain bundle, and the neurons of the LHN. The neuropil interposed between these two major components is richly populated with a variety of synaptic terminals. Ultrastructural analysis of the LHA demonstrates three major components; the neuronal perikarya of the lateral hypothalamic nucleus, the axons of the medial forebrain bundle, and a complex neuropil composed chiefly of a varied array of synaptic terminals. The following description of the ultrastructural organization will emphasize those electron microscopic features that are distinctive to the normal LHA, since the subcellular structure of the area is similar in most respects to other regions of mammalian brain (Peters, Palay and Webster, 1970). This is followed by an analysis of the effects of rostral and caudal MFB lesions on the organization of the neuropil of the tuberal LHA.
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Lateral Hypothalamic Nucleus The cell bodies of the lateral hypothalamic neurons and their processes f o r m an almost continuous spectrum of cell morphology from the large neurons (35-50 Ixm in diameter) that contain abundant Nissl substance to small neurons (12-15 I~m in diameter) with extremely sparse Nissl substance (Fig. 2). In general, the cell bodies o f these neurons tend to be rounded or fusiform with the perikaryon occupied by a large nucleus which exhibits a rounded or slightly oval contour. The nuclear envelope has a typical, double-layered structure with periodic nuclear pores and shows a remarkable tendency toward invagination (Fig. 2) similar to that seen in other hypothalamic neurons (Clementi and Ceccarelli, 1970). A conspicuous, dense spherioidal nucleolus is surrounded by evenly dispersed delicate nuclear chromatin. Cytoplasmic organelles are identical to those in all neurons save for their distribution. The pattern of Nissl substance in the small and medium size neurons is characterized by only a few slender cisternae of granular endoplasmic reticulum which become less conspicuous in the smaller neurons (Fig. 4). Large neurons, on the other hand, display large rhomboid stacks of granular endoplasmic reticulum (Fig. 3) that are often disposed in a crescent shape about the nuclear envelope. Nissl substance is not confined to the perikaryon and is often observed extending into the main dendritic trunks. N u m e r o u s ribosomes and polyribosomal aggregates are particularly abundant in the smaller neurons where they lie free in the cytoplasm and are not closely associated with the surface o f the endoplasmic reticulum (Fig. 4). The Golgi apparatus, however, is very prominent in all neurons, but in the smaller neurons these stacks of broad, flattened cisternae are often more numerous than the Nissl bodies and are regularly present in the proximal dendrites (Fig. 2). M a n y small vesicles adjacent to the Golgi apparatus also extend f r o m the perikaryon into the proximal dendrites. Nearly all L H A neurons contain 1200-1500 diameter, smooth-walled vesicles (Fig. 3) with dense granules that are noted in the dendritic cytoplasm as well. Multivesicular bodies are occasionally situated in the Golgi region or lying free within the dendrites. In m a n y cells there are 600-700 A diameter coated or alveolate vesicles distinguished by radially arranged bristles projecting f r o m their surface. Although lipofuscin granules are inconspicuous, numerous lysosomes are contained within the neuronal cytoplasm. Subsurface cisternae with narrow channels closely applied to the cytoplasmic surface are evident in a few neurons where they are typically located adjacent to areas of p l a s m a l e m m a free o f axosomatic synaptic terminals (Fig. 2).
Fig. 1 a--d. Photomicrographs of the lateral hypothalamic area. a Coronal section of the tuberal hypo-
thalamus at the level analyzed in this study. Abbreviations: A, arcuate nucleus; F, fornix; IC, internal capsule; LH, lateral hypothalamic area; ME, median eminence; OT, optic tract; VM, ventromedial nucleus; VTA, ventral tuberal area. Cresyl violet stain, marker bar = 750 pro. b Horizontal section through lateral hypothalamic area showing rows of interfascicular oligodendroglia with interspersed neurons of the lateral hypothalamic nucleus. Cresyl violet stain, marker bar = 25 ~tm. c High power photomicrograph to illustrate small neurons of the area. Cresyl violet stain, marker bar = 8 rim. d Large neuron of the lateral hypothalamic area. Cresyl violet stain, marker bar = 8 ~tm
Fig. 2. Normal tuberal LHA, coronal plane. Typical small neuron contains sparse Nissl substance, abundant Golgi apparatus, subsurface cisternae (thin arrows) and a nucleolus-like body (boldarrow) in the cytoplasm. No axosomatic synaptic terminals are evident. Surrounding neuropil composed of myelinated and unmyelinated MFB axons, dendrites and synaptic terminals, x 10,250 Inset: Higher magnification of a cytoplasmic nucleolus-like body demonstrating a fenestrated appearance and the predominantly granular component of the matrix, x 22,000
Fig. 3. Cytoplasm of large LHA neuron illustrating large Nissl bodies (ER), Golgi apparatus (G), and rare dense granular vesicles (thin arrows). • 4,800 Fig. 4, LHA coronal plane. Bundle of unmyelinated MFB axons (center) is tightly compacted in the intercellular space between two small neurons. Neuronal cytoplasmic contents similar to Figure 2. N, nucleus; G, Golgi apparatus; MVB, muilivesicular body. x 26,000
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A conspicuous feature of some LHA neurons is the presence of a distinctive cytoplasmic inclusion body. These nucleolus-like bodies (NLB) measure up to 2 lxm in diameter and are exclusively found in the neuronal cytoplasm (Fig. 2). At higher magnification the NLB are composed of two principal components; moderately dense 150-200 A diameter granules and occasional filaments approximately 70 A in diameter (Fig. 2, inset). Frequently NLB's exhibit electron lucent clearing in the matrix of the dense granules producing a fenestrated appearance (Fig. 2, inset). Ribosomes and mitochondria may be seen adhering to the outer surface of many NLB's.
