Sympathetic preganglionic neurons: properties and inputs.

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Sympathetic Preganglionic Neurons: Properties and Inputs Susan A. Deuchars*1 and Varinder K. Lall1 ABSTRACT The sympathetic nervous system comprises one half of the autonomic nervous system and participates in maintaining homeostasis and enabling organisms to respond in an appropriate manner to perturbations in their environment, either internal or external. The sympathetic preganglionic neurons (SPNs) lie within the spinal cord and their axons traverse the ventral horn to exit in ventral roots where they form synapses onto postganglionic neurons. Thus, these neurons are the last point at which the central nervous system can exert an effect to enable changes in sympathetic outflow. This review considers the degree of complexity of sympathetic control occurring at the level of the spinal cord. The morphology and targets of SPNs illustrate the diversity within this group, as do their diverse intrinsic properties which reveal some functional significance of these properties. SPNs show high degrees of coupled activity, mediated through gap junctions, that enables rapid and coordinated responses; these gap junctions contribute to the rhythmic activity so critical to sympathetic outflow. The main inputs onto SPNs are considered; these comprise afferent, descending, and interneuronal influences that themselves enable functionally appropriate changes in SPN activity. The complexity of inputs is further demonstrated by the plethora of receptors that mediate the different responses in SPNs; their origins and effects are plentiful and diverse. Together these different inputs and the intrinsic and coupled activity of SPNs result in the rhythmic nature of sympathetic outflow from the spinal cord, which has a variety of frequencies that can be altered in different conditions. © 2015 American Physiological Society. Compr Physiol 5:829-869, 2015.

Introduction The sympathetic nervous system comprises one half of the autonomic nervous system and participates in maintaining homeostasis and enabling organisms to respond in an appropriate manner to perturbations in their environment, either internal or external. This branch contributes to the activity of many end organs, including the cardiovascular system, brown adipose tissue (BAT), gastrointestinal tract, bladder, bowel, and sex organs, thus, it is critical that selective and coordinated changes in this branch are possible to elicit the relevant response or compensate for specific environmental changes. The sympathetic preganglionic neurons (SPNs) form the last central component of this system; their cell somata lie within the lateral horn and central regions of the spinal cord mainly at the thoracic and lumbar levels, although some are found at cervical level 8. Their axons predominantly innervate postganglionic neurons or directly innervate the adrenal medulla and accessory nerves in the periphery and thus these SPNs form the final point within the CNS at which descending and local influences can shape the sympathetic output to the many different end organs. For much of the early research, SPNs were considered simple relay neurons, sending on information without much integration or delicate control. However the degree of complexity of circuits within the spinal cord is now understood to be much greater. This review article will focus on the characteristics of these neurons [also reviewed by (113, 147, 193, 194, 255)] and also the interneurons in the

Volume 5, April 2015

spinal cord that form part of the circuits involved in sympathetic control (83, 84), thus bringing together in one review, our understanding of the two types of neuron that are major contributors to the spinal circuitry underlying sympathetic control. We will consider how these neurons are placed in the spinal cord, how they may subserve different functions and how they are influenced by different inputs that shape their activity (Fig. 1). Since we know that SPNs serve to control a multiplicity of functions, we will consider what enables us to identify functions of SPN and the issues involved in this unequivocal identification.

Location and Morphology of SPNs Techniques to study SPN anatomy Our earliest understanding of how SPNs may be arranged comes from studies in the 1800s showing that SPNs were found in the lateral border of the grey matter in the thoracolumbar region of the spinal cord (65). Since that time, a number of techniques have been utilized to enable more * Correspondence

to [email protected] of Biomedical Sciences, University of Leeds, Leeds, United Kingdom Published online, April 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.c140020 Copyright © American Physiological Society. 1 School

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Overview of inputs and outputs of SPNs. SPNs synapse on sympathetic postganglionic neurons, which themselves synapse on the target organ, enabling reflex adjustments of the internal environment. Sensory information is conveyed to the thoracolumbar spinal cord by afferents with their somata in the dorsal root ganglion. Afferents communicate with SPNs in the spinal cord via local interneurons. Afferents additionally synapse onto ascending neurons which communicate with autonomic, limbic, and endocrine circuits in the brainstem and forebrain. Supraspinal pathways provide extensive innervation of SPNs. This enables higher order, integrative responses to changes in the internal environment. SPN: sympathetic preganglionic neuron; SPGN: sympathetic postganglionic neuron; DRG: dorsal root ganglion.

comprehensive labeling. Petras and Cummings (284) localized SPNs by carrying out thoracic or abdominal sympathectomies in the rhesus monkey to cause chromalysis of neurons in distinct groups of the spinal cord; these groupings are described below. The advent of retrograde tracers enabled labeling of SPNs contributing to specific nerves or innervating ganglia in a variety of species, including the rat [superior cervical ganglion (SCG) (300); adrenal medulla (17, 308); SCG, stellate ganglion or adrenal medulla (297)]; guinea pig [single white rami from T4-8 (305)]; cat [stellate ganglion (62, 63, 276); hypogastric nerve (26); lumbar sympathetic chain and splanchnic nerves (148); SCG or adrenal medulla (299)]; and dog [single white ramus (29)]. Use of cholera toxin B conjugated to horseradish peroxidase (CB-HRP) as a retrograde tracer [SCG (348)] revealed more extensive dendritic arborizations than those observed with HRP. More recent studies have also considered the sympathetic innervations in teleosts, carrying out injections of HRP into the sympathetic trunk or celiac ganglion of puffer fish (100). This technique labels discrete groups of SPNs innervating ganglia serving specific body regions; however, to gain a more comprehensive picture, researchers have exploited the fact that SPNs are cholinergic by using antibodies specific to choline acetyltransferase (ChAT), which is an enzyme involved in the synthesis of acetylcholine (21). Immunohistochemistry

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carried out with this antibody will naturally also label all motoneurons but since these lie more ventral to the SPNs, they are easily distinguished and discounted from study. More critical is the fact that cholinergic interneurons around the central autonomic regions will also be labeled with this method so care must be taken with interpretation of these data, such that definitive identification of an interneuron would be one that was not retrogradely labeled by other tracing techniques. One technique has proven one of the most reliable and easy methods of labeling SPNs; this involves intraperitoneal injections of the tracer Fluorogold which in a short time (1-2 days) labels all SPN (5, 8). Many researchers have since exploited this method to good effect, combining it with other tracers to look at autonomic circuits; this research will be considered at later points in the article. Fluorogold can also be injected into specific ganglia or nerves to examine the location of SPN (336). These techniques are all excellent ways in which to gain an understanding of how whole or limited populations of SPNs are clustered and in which directions dendrites and axons are orientated. However, such methods are more limited when considering the morphological properties of single SPN, to understand the extent and complexity of each SPN and its neurites. For this, single cell recording and filling is used, first pioneered (79) in the cat and used subsequently in the cat (289), neonatal rat (99), and pigeon (45). Interestingly, despite the diverse number of approaches, very few studies have given an estimation of the total number of SPN in any one animal; one such (130), counted thionin stained SPNs and reported a range of 32,790 to 53,340 neurons in the intermediolateral cell column extending from C8-L4 in eight cats. A statistically significant difference in SPN numbers in male and female cats was also reported in this study.

Arrangement of SPNs in the spinal cord All of these studies have given an insight into how the SPNs are arranged in the spinal cord. These neurons are located in four topographically distinct groups, known as the autonomic nuclei; the intermediolateral cell column, nucleus intermediolateralis thoracolumbalis pars funicularis, intercalated nucleus, and central autonomic area (Fig. 2). The majority [75% in rat (300); 78.2% in cat (62)] of these neurons are located in the intermediolateral cell column (IML, nucleus intermediolateralis thoracolumbalis pars principalis) which sits at the lateral edge of the gray matter, forming a prominent peak at the thoracolumbar level but almost invisible at more rostral and caudal regions where there are no SPNs. SPNs form bilateral columns within the IML but these columns are not uniform in shape, instead SPNs cluster in groups of 20 to 150 neurons known as nests. The size of the clusters and distance between nests (100500 μm) vary according to species (62, 276, 284) and rostrocaudal level within the spinal cord since the distance between nests is smaller at more lumbar levels (100-300 μm). Within the IML, SPN comprise the majority of somata present and it seems that the remaining cells are interneurons associated


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Autonomic nuclei in the spinal cord. (A) Spinal cord section with immunohistochemical identification of cholinergic SPNs and ventral horn motoneurons (using an antibody to choline acetyltransferase). (B) Line diagram of a transverse section of spinal cord showing the four groups of SPNs DH: dorsal horn; IML: intermediolateral cell column (nucleus intermediolateralis thoracolumbalis pars principalis); Ilf: nucleus intermediolateralis pars funicularis; IC: intercalated nucleus (nucleus intercalatus); IPPe: central autonomic area (nucleus intercalatus pars paraependymalis); VHMNs: ventral horn motoneurons.

with autonomic control (see below). These SPNs have multipolar (triangular) or fusiform (spindle-shaped) cell bodies with a small number of neurons also exhibiting round or elongated somata (276,297,299). The proportion of each neuronal soma type is not uniform along the rostrocaudal axis since multipolar somata are more common in the first and second thoracic segments while in more caudal sections, they are generally fusiform (276). The dendritic trees of these neurons lie mainly in the rostrocaudal direction with less prominent dorsoventral arborization and these dendrites can extend over several nests of SPN with trajectories reaching 1.5 to 2.5 mm in the cat (79). More recent studies in the rat and cat revealed extensive mediolateral dendritic profiles reaching medially to the central canal and laterally to the white matter (297, 299, 349), indicating that the four SPN groups have the capacity to receive information from widespread yet similar sources, thus providing a clue as to the complexity of the spinal circuits controlling sympathetic outflow. Indeed, the report by Tang et al. (349) of a subependymal dendritic plexus raised the intriguing possibility that the SPN may be influenced by substances within the cerebrospinal fluid itself. Lying just lateral to the IML in the white matter of the spinal cord is the second group of SPNs, the nucleus intermediolateralis thoracolumbalis pars funicularis (Ilf). This region contains the majority of the remaining SPNs, although the extent of this varies depending on animal and projection targets—Ilf neurons make up around 8% of SPNs projecting to the adrenal medulla or SCG of the cat (299) but comprise a higher proportion of the total SPNs projecting to these targets in the adult and neonate rat [18%-23% (297)]. Numbers of Ilf neurons are sparse at more caudal regions where the neurons almost merge with those of the IML but more rostrally, these large mainly fusiform or multipolar neurons are scattered throughout the lateral funiculus (62, 336). The dendritic bundles of these cells extend mainly mediolaterally (21,284), mingling with those from the IML in the white matter.


Figure 3 shows the dendritic arborization patterns of SPNs from all four groups. The nests of SPNs in the IML are often associated with SPNs in the third grouping, namely the intercalated nucleus or nucleus intercalatus (IC) which, together with the fourth group may account for only 0.5% to 8% of SPNs (299). These neurons form transverse bands stretching across from the IML to the central canal but, although at times, they continue on from clusters of IML SPNs, this relationship is not 1:1. These cells are mainly fusiform and their dendrites run in a mediolateral plane. The fourth grouping of SPNs is in the central autonomic nucleus or nucleus intercalatus pars paraependymalis (IPPe). Once more, these are fusiform or multipolar with mediolaterally orientated dendrites that sometimes cross the central canal to the contralateral spinal cord, enabling some degree of interaction between the two sides. Figure 3 shows the dendritic orientation of the SPNs in the 4 different regions.

Can the location of SPNs in the spinal cord be associated with the function of these neurons? To determine whether SPNs innervating diverse postganglionic nerves arise from different autonomic nuclei, the distribution of SPNs retrogradely labeled from a number of sympathetic nerves or ganglia was studied (9, 148, 336). SPNs labeled from more rostral ganglia are located mainly in the IML and Ilf with only a small percentage in the IC and IPPe; however, SPNs labeled from the inferior mesenteric ganglia are predominantly (up to 80%) located in the IPPe. McLachlan (241) compared the spatial distribution of SPNs projecting to the hypogastric nerve, that supplies the viscera of the pelvic cavity, with those SPNs in the lumbar sympathetic trunk that are associated with vasomotor outflow to the lower body. Thus, SPNs with a mainly vasoconstrictor function are located in the lateral region of the IML or in

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Longitudinal section of mouse spinal cord with immunohistochemical labeling of SPNs using NADPH diaphorase which enables good dendritic visualization. Main picture at low magnification shows the different groups of SPNs with each panel illustrating the dendritic arborizations of each group of SPN. IML: intermediolateral cell column (nucleus intermediolateralis thoracolumbalis pars principalis); Ilf: nucleus intermediolateralis pars funicularis; IC: intercalated nucleus (nucleus intercalatus); IPPe: central autonomic area (nucleus intercalatus pars paraependymalis).

the Ilf with fewer in other autonomic regions, in stark contrast to the more widespread distribution of SPNs innervating the hypogastric nerve. Simultaneous retrograde labeling of SPNs from the stellate ganglion, SCG, and adrenal medulla (298) further proved that SPNs are arranged in target specific columns in the thoracic spinal cord (Fig. 4). Differences in the contributions of SPNs to specific functions in the two sides of the spinal cords are also evident from studies using electrical IML stimulation or small microinjections of glutamate into the IML of either the left or right sides. Activation of the right IML significantly increased heart rate, while left IML activation had few chronotropic effects but significantly increased contractility (129, 344). Moreover, electrical stimulation of the right inferior cardiac nerve increased heart rate by 67 beats/min while left nerve stimulation only elicited a change of 43 beats/min (161). These studies also demonstrated that the major influence on heart rate and contractility is due to SPNs arising from the IML, rather than the more medially sited SPNs in the IC and IPPe (267).

Projections of SPNs From the anatomical studies mentioned above, we know that SPNs are arranged in bilateral and symmetrical arrangements

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and neurons mainly project to end organs on the side in which they are located in the spinal cord. Obviously, where these organs are not unilaterally organized (such as the gut), this is not the case. SPNs have influence over many end organs, yet only project directly (i.e., monosynaptically) to one, the adrenal medulla. In all other cases, the SPNs project to sympathetic ganglia where they synapse directly onto postganglionic neurons. Indeed, there is a great deal of divergence of SPNs onto postganglionic neurons: in humans, a single SPN can innervate 200 postganglionic neurons (150), although the ratio of pre- to postganglionic neurons in rats is 1:15. On the whole, SPNs project in an ipsilateral fashion to thoracic ganglia (61, 179, 284, 285) and the adrenal gland (73, 308) although SPNs in lumbar spinal segments may send some contralateral projections to the lumbar sympathetic trunk (94), lumbar splanchnic nerve (27), and hypogastric nerve (26). Controversy also surrounds the distance in which an SPN axon travels in the spinal cord before exiting via a dorsal root; some studies suggest long intraspinal projections with axons exiting the spinal cord some distance from the site of origin (62, 94). However, one of the clearest indications that most SPNs exit at the segmental level of their cell somata used HRP labeling restricted to specific spinal levels to prevent spillage or leakage; selective labeling of a T9 spinal nerve led to 95%


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Figure 4

Target-specific location of SPNs. (A) Transverse sections of the upper thoracic spinal cord of the adult rat at the T3 segmental level showing SCG-SPN labeled with Fluorogold (bright gold) and SG-SPN labeled with Fast Blue (bright blue). The groups of neurons have discrete occupancies in the IML. IML: nucleus intermediolateralis pars principalis; Ilf: nucleus intermediolateralis pars funicularis; DH: dorsal horn. (B) Diagram of the gray matter of the right side of spinal cord indicating the location in the IML of the different groups of SPN as they would appear at T5 thoracic segment. SCG: SPN projecting to superior cervical ganglion; SG: SPN projecting to stellate ganglion; SCG + SG: region containing both groups of SPN; AM: SPN projecting to the adrenal medulla. At more rostral levels, there would be very little AM representation whereas at more caudal levels such as T10 there would be very little representation of SG and no SCG. Adapted, with permission, from Pyner and Coote 1994 (298) Abbreviations: AM: adrenal medulla; SCG: superior cervical ganglion; SG: stellate ganglion.

of labeled SPNs in the T9 section of the spinal cord with the remaining 5% located in the caudal most pole of T8 (179). These authors concluded that the previous studies had contaminated the sympathetic chain and this idea has been further corroborated (305) with similar restrictions of SPNs innervating a single white ramus in the cat, hamster, and guinea-pig. Single cell fills of SPNs in neonatal rat (99) or pigeon (34) show axon collateral from some SPN extending intraspinally but the role and extent of these is not really known and the authors stated that these were only observed in a minority of SPNs. Thus there is a distinct topographic organization of SPN within the spinal cord. However, within any one segment of the spinal cord, there are SPNs innervating different ganglia. Furthermore, retrograde labeling of more than one ganglion or the adrenal medulla can occasionally lead to double labeling of single SPNs, suggesting that these project to more than one


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ganglion (298), although others (11) do not report any double labeled SPNs after injections into the cervical sympathetic trunk and adrenal medulla. One major breakthrough on the topography of these neurons came with the use of transneuronal tracing studies, pioneered by Strack and Loewy, which enabled precise and discreet labeling of SPNs innervating different end organs (334, 335). Now it was possible to selectively label the whole circuit controlling just one end organ and thus determine the contribution of SPNs within different segmental levels (Fig. 5). With these also came further proof of the paucity of neurons that appear to be influencing more than one end organ. Thus injection of different tracers into two targets, such as the adrenal medulla or stellate ganglion, labeled SPNs in similar locations, but there is little or no (less than 1%) double labeling, indicating specific functional assignments for SPNs (153, 298). Traditional retrograde tracers injected into the stellate ganglion (which contains sympathetic postganglionic neurons innervating the heart and BAT) labeled SPNs at levels C8 to T9 (49, 297, 298); however, transneuronal tracing enables a clearer picture of the restricted location of SPN involved in these two separate functions. PRV injections into BAT infected SPN at early stages post inoculation in IML clusters at T4 and T5 with fewer neurons infected in T3 and T6 to T8 levels (49). Inoculations of the ventricular myocardium infected SPN at levels T1 to T6/7 (353). SPNs from both sides of the IML innervate the ventricular myocardium, even though single SPN innervate sympathetic ganglia on the ipsilateral side. There is further topographical organization of the SPN controlling the heart since those labeled from infections of the left ventricle were located more medially in the IML than those infected by right atrial injections. Furthermore, it appeared that those SPN influencing the right atrium (and thus likely to be contributing most to the chronotropic effects of sympathetic activation) were located in the left IML, that is, the contralateral side (353). This is somewhat at odds with the functional studies analyzing the contributions of left and right IML to heart rate, since microinjections of glutamate into left IML caused little change in heart rate while stimulation of the right stellate ganglion or injections of glutamate into the right IML had profound effects on heart rate, as reported above (343, 344). The reasons for these differences are not clear but may be due to the level of infection of SPNs in the transneuronal tracing. Inoculations of the kidney with PRV caused infection of SPNs at the level of T6-L1, with a mode at T11 (137) although Schramm (310) reported the highest number of infected cells at T10, with a rostral extension to T5. Four groups of SPNs all contributed a similar proportion of cells at each level, with the majority occurring in the IML, as expected. Dual inoculations of left and right kidney with different PRV strains further confirm lateralization of outputs from the spinal cord since, at early stages postinoculation, only ipsilateral SPNs are infected (48). As expected, the further rostral the target, the more rostral the location of SPNs innervating that target: those infected

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Figure 5 Segmental localizationof SPNs projecting to different targets. (A) Photomicrograph of transverse spinal cord section in rats following pseudorabies (PRV) injection into the left kidney and a 4-day survival postinfection. Arrows indicate the processes of infected neurons in the IML. (B) Number of PRV-infected neurons in each segment 4 days after kidney infection. The arrow indicates the spinal cord segment that contains the most infected cells. Abbreviations: IML: the intermediolateral nucleus; LF: the lateral funiculus; IC: the intercalated nucleus; CA: central autonomic area. Adapted, with permission, from Huang and Weiss (137). (C) Summary of levels in the spinal cord where PRV-infected SPNs were located following PRV injections into various end organs. Data used, with permission, from (50,105,106,226-228,281,310,334,337,342,353,354,363-365).

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after labeling of the SCG were found at C8 to T5 with the majority in T1 to T3 (349), see Figure 5. So overall, these studies have shown a clear degree of topographical organization of SPN, with rostrocaudal location and grouping of SPN giving some clues as to their functional role. However, none of these studies have proved that it is possible to identify the function of an SPN merely by morphology or location. The next section discusses other characteristics that may yield further information regarding functional identification of classes of SPN.

Neurochemistry of SPNs We now consider whether populations of SPNs regulating different target tissues have unique neurochemical profiles. Studies combining retrograde tracing with immunohistochemistry for specific markers have shown differences in expression patterns of these markers. All SPNs are cholinergic but the coexpression of other modulators has been associated with the function of those SPN. The majority of SPN are also considered by some to be nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase positive which indicates that many SPN synthesise nitric oxide (7). However, there was a degree of topography since NADPH diaphorase positive SPN were located in the more lateral regions of the IML. The expression of nitric oxide synthase (NOS) may correlate with SPN targets since combining retrograde tracing with NOS immunoreactivity revealed that only 54% of celiac ganglionprojecting SPN were NOS immunopositive compared with 98% of adrenal medulla-projecting SPNs [Fig. 6 (131)]. Differences in the locations within the autonomic nuclei and in the size of NOS-positive SPN were also noted, suggesting a more subtle expression of NOS than originally thought. One common protein group used for chemical coding to enable identification of specific neuronal subtypes is that of the calcium-binding proteins. These proteins are critical in maintenance of appropriate calcium levels in neurons and there are a number of common calcium-binding proteins in neurons that are expressed in distinct subsets of neurons. SPNs synapsing on the secretomotor postganglionic neurons innervating the submandibular salivary gland express calretinin (118) while those SPN innervating postganglionic neurons that influence the lacrimal gland, thyroid gland, anterior chamber of the eye, or the skin of the forehead do not express this protein. The presence of calbindin is also restricted to specific subsets of SPNs, with higher proportions of immunopositive SPN in rostral (T2-4) and caudal (T13-L2) regions than the mid-thoracic segments. No SPNs retrogradely labeled from the adrenal gland and few from the inferior mesenteric ganglion are calbindin immunopositive yet over a quarter of stellate ganglion projecting SPN express calbindin (119). The majority of these are also NOS immunoreactive; however, those few that are not double-labeled are in the mid-thoracic region and thus likely to project to the abdominal viscera. It is also likely that those calretinin-expressing SPNs innervating



the salivary gland are also calbindin positive. Further research identified that SPNs synapsing on postganglionic neurons innervating the iris of the eye are calcitonin-gene related peptide immunopositive (120). Even within subsets of SPNs influencing a specific end organ, there is distinct chemical coding: SPN innervating noradrenergic chromaffin cells in the cat are calretinin positive (93) while those that target adrenergic chromaffin cells express enkephalin (133). Proteins involved in signal transduction are also differentially expressed in neurons and this provides another useful tool in the analysis of the roles of specific groups of SPNs since extracellular signal-related kinases (ERK) or cyclic AMP response element binding protein (CREB) become phosphorylated upon activation. If these are only expressed in subsets of neurons, these could be used to identify which neurons are activated and thus involved in mediating a response to a particular perturbation or stimulus. Phosphorylated ERK1/2 immunopositive SPNs are located throughout the IML from C8 to L3 with the highest proportion at the rostral and caudal levels rather than mid thoracic, similar to that pattern observed with calbindin-positive SPNs (326). However, here the similarity stops and it is clear that these are not the same SPNs since 64% of adrenal SPNs were p-ERK positive (and all of these were also NOS-positive), compared to no calbindin positive adrenal-SPNs (119). This widespread presence of phosphorylated ERK suggests that high proportions of SPNs are tonically active, even taking into account the methods used to harvest the tissue.

Neuropeptide content of SPNs It is also possible to identify subsets of SPNs based on their neuropeptide content but at times, these experiments have proved troublesome since intrathecal injections of colchicine are required to inhibit transport of these neuropeptides to the terminals and thus maintain levels in the cell body for detection. Some experimenters used terminal labeling of SPN and studied neuropeptide content within the axon terminals in the ganglia but recent work has utilized in situ hybridization to bypass these issues. Enkephalin, somatostatin, neurotensin and substance P immunoreactivity is present in SPNs at all levels of the cat spinal cord (173,174). The extent of coexistence of these peptides varies considerably, with substance P coexisting with all other peptides, while enkephalin only coexpresses with substance P (173, 174). The extent of coexistence is highest in the IML with little or no coexistence of neuropeptides in the Ilf and CA (173, 174). Corticotrophin-releasing factor and vasoactive intestinal peptide but not luteinizing hormonereleasing hormone are highly expressed in upper thoracic and lumbar levels (172). Corticotrophin-releasing factorimmunopositive SPN are those synapsing on the cholinergic postganglionic neurons innervating the sweat glands (316), thus these SPN control sudomotor function. The importance of this chemical coding is revealed by studies into the significance of enkephalin and pituitary

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Nitric oxide synthase (NOS) is found in select groups of SPNs retrogradely labeled from the adrenal medulla (AM), superior cervical ganglion (SCG), coeliac ganglion (CG), and major pelvic ganglion (MPG). (A and B) SPN that project to the AM (arrows) contain NOS immunoreactivity. (C and D) SPN that project to the SCG (arrows) from the ventral IML (vIML) lack NOS immunoreactivity. (E and F) A sympathoadrenal neuron in the dorsolateral funiculus (DLF) that is NOS positive. (G and H) Most of the SPN that project to the MPG from the CAA are NOS positive (arrows) but one MPG-projecting SPN (arrowhead) is NOS negative. (I and J) NOS-immunoreactive SPN that send axons to the MPG (arrows) from the intercalated nucleus (ICN). (K and L) An NOS-negative SPN (arrow) that supplies the CG from the ICN. A nearby NOS-immunoreactive (arrowhead) neuron is not retrogradely labeled. (M and N) An NOS-negative SPN that projects to the SCG from the central autonomic area (CAA) lies near a group of NOS-positive CAA neurons that are not retrogradely labeled. Scale bars = 25 μm. Taken, with permission, from Hinrichs and Llewellyn-Smith (131).

adenylate cyclase activating polypeptide (PACAP) expression in adrenally projecting SPN (178). A high proportion (80%) of total unlabeled SPNs at the level of T4 to T10 segments expressed PACAP, while only around 5% of SPN expressed enkephalin, these are likely to include a proportion of those innervating chromaffin cells (133). Further investigation revealed that almost all adrenally projecting SPN were PACAP expressing while around half contained

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enkephalin (178). Next, the neurochemical identity of those SPNs activated by glucoprivation was determined (283) since glycogenolysis would be promoted by discrete activation of SPNs innervating the adrenal gland (to release adrenaline) and the celiac ganglion (influencing pancreatic glucagon release). Seventy percent and 37% of SPNs innervating the adrenal gland and celiac ganglion respectively were activated by glucoprivation (shown by the presence of c-fos, a marker used


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Neurochemical coding of SPNs innervating the adrenal or coeliac ganglia and activated by glucoprivation Using cFos immunoreactivity to label SPNs activated by glucoprivation (A), the retrograde tracer CTB conjugated to different fluorescent labels to retrogradely label either adrenal projecting (B) or coeliac projecting (C) SPN and in situ hybridization to show the presence of mRNA for preproenkephalin (D) it is clear that some of the activated SPN that project to these target organs are enkephalinergic [arrows in merged image (E)]. The group data are shown in F. Taken, with permission, from Parker et al. (283).