Medial Forebrain Bundle The second major division of the LHA is the medial forebrain bundle which contributes large groups of axons to the neuropil that are principally fibers of passage (Millhouse, 1969). Two populations of axons can be recognized in the medial forebrain bundle. Small, unmyelinated axons are grouped in fascicles which are intermingled with individual myelinated axons (Fig. 5). The strikingly homogeneous group of small unmyelinated axons traverses the MFB in parallel bundles that run in a rostro-caudal plane. In the coronal sections these axons measure 100-400 nm in diameter and exhibit the full range of size variability within any one bundle (Fig. 5). Neurofilaments, microtubules, smooth endoplasmic reticulum, and occasional mitochondria are the most common axoplasmic contents. Frequently, 800-1000 A diameter, dense granular vesicles may be found within the axoplasm or collected near terminal enlargements of synaptic endings (Fig. 5, inset). In sagittal sections, the individual axons are tightly grouped and and have extensive, regular linear contours without branching points. It is only on rare occasions that these small axons are observed to expand into synaptic terminal enlargements within the neuropil. A second prominent component of the MFB is the presence of individual myelinated axons 0.5 to 2 Ixm in diameter (Fig. 5). These axons, unlike the unmyelinated fibers, are present individually scattered among the other axons, often in company with oligodendrocytes. A thin to moderately thick myelin sheath surrounds the axolemma and axoplasmic contents, the latter mainly being formed by numerous neurofilaments and microtubules together with variable smooth endoplasmic reticulum and mitochondria. In contrast to the small unmyelinated axons, dense granular vesicles or other neurosecretory vesicles are not a conspicuous feature of individual myelinated axons. On rare occasions, single 1000 A diameter dense granular vesicles may be found within the axoplasm of these large axons.
The LHA Neuropil The neuropil contains a substantial dendritic population which takes the form of large dendritic trunks occasionally observed to be in continuity with neuronal perikarya (Fig. 2). In addition, the neuropil is densely populated with the more numerous secondary and tertiary dendritic branches that contain projections
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Fig. 5. LHA neuropil, coronal plane. Prominent components include myelinated and unmyelinated axons of MFB, some of which contain 800-100 A diameter dense granular vesicles (arrows), synaptic terminals, and dendrites (D). Cytoplasm of two small neurons appears below and at lower left. x 26,400 Inset: Higher magnification view of synaptic terminal that contains abundant 800-1000 A diameter dense core vesicles mixed with lucent core vesicles. • 45,200
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Fig. 6. Representative axosomatic and axodendritic synaptic terminal types encountered in the analysis of LHA neuropil. Axosomatic dense core vesicle-containing terminals (DCIO are mingled with terminal (center) containing pleomorphic spherical or elliptical lucent core vesicles (L C V). Axodendritic terminal (upper left) contains only lucent spherical vesicles, x 45,000 Fig. 7. This axodendritic terminalillustrates the distinctive crystalline-like array of compactly organized lucent vesicles, x 37,000
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and spines. Large dendrites are frequently seen lying at right angles to the longitudinally oriented axons of the medial forebrain bundle. That an extensive dendritic arborization is present is evident because of the presence of numerous, slender dendritic branches measuring 0.4 to 1.8 gm in diameter. Abundant synaptic terminals are present in the neuropil and these make contact principally with dendritic branches (Figs. 2 and 5). Compared with the visual cortex (Valverde, t967) and other areas of cortex (Peters, Palay and Webster, 1970), dendritic spines are relatively sparse in their distribution, even on the most terminal dendritic branches. Spines measuring 0.4 to 1.4 gm in greatest dimension are distinguished from dendritic branches by their lack of cytoplasmic organelles, especially microtubules, and the presence of fine, fibrillar material (Fig. 8 a) characteristic of dendritic spines even in the absence of a spine apparatus (Peter, Palay and Webster, 1970). Another component of the neuropil is the rich network of synaptic terminals. Two populations of synaptic endings are recognized in the LHA; axosomatic synapses terminating upon cell bodies and axodendritic synaptic terminals present in the dendritic field (Fig. 6). Axosomatic synapses are, in general, infrequent in the LHA. Less than 10~ of the cell body surface appears to be occupied by such terminals since individual neurons rarely show more than four cell surface terminals in a coronal section and it is more common that no axosomatic ending are evident in a single section (Fig. 2). Two types of axosomatic synaptic terminals are encountered and, in a count of 100 of these terminals, the two groups appear to occur with equal frequency. The first type is terminals containing 800-1000 A diameter, dense-core vesicles among greater numbers of 400-600 A lucent spherical vesicles (Fig. 6). A second type contains only lucent core spherical vesicles or pleomorphic spherical and slightly elliptical vesicles (Fig. 6). The pre- and postsynaptic specializations and cell organelles in the subjacent neuronal cytoplasm do not differ structurally from chemical axosomatic synapses described in other regions of mammalian brain (Peters, Palay and Webster, 1970). Axodendritic synaptic terminals comprise the majority of terminals in the neuropil and are represented by three common types (Table 1). Approximately 50~o of 1000 axodendritic synaptic terminals counted in the LHA, contain one or more 800-1000 A diameter dense granular vesicles amid a more numerous population of 400-600 A diameter, lucent spherical vesicles (Fig. 8 a). A second type of terminal contains a compactly organized array of slightly smaller 300-500 A diameter, spherical vesicles that have the appearance of crystalline-like structure (Figs. 7 and 8 b). This terminal type is infrequent and contributes less than 3~o to the total population of axodendritic terminals. The remainder of the terminals (47~) belong to the third type which contains only spherical or pleomorphic spherical and slightly elliptical vesicles distributed throughout the terminal (Figs. 6 and 8 a). Like many other sites of chemical synapse in the mammalian brain, the pre- and post-synaptic membrane specializations can usually be separated into asymmetrical synapses of the Gray type I and symmetrical synapses of the Gray type II. In the tuberal LHA, the Gray type II terminals comprise about 7 0 ~ of the synaptic specializations primarily at axosomatic and axodendritic contacts (Fig. 6). This is especially evident on the larger dendritic branches although type II specializations were occasionally encountered on smaller dendritic branches.
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Table 1. Relative distribution of synaptic terminal types
Normal LHA DCV terminals LCV terminals Compact V terminals
50~ 47~ 3~
Following rostral lesions
DCV terminals LCV terminals Compact V terminals
Acute
Chronic
48% 49% 3%
45-50~o 45-50% 1- 2%
Acute
Chronic
Following caudal lesions
DCV terminals LCV terminals Compact V terminals
37~ 61 ~ 2~/o
40~ 58~ 2~
Abbreviations: DCV = Dense Core Vesicle; LCV = Lucent Core Vesicle; V = Vesicle. See text for definitions of lesions and terminal types
The Gray type I synapses make up the remaining 30~ and are found mainly on dendritic spines and terminal dendritic branches (Fig. 8 a) but occasional exceptions to this rule are encountered. This ratio of Gray I to Gray II synaptic types is similar to that observed in the rat suprachiasmatic nucleus (Giildner, 1976). Dendrodendritic, dendrosomatic and axoaxonal synapses were not observed in the normal LHA although the former two types of synapses have been described in the suprachiasmatic nucleus (Giildner, 1976).
Neuroglia Nearly the whole of the neuroglial population is composed of oligodendrocytes while the remainder is represented by widely spaced protoplasmic astrocytes. Oligodendrocytes are the most numerous among the myelinated fibers of the medial forebrain bundle but they are also distributed as perineuronal satellite cells. A few protoplasmic astrocytes occur in the LHA and are easily distinguished by their intracytoplasmic fascicles of 80-90 A diameter glial filaments. Although astrocytic cell bodies appear to be infrequent, abundant small astrocytic processes are distributed throughout the neuropil in such a manner as to isolate or surround the receptive surfaces of neurons and synaptic terminals. It is within the neuropil that these slender isolating glial processes are connected by a system of membrane specializations that have the features of gap or nexus junctions (Fig. 8 e) (Brightman and Reese, 1969; Sotelo and Llin~ts, 1972; Weinstein and McNutt, 1972).