commonly to indicate whether a neuron has been activated by a specific perturbation or stimulus) and 78% of total SPN activated in the 4th to 11th thoracic segments were enkephalinergic. Further analysis revealed that 57% and 68% of activated SPNs innervating the adrenal gland and celiac gland, respectively, were enkephalinergic (Fig. 7), indicating that these SPN are critical in producing the appropriate responses to glucoprivation. A potential chemical coding for SPNs with a cardiovascular function was revealed (115) which could be a useful tool since, until recently, it has not been possible to definitively isolate cardiovascular SPN based solely on their neurochemistry. Using retrograde tracing, transneuronal tracing and immunohistochemistry, cocaine and amphetamine-regulated transcript peptide (CART) was present in SPN targeting


vasoconstrictor and cardiac-projecting postganglionic neurons but was absent from SPN with a noncardiovascular role. Furthermore, when rats were challenged with systemic hypoxia, a high proportion of SPNs that were activated (shown using c-fos staining) were also CART-immunopositive (115). SPN innervating noradrenergic chromaffin cells are also CART positive (97, 115); however, there is still some discrepancy between these two studies as to the “exclusive” expression of CART in cardiovascular SPN, Fenwick and Llewellyn-Smith maintain that CART is more ubiquitous in SPN (97). So a modicum of caution must be applied in using this marker to identify cardiovascular SPN. Rather interestingly, the chemical coding for SPNs seems to be influenced by the target tissue since transplanting of the adrenal gland into the position normally occupied by the SCG

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resulted in the preganglionic neurons that would normally have innervated this ganglion instead innervating the adrenal gland and expressing the same neurochemical code as adrenal SPNs (266). Reviewing all the evidence from above leads one to conclude that there is no clear chemical coding that would unequivocally identify an SPN involved in a specific function; rather that the contribution of certain modulators may differ in the sympathetic control of specific end organs.

Intrinsic Properties of SPNs Whole cell patch clamp and intracellular recordings from SPNs in the spinal cord slice, working heart brain stem preparation and in vivo have enabled an in depth understanding of the intrinsic properties of SPNs. Some properties of SPNs, such as their input resistance, are clearly influenced by the recording configuration. Thus intracellular recordings, with a low resistance seal around the electrode, produce readings of input resistance as low as 23 MΩ [cat, in vivo (80) and up to 110 MΩ (rat and cat spinal cord slices (92,380)]. Patch clamp recording does not require such a large hole to be made in the membrane around the electrode and thus produces values for input resistance of up to 1 GΩ (86, 322); however, other electrophysiological properties are similar, regardless of the techniques utilized. Thus, resting membrane potential is reported as around −60 mV in adult SPNs (380) but is more depolarized in neonatal SPNs (86, 322). Many studies have noted the presence of multiple potassium currents in SPNs, one notable experiment (251) which recorded from retrogradely labeled SCG SPNs listed five such currents, a delayed rectifier (IK), a fast 4-aminopyridine sensitive transient outward rectifier (IA), inward rectifying current (IIR), and two Ca+ -dependent currents—a sustained one (IAHP) and a low voltage-activated T-type calcium conductance. Transient A currents play a crucial role in the firing properties of SPNs. In voltage clamp, this current activates at around −45 mV with a monoexponential time course of decay (35). When the membrane potential is depolarized from a potential that is negative to resting values, this current is activated then slowly inactivates. If an SPN is firing, then during the afterhyperpolarizaton (AHP), this current may be removed from its inactivation and thus contribute to the interspike interval of the firing of SPNs. Once SPNs reach the threshold for firing, they respond with an action potential which is broad in nature [(1.7 ms in guinea pig, (141), intracellular recordings] associated in all studies with a prominent sag on the decay phase. The action potential has both tetrodotoxin (TTX) sensitive and insensitive components; the TTX-resistant component is sensitive to cobalt and therefore due to opening of calcium channels (379, 380). The action potential repolarization phase is sensitive to caesium and barium, indicating involvement of an Ik. Following the action potential is a long lasting AHP [duration of 2.8 s in cat spinal cord, intracellular recordings (379); duration of 1.9 s in guinea pig spinal cord (141); duration of 0.5-4 s in rat spinal

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cord, intracellular recordings (322)] with two main components: a fast AHP which lasts 150 to 600 ms and a slow AHP which can be distinguished pharmacologically. The fast AHP is suppressed by intracellular caesium (379) or high (20 mM) concentrations of the potassium channel blocker, tetraethylammonium chloride (322) and may be due to the slow inactivation of delayed rectifier potassium channels with a possible contribution of the A current (80, 379). The slow component involves activation of calcium-activated potassium channels since it is attenuated by cobalt or low calcium concentrations (294). The AHP measured in vitro (379) is longer in duration than that observed in vivo (70,240) and these differences may be due to factors regulating the calcium-activated potassium channels, such as the intracellular calcium concentration, the levels of ongoing synaptic activity or simply due to greater damage inflicted on the neurons with intracellular recordings in vivo.

Axonal conduction velocities of SPNs Axonal conduction velocities of SPNs vary considerably across the species and reflect the degree of myelination of the axons. The axon actually arises from the cell somata or proximal dendrite; seldom do they arise from second order dendrites (45,79). In the cat, around 60% of axons are myelinated (66) but this proportion is much reduced in the rat; almost all rat SPN axons are unmyelinated (111, 112). These species differences lead to measurements of axonal conduction velocities ranging from 0.6-15 m/s in the cat (15, 145, 240, 380) to 0.2-3.3 m/s in the rat (111, 112, 237, 239, 259, 262).