Fig. 8. a Representative Gray I synapse (lower left) upon dendritic spine with terminal that contains several 800-I000 ~, diameter DCV mixed with more numerous spherical LCV. Terminal (upper right) contains pleomorphic spherical and slightly elliptical LCV. • 25,000. b Terminal containing compactly organized array of slightly smaller 400-500A diameter lucent spherical vesicles. • 35,000. c Degenerating Gray I axodendritic synaptic terminal 4 days following a caudal MFB lesion (midbrain hemisection). • 46,000. d Degenerating synaptic terminal following acute caudal MFB lesion. Terminal contains at least two dense core vesicles (arrows). x 58,700. e Gap junction (center) joins two slender isolating astrocytic processes in the neuropil. S, synaptic terminal, x 183,700
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These junctions are formed by the outer leaflets of the glial membrane converging to produce a cleft or gap 20-30 A wide between the apposed membranes. In material stained "en bloc" with aqueous uranyl acetate, a lattice-like structure with transverse dense lines repeating every 80-90 A is evident in the junction (Fig. 8e). The overall width of the septalaminar junction is 160 A. In tangential sections the gap junctions appear to be composed of polygonal subunits with 90 center to center spacing.
Effects of Lesions on the Structural Organization of the LHA Lesions placed either in the rostral LHA in the preoptic area or caudally at the junction between the midbrain and diencephalon, allowed analysis of both acute and chronic effects of denervation on the structure and synaptic organization of the LHA (Table 1).
Acute Caudal Lesions Electron microscopic study of the LHA 2 and 4 days following midbrain hemisection reveals numerous degenerating synaptic terminals and axons (Fig. 8c and d). A count of degenerating axons indicates that 7 5 ~ are myelinated and 25~o unmyelinated. Of the degenerating synaptic terminals that could be classified according to their contents, 6 0 ~ contain at least one dense core vesicle (Figs. 8 c and d), 5 ~ contain lucent core vesicles and the remaining 3 5 ~ cannot be classified with certainty due to the marked electron density of the degenerating terminal matrix. While numerous degenerating axons and terminals are evident at both 2 and 4 days after interruption of all ascending input into the LHN, it should be emphasized that these represent only a relatively small proportion of the total axons and terminals in the LHN.
Chronic Caudal Lesions Two animals with caudal lesions were allowed to survive 4 months before examination of the synaptic organization in the LHA. In these animals there appears to be significant rearrangement of the synaptology resulting in an alteration in the distribution of terminal types. Terminals with one or more dense granular vesicles comprised only 40~o in animals with chronic caudal lesions compared to 50~o in the normal LHA. The contribution of terminals with lucent core vesicles of either the spherical or elliptical types increased to 5 8 ~ in the LHA of animals with chronic caudal lesions in contrast to the normal distribution of47~o. The presence of terminals with compact lucent vesicles remained unchanged at 2 ~ in these lesioned animals. In the chronically denervated LHA, no change in LHA is evident other than the change in frequency of terminal types. In particular, no increase in vacant dendritic space or other aspects of LHA organization is apparent.
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Acute Rostral Lesions
Animals subjected to rostral transection of the LHA in the preoptic area and sacrificed after 2 to 4 days exhibit relatively few degenerating terminals or axons in contrast to the prominent short term degeneration observed in animals with caudal transection. Chronic Rostral Lesions
Two animals surviving 8 months with a rostral lesion were studied. These showed no evident alteration of the terminal distribution pattern as observed in animals with chronic caudal lesion. In the animals with a rostral lesion, approximately 50% of the terminals contained at least one dense core vesicle, 49% contained only lucent core vesicles and approximately 1~ were terminals with compactlyorganized vesicles in a crystalline-like fashion. This distribution of terminal type conforms closely to that described for normal control animals above in which 50% of the terminals counted contained large, dense-core vesicles, 47% contained spherical or pleomorphic vesicles and 3 ~ the compactly-organized vesicles.
Discussion
Investigations of the ultrastructure of the hypothalamus have concentrated on the medial hypothalamic nuclei and, particularly, those concerned with hypothalamic-pituitary relations (cf. Clementi and Ceccarelli, 1970; Knigge and Silverman, 1974, for reviews). It has been evident for some time, however, that the LHA serves not only as a conduit for the fibers of the MFB but contains a prominent nucleus with numerous connections and a complex synaptic architecture. In addition, there is substantial evidence (cf. Szent~igothai et al., 1968; Millhouse, 1969; Nauta and Haymaker, 1969; Raisman, 1971, for reviews) that the lateral hypothalamic nucleus is uniquely situated in that it receives input both from ascending and descending components of the MFB and the medial hypothalamic nuclei, and projects upon the medial hypothalamic nuclei, as well as into the ascending and descending MFB. On this background it appeared important to undertake an analysis of the LHA ultrastructure as a basis for understanding its functional organization. The present study represents an initial step in this analysis. Its intent was to provide a description of the LHA at the tuberal level. This level was chosen because of its morphological relationship to the medial hypothalamic areas which constitute the "hypophysiotrophic" area of the hypothalamus (Szent~igothai et al., 1968). The description has included the neurons of the LHA, the neuropil, the glia and the effects of acute and chronic denervation on the synaptic organization of the LHA. L H A Neurons
The ultrastructural features of the lateral hypothalamic area are similar in many respects to other brain areas and, particularly, to other hypothalamic nuclei,
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Neurons of the LHA have the characteristics of non-neurosecretory hypothalamic cells as detailed by Clementi and Ceccarelli (1970) in that they lack the large 1800-2400 A diameter cytoplasmic neurosecretory granules that are prevalent in the supraoptic-paraventricular complex and arcuate nucleus (Subro and Pellegrino de Iraldi, 1969; Clementi and Ceccarelli, 1970; Bandaranayake, 1971; Kalimo, 1971; Adamo, 1972). The presence of well-developed Golgi apparatus and only a few cisternae of granular endoplasmic reticulum together with scattered 8001200/~ diameter dense granular cytoplasmic vesicles are features that have been described in neurons of the anterior and suprachiasmatic nuclei (Clementi and Ceccarelli, 1970). Many large, amorphous, dense membrane bound bodies are present in the neuronal cytoplasm and are similar in appearance to lysosomes described in other hypothalamic neurons (Clementi and Ceccarelli, 1970). Subsurface cisternae that appear unrelated to axosomatic terminals are comparable to those extensions of endoplasmic reticulum seen in cortical neurons and in Purkinje cells (Peters, Palay and Webster, 1970). The distinctive, nucleolus-like bodies (NLB) observed in the present study are similar in every respect to the NLB's reported in neurons of several mammals and lower vertebrates (Santolaya, 1973). Since the first reports of NLB in neurons of the rat hypothalamus (Shimizu and Ishii, 1965), nearly all hypothalamic nuclei have been found to contain these bodies (Adamo, 1972; Santolaya, 1973; Anzil et al., 1973) and, as shown in this study, the LHA is no exception.
LHA Neuropil Ultrastructurally, the organization of the LHA differs significantly from other hypothalamic regions in the following respects. First, as noted above, the neurons of the lateral hypothalamic nucleus do not contain the characteristic organelles of secretory neurons (Clementi and Ceccarelli, 1970). Secretory granules are only infrequently encountered in the neuronal cytoplasm and are not a constant feature of either the small or large neurons. Second, a distinctive feature of the LHA is the compact arrangement of the rostro-caudally oriented MFB fibers among the neuronal cell bodies. Although small, unmyelinated fibers are a very common finding in many hypothalamic nuclei, their arrangement into compactly organized parallel bundles mingled with individual myelinated axons is characteristic of the large MFB pathway and is similar to the arrangement illustrated by Adamo (1972) in the lateral preoptic area (LPA) and described in the medial preoptic area (MPA) by Prince and Jones-Witters (1974). This is in accord with the view that the LPA is a rostral continuation of the LHA (Bleier, 1963; Crosby and Showers, 1969) and, thus, exhibits a similar structure. In contrast to other major hypothalamic regions, the LHA neuropil has the appearance of a more regularly organized structure due to the presence of MFB axons arranged in bundles. Finally, the dendritic arborization of the lateral hypothalamic path neurons is generally oriented perpendicular to the axon bundles of the MFB (Millhouse, 1969). These differences from other hypothalamic nuclei are important factors that contribute to the distinctive fine structural organization of the LHA. In the present study, analysis of the synaptic types in the LHA has shown three main variations. The synaptic types are not unique to the LHA, for similar terminals have been illustrated in many areas of the CNS including other hypothalamic
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nuclei (Clementi and Ceccarelli, 1970; Ifft and McCarthy, 1974). We recognize, as do others, that division of synaptic types on the basis of morphology alone must he undertaken with caution. However, the striking differences between synaptic terminals in the LHA suggests that a functional differentiation may correlate with their morphology. The most unusual synaptic type is so infrequent that it comprises less than 3 ~ of the available axodendritic synaptic terminals. In this type, numerous lucent spherical vesicles are compacted tightly together, usually in the central portion of the terminal, to form the appearance of a crystalline-like array. Although the morphology of the lucent vesicles is not usual, they are of uniformly smaller size than lucent vesicles noted in other types of terminals. Synaptic endings with a nearly identical vesicle population have been illustrated by Clementi and Ceccarelli (1970) in the anterior hypothalamic nucleus, by Ifft and McCarthy (1974) in the supraoptic nucleus and by Gfildner (1976) in the suprachiasmatic nucleus. The presence of this terminal type in more than one hypothalamic nucleus suggests that the synaptic endings may have a uniform function in the areas in which they are present but the details of functional aspects of this type of terminal remain to be elucidated. A similar, tightly-compacted synaptic terminal containing a population of dense granular vesicles and lucent spherical vesicles has recently been described in the superior cervical ganglion of cats (Birks, 1974). Unlike the compactly organized terminals in the LHA, many superior cervical ganglion terminals, presumed to contain acetylcholine, also contain dense granular vesicles. The identification of this type of terminal in the superior cervical ganglion and the changes occurring with stimulation (Birks, 1974) suggests that it may represent a functional variant of a more typical lucent core vesicle terminal. Our observations indicate that axodendritic terminals with 800-1000 A diameter dense granular vesicles occur with a frequency of 50~. Approximately 4 7 ~ of the axodendritic terminals contain lucent vesicles of variable shape. Since histochemical studies have shown that the MFB is a major ascending pathway of monoamine neuron systems (cf. Anden et al., 1966; Ungerstedt, 1971, for reviews) and these pathways appear to give off terminals in the LHA (Ungerstedt, 1971; Lindvall and Bj6rklund, 1974), the question arises as to whether some of these dense core-vesicle-containing terminals arise from monoamine neurons. This cannot be stated with certainty from the present material but it is likely that some do fall into this class. Bloom (1972) has emphasized the difficulty of correlating vesicle morphology with neurotransmitter content. Nevertheless, large numbers of monoamine axons are traversing the area studied and Descarries and his associates (Descarries et al., 1975, 1977) have demonstrated that large dense core vesicles are a uniform feature of the terminals of serotonin and noradrenaline neurons in the central nervous system. Thus, both the lucent core and large dense core containing terminals undoubtedly represent axons arising from a wide variety of sources and employing a number of substances as neurotransmitters.
L H A Glia
Neuroglial elements in the LHA differ substantively from those of other hypothalamic nuclei in one important respect. This is the finding of numerous slender
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glial processes connected by gap junctions and interposed between the active surfaces of neurons, dendrites and synaptic terminals. Such a distribution of asstrocytic processes is well known in different regions of the nervous system (Peters and Palay, 1965; Peters, Palay and Webster, 1970). Throughout the LHA these glial processes are interconnected by gap or nexus junctions of the type encountered in the central nervous system but only recently found to be a feature of isolating astrocytic processes (Morales and Duncan, 1975; Sipe and Moore, 1976).
Degeneration Studies Degeneration studies offer the opportunity to study not only alterations in the distribution of synaptic terminals in the area but also to identify the sources of afferent and efferent projections. Because the number of afferents to the LHA is so great and their sources so diverse, it was beyond the scope of this introductory study to attempt to identify sources of input to the LHA and the location and types of their terminals on LHA neurons. This promises to be a difficult task for the future because of the limited number of synaptic terminal types evident in routine electron microscopic study and the known variety in their sources of origin. The preliminary degeneration experiments carried out in the present study, however, have indicated that this method can contribute to our understanding of the organization of the LHA neuropil. Studies employing rostral and caudal lesions have resulted in two different patterns of terminal degeneration in the acute phase. Rostral preoptic lesions produced little degeneration of synaptic terminals in the tuberal LHA. This failure to produce more evidence of degenerating terminals and axons may reflect several factors. First, many of the descending projections in the MFB terminate quite rostrally (Raisman, 1971). Second, the postoperative survival periods selected may not be optimal for the descending systems. And, although more degenerating terminals are noted within the caudal lesions (see below), this may represent a general problem with the degeneration studies. The medial forebrain bundle is made up almost entirely ofunmyelinated axons and such axons have been shown at least in the periphery, to undergo a very rapid degeneration which is difficult to visualize (Roth and Richardson, 1969). With acute midbrain hemisection, however, numerous degenerating axons and synaptic terminals were observed in the tuberal LHA. Up to 60~o of these degenerating synaptic terminals contained at least one large dense core vesicle. As discussed above, it would appear likely that at least some of the degenerating terminals with large dense core vesicles in animals with acute caudal lesions represent ascending brainstem monoamine neuron axons in the MFB terminating on neurons of the LHA. Animals allowed to survive 4 to 8 months following both rostral and caudal lesions showed alterations of the synaptic terminal population when compared to normal controls. In animals with long-standing caudal hemisection, the relative number of synaptic terminals containing at least one dense core vesicle dropped to 40~o compared to 50~o in normal animals while the relative number of lucent vesicle terminals rose to 57~o compared to 47~o in normal controls. However, in animals with chronic rostral lesions, the quantitative distribution of terminal types was essentially unchanged from normal controls. The problem of determining whether or not an actual reorganization of syn-
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aptic terminal space occurs in animals with chronic caudal lesions cannot be resolved at this time since the quantitative differences in terminal types may only reflect terminal loss without reorganization. For example, analyses of vacant terminal space in the dendritic arborization of normal LHA neurons, as well as lesioned animals would need to be undertaken in order to determine if changes in synaptic organization could be related to significant changes in available synaptic terminal space. Nevertheless, the chronic alteration in the proportion of terminals with large dense core vesicles compared to those with lucent core vesicles following caudal lesions indicates that a significant proportion of the dense core vesicle terminals arise from brainstem neurons. The present study was devoted to characterizing the ultrastructure of the LHA in the normal animal and in the animal with acute and chronic denervation of the area. It is meant as a preliminary investigation to provide a background for subsequent studies into the organization of this complex area. The morphological analysis of the LHA appears to be a worthwhile undertaking in that it forms a background for understanding the participation of this region in such important neural functions as the mechanisms of reward (Olds, 1972; Olds, 1974, 1975; Rolls, 1975). References Adamo, N.J.: Ultrastructural features of the lateral preoptic area, median eminence, and arcuate nucleus of the rat. Z. Zellforsch. 127, 483491 (1972) Anden, N.-E., Dahlstr/Sm, A., Fuxe, K., Larsson, K., Olson, L., Ungerstedt, U.: Ascending monoamine neurons to the telencephalon and diencephalon. Acta physiol. Scand. 67, 31 3.326 (1966) Anzil, A.P., Herrlinger, H., Blinzinger, K. : Nucleolus-like inclusions in neuronal perikarya and processes: phase and electron microscopic observations on the hypothalamus of the mouse. Z. Zellforsch. 146, 329-337 (1973) Bandaranayake, R.C.: Morphology of the accessory neurosecretory nuclei and of the retrochiasmic part of the supraoptic nucleus of the rat. Acta anat. (Basel) 80, 14-22 (1971) Birks, R.I.: The relationship of transmitter release and storage to fine structure in a sympathetic ganglion. J. Neurocytol. 3, 133-160 (1974) Bleier, R.H.: The hypothalamus of the cat. Baltimore: John Hopkins Press 1961 Bloom, F.: Localization of neurotransmitters by electron microscopy. Res. Publ. Ass. nerv. ment. Dis. 50, 25-57 (1972) Brightman, M.W., Reese, T.S. : Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 40, 648-677 (1969) Christ, J.E. : Derivation and boundaries of the hypothalamus with atlas of hypothalamic grisea. In: W. Haymaker, E. Anderson, W.J.H. Nauta (eds.), The hypothalamus, pp. 13.60. Springfield, Illinois: C.C. Thomas 1969 Clementi, F., Ceccarelli, B.: Fine structure of rat hypothalamic nuclei. In: L. Martini, M. Motta, F, Fraschini (eds.), The hypothalamus, pp. 1743. New York: Academic Press 1970 Crosby, E.C., Showers, M.J.C.: Comparative anatomy of the preoptic and hypothalamic areas. In: W. Haymaker, E. Anderson, W.J.H. Nauta (eds.), The hypothalamus, pp. 61-135. Springfield, Illinois: C.C.Thomas 1969 Descarries, L., Raudet, A., Watkins, K.C.: Serotonin nerve terminals in adult rat neocortex. Brain Res. 100, 563.588 (1975) Descarries, L., Watkins, K.C., Lapierre, Y.: Noradrenergic axon terminals in the cerebral cortex of the rat. III. Topometric ultrastructural analysis. Brain Res. (In press, 1977) Epstein, A.N.: The lateral hypothalamic syndrome: its implications for the physiological psychology of hunger and thirst. Progr, physiol. Psychol. 4, 263.317 (1971) Gtildner, F.-H.: Synaptology of the rat suprachiasmatic nucleus. Cell Tiss. Res. 165, 509-544 (1976) Guillery, R.W. : Degeneration in the hypothalamic connections of the albino rat. J. Anat. (Lond.) 91, 91-115 (1957)
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Gurdjian, E.S. : The diencephalon of the albino rat. Studies on the brain of the rat, No. 2. J. comp. Neurol. 43, 1-114 (1927) Ifft, J.D., McCarthy, U : Somatic spines in the supraoptic nucleus of the rat hypothalamus. Cell Tiss. Res. 148, 203-211 (1974) Kalimo, H.: Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat. Z. Zellforsch. 122, 283-300 (1971) Knigge, K.M., Silverman, A.J.: Anatomy of the endocrine hypothalamus. In: E. Knobil, W.H. Sawyer (eds.), The pituitary gland and its neuroendocrine control, Part 1, pp. 1-32. Washington: American Physiological Society 1974 Lindvall, O., Bj6rklund, A.: The organization of the ascending catecholamine neuron system in the rat brain. Acta physiol, scand., Suppl. 412, 1-48 (1974) Millhouse, O.E.: A Golgi study of the descending medial forebrain bundle. Brain Res. 15, 341-363 (1969) Morales, R., Duncan, D. : Spezialized contacts of astrocytes with astrocytes and with other cell types in the spinal cord of the cat. Anat. Rec. 182, 255-266 (1975) Nauta, W.J.H., Haymaker, W.: Hypothalamic nuclei and fiber connections. In: W. Haymaker, E. Anderson, W.J.H. Nauta (eds.), The hypothalamus, pp. 136-209. Springfield, Illinois: C.C. Thomas 1969 Olds, J.: Hypothalamic substrates of reward. Physiol. Rev. 42, 554-604 (1962) Olds, M.E.: Unit responses in the medial forebrain bundle to rewarding stimulation in the hypothalamus. Brain Res. 80, 479-495 (1974) Olds, M.E. : Short-term changes in the firing pattern of hypothalamic neurons during Pavlovian conditioning. Brain Res. 58, 95-116 (1973) Peters, A., Palay, S.L.: An electron microscope study of the distribution and patterns of astroglial processes in the central nervous system. J. Anat. (Lond.) 99, 419 (1965) Peters, A., Palay, S.L., Webster, H.D.: The fine structure of the nervous system. New York: Harper and Row, Publishers, Inc., 1970 Prince, F.P., Jones-Witters, P.H.: The ultrastructure of the medial preoptic area of the rat. Cell Tiss. Res. 153, 517-530 (1974) Raisman, G.: Some aspects of the neural connections of the hypothalamus. In: L. Martini, M. Motta, F. Fraschini (eds.), The hypothalamus. New Yorl~: Academic Press 1970 Rolls, E.: The brain and reward. New York: Pergamon Press 1975 Roth, C.D., Richardson, K.C.: Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy. Amer. J. Anat. 124, 341-360 (1969) Santolaya, R.C.: Nucleolus-like bodies in the neuronal cytoplasm of the mouse arcuate nucleus. Z. Zellforsch. 146, 319-328 (1973) Shimizu, N., Ishii, S.: Electron-microscopic observations on nucleolar extrusion in nerve cells of the rat hypothalamus. Z. Zellforsch. 67, 367-372 (1965) Sipe, J.C., Moore, R.Y.: Astrocytic gap junctions in the rat lateral hypothalamic area. Anat. Rec. 185, 247-252 (1976) Sipe, J.C., Vick, N.A., Schulman, S., Fernandez, C. : Plasmocid encephalopathy in the Rhesus monkey: a study of selective vulnerability. J. Neuropath. exp. Neurol. 32, 446-457 (1973) Sotelo, C., Llin~ts, R.: Specialized membrane junctions between neurons in the vertebrate cerebellar cortex. J. Cell Biol. 53, 271-289 (1972) Stricker, E.M., Zigmond, M.J.: Recovery of function after damage to central catecholamine-containing neurons: a neurochemical model for the lateral hypothalamic syndrome. Progr. Psychobiol. Physiol. Psych. 6, 121 188 (1976) Suburo, A.M., Pellegrino de Iraldi, A.: An ultrastructural study of the rat's suprachiasmatic nucleus. J. Anat. (Lond.) 105, 439-446 (1969) Szentfigothai, J., Flerk6, B., Mess, B., Halfisz, B.: Hypothalamic control of the anterior pituitary. Akadrmiai Kiad6 (Budapest) 1968 Ungerstedt, U.: Stereotaxic mapping of the monoamine pathways in the rat brain. Acta physiol. scand., Suppl. 367, 1-48 (1971) Valverde, F.: Apical dendritic spines of the visual cortex and light deprivation in the mouse. Exp. Brain Res. 3, 337-352 (1967) Weinstein, R.S., McNutt, N.S.: Cell junctions. New Engl. J. Med. 286, 521-524 (1972)
Accepted December 20, 1976