Do subgroups of SPNs differ according to their intrinsic properties? To date, all SPNs have been considered together with respect to their intrinsic properties; however, it is worthwhile considering differences and their significance. SPNs recorded intracellularly from the cat can be grouped according to their active and passive electrophysiological properties into three categories. The first group had a relatively negative resting membrane potential and low input resistance while the second group of SPNs were more depolarized at rest and exhibited higher input resistances. These neurons also displayed afterdepolarizations and had low and irregular patterns of ongoing action potential firing but could reach rates of 100 spikes/s if depolarized by a positive current pulse. The last grouping had characteristics similar to group 2 but did not exhibit afterdepolarizations (80). These researchers were thus one of the first to suggest that there were differences in intrinsic properties that may be attributed to function but did not assign that function. Zimmerman and Hochman (387) have recently used an enhanced green fluorescent labeled transgenic mouse line where HB9 homeodomain protein expressing SPNs are easily visualized and targeted for recording. These neurons had membrane potentials and input resistances similar to those observed in rat spinal cord slices and other species but they


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reported that it was possible to group these SPNs into four different groups based on cluster analysis of various passive and active membrane properties. They hypothesized that these differences may reflect the different functional roles of SPNs in the IML but for obvious reasons, again could not assign that function to SPNs in an isolated spinal cord slice. Other researchers have approached the problem from a different angle by examining whether SPNs with a specific function have different intrinsic properties. One effort at recording from SPNs previously infected from pseudorabies viral inoculation of the kidney has shown that these SPNs exhibited lower membrane potentials than previously recorded in adult rats (−51 mV) and lower input resistances, which may be due to the infection, but these SPNs had clear ongoing synaptic activity (81). Spanswick and co-workers (372) reported values for passive and active membrane properties for adrenal gland labeled SPNs that overlapped with those of SPNs with different functions (Fig. 8). It is important to note that injections into the adrenal gland will label SPNs innervating blood vessels supplying the adrenal gland as well as SPNs that may have different adrenal functions, such as secretory. This may account for the fact that even though all SPNs were retrogradely labeled from a single end organ, there was diversity within this group with respect to membrane properties. Janig and colleagues (149) have also considered differences in SPN intrinsic characteristics that may reflect their functional role: vasoconstrictor SPNs were more likely to exhibit ongoing activity then other SPNs which may reflect the nature of their role, maintaining appropriate basal tone—other SPNs innervating end organs that do not require constant activity exhibited lower levels of synaptic activity. There was little correlation between conduction velocity and function, suggesting that this characteristic is not a reliable indicator of function. In a recent study (327) using the working heart brainstem preparation, recorded vasoconstrictor SPNs were further classified as cutaneous vasoconstrictor (CVC) and muscle vasoconstrictor (MVC)—like SPN using specific criteria such as the responses to peripheral chemoreceptor activation. MVC-like SPNs had significantly lower input resistances, shorter AHP durations and higher firing frequencies than the CVC-like SPNs. From that same study, although most of the MVC-like SPNs displayed both anomalous and transient rectification, only around half of the CVC-like SPNs exhibited anomalous rectification while just one third showed transient rectification. These discrete differences in intrinsic properties may be related to still further subgroupings of these vasoconstrictor SPNs. Although these results were obtained in young rats (5-16 days), similar studies show that sympathetic reflexes are well established by that age (320) and the proportions of MVC- and CVC-like SPNs were very close to those reported in the adult anaesthetized cat (32). This is important since it suggests that use of younger preparations, which are used in many of the studies in this review, can provide physiologically relevant data that is hard to obtain in the adult in vivo situation. So it seems that a range of properties within a particular functional group reflects further


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sub-classifications—one suggestion by Pickering et al. (320) is that their MVC-like SPNs include vasoconstrictors to the viscera or cardiac SPNs and thus it is unlikely that it would be possible to be able to assign an SPN to a particular function based solely on its intrinsic properties. It is perhaps likely that with such a complex complement of membrane conductances, the role or contribution of particular conductances is aligned to the physiological or environmental demands on the neurons. The individual membrane conductances of the SPN classes and their level of involvement in different conditions will “filter” the incoming descending and afferent synaptic inputs appropriately to shape the final firing patterns of the sympathetic outflow from the spinal cord.

Sympathetic preganglionic neurons and gap junctions In the following section, we shall consider the presence and role of specialized electrical synapses, known as gap junctions in SPNs. Gap junction coupling allows populations of SPNs to produce coordinated activity. This is valuable when coordinated responses are required as membrane potential changes can be reproduced almost simultaneously in multiple postsynaptic neurons.

Electrophysiological evidence for electrotonic coupling between SPN Electrically coupled SPNs participate in synchronized spiking and rhythmic firing patterns; three types of rhythmic behavior have been described from in vitro studies (322): burst firing, tonic beating and subthreshold oscillations in membrane potential (continually occurring spikelets). The type of behavior observed in coupled SPNs is dependent on the membrane potential. Some SPNs are able to display spontaneous rhythmic oscillations in their membrane potentials; this activity is mediated by action potential discharge in electrically coupled neurons and can give rise to bursting or beating spike discharge (107). These oscillating neurons display tonic beating when depolarized above threshold and further depolarization results in burst firing of neurons (322). Subthreshold oscillations are derived from within the SPNs either from the distal dendrites or in neighboring electrotonically coupled SPNs allowing SPNs to generate highly synchronized activity patterns (107, 210). The spontaneous membrane potential oscillations in a population of SPNs have been described further in slices of rat thoracolumbar spinal cord (318, 322). These on-going spontaneous membrane potential oscillations were not abolished by a calcium free solution or antagonists to excitatory and inhibitory amino acid transmitters, and they exhibited a relative insensitivity to changes in membrane potential. This suggested that these membrane potential oscillations were generated intrinsic to SPNs or in neighboring electrotonically coupled cells (318). Indeed, electrotonic coupling has been described in the mesencephalic nucleus (20), the inferior

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Properties of adrenal medulla-projecting SPNs. (A) Longitudinal section of thoracolumbar spinal cord showing the distribution of SPN innervating the adrenal medulla as revealed by retrograde labeling of SPN with Rhodamine Dextran Lysine (RDL). [B(a)]) Transverse section of thoracic spinal cord showing SPNs labeled with RDL after injection into the adrenal medulla. The neuron indicated with the arrow in (a) was also filled (b) with Lucifer Yellow (LY) from the recording pipette, identifying its axonal projection to the adrenal medulla. Note another LY-labeled neuron that lacked RDL, therefore not considered among the AD-SPN population data. (C) Frequency histograms summarising the distribution of passive membrane properties of AD-SPN. Top, resting membrane potentials (RMP; mV). Bottom, input resistances (MΩ). (D) Whole-cell current-clamp recordings (holding potential −50 mV) illustrate instantaneous inward rectification (∗) activated during the membrane response to large amplitude hyperpolarising current pulses (not shown), and transient outward rectification (■) observed as a delayed return to rest of the membrane responses. Also shown in the inset is an action potential on a faster time base to illustrate the pronounced shoulder on the repolarising phase (+). Adapted, with permission, from Wilson et al. (372).

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olive (207), and in motor neurons (367) to name but a few. In whole cell patch clamp recordings from rat spinal cord slices, SPNs were also electrotonically coupled (210). Around 26% of these SPNs displayed spontaneous biphasic membrane potential oscillations termed “spikelets.” The injection of depolarizing current pulses past the threshold for spike discharge produced bursting or beating action potential discharge in these SPN revealing that these SPN are rhythmically active and show oscillatory activity. Oscillations were blocked by bath application of TTX (200 nM) but were unaffected by the voltage gated sodium channel blocker QX-314 in the electrode and various other neurotransmitter antagonists. As spikelet activity is not affected by low calcium and high magnesium solutions (322), together these data suggest that spikelets are not postsynaptic potentials resulting from the release of neurotransmitters and that spikelets require voltage gated sodium channel activity that does not originate in the recorded neuron (210). Further, paired whole cell recordings revealed that the activation of a pair of SPNs following antidromic ventral root stimulation resulted in spikelet production in one SPN pair occurring in synchrony with the action potential discharge of the electrotonically coupled, neighboring SPN (Fig. 9). The magnitude of the spikelet observed in the recorded neuron was larger when the electrically coupled SPN discharged an action potential rather than a spikelet. Following pipette application of QX-314 in the recorded neuron, action potential firing but not spikelet production was blocked. Therefore, oscillations are thought to result from action potentials fired in neighboring neurons that are conducted via electrical synapses (273). As spikelets can be abolished in SPNs (288), by the general gap junction blocking drug carbenoxolone, this provides further evidence that spikelets are conducted via gap junctions.


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Anatomical evidence for gap junction communication between SPNs As well as electrophysiological evidence, anatomical evidence suggests that SPNs are electrically coupled. At least some SPN-SPN gap junctions contain the gap junction subunit connexin 36 (Cx36) (223). Dual immunohistochemical labeling for Cx36 and ChAT in adult rat spinal cord revealed Cx36 positive punctate labeling in the IML, intercalated cell group and central autonomic area of the lower thoracic spinal cord along the dendrites and somata of the majority of ChAT positive neurons (223). Cx36 labeling was observed along dendritic processes where the dendrites were in close contact with other cell bodies in the IML. As paired recordings demonstrate synchronization of action potential firing and spikelets between electrotonically connected SPNs (210), the presence of Cx36 here may represent a potential site for gap junctional coupling between dendrites of the neighboring SPNs and somatic neurons. This may allow synchronization of action potential firing between electrically coupled cells. By increasing the synchronicity of discharges in SPNs, gap junctional communication may play a vital role in the generation of rhythmic sympathetic activity.


Figure 9

Gap junctions in SPNs (A) The schematic shows the components of the gap junctions. (B) Upper trace shows regular action potentials recorded from an SPN in rat spinal cord slice (7-22 days). If the membrane potential is hyperpolarized, these subthreshold oscillations in membrane potential are observed. (C) With Qx314 in the patch pipette to block sodium action potentials in the recorded cell, SPNs in the rat spinal cord slice are antidromically activated, producing an action potential in an SPN which is coupled through gap junctions to the recorded SPN. The oscillation then observed in the recorded SPN is the filtered action potential passing through the gap junction. (D) Simultaneous recordings from two electrotonically coupled SPNs demonstrate conduction of membrane potential changes from cell 1 to cell 2. A series of rectangular-wave current steps (amplitude, −160, −80, and 40 pA; duration, 800 ms) injected into cell 1 elicited corresponding membrane potential responses in both neurons. Recordings from the same pair of neurons also show conduction of membrane potential changes from cell 2 to cell 1, following injection of current steps (amplitude, −160, −80, and 40 pA; duration, 800 ms) into cell 2. Taken, with permission, from Logan et al. (210) (B and C) and Nolan et al. (273) (D).

The importance of gap junctions in SPNs The sympathetic nervous system requires coordination of functionally related activity across a large column of neurons and gap junction expression may be consistent with this functional specialisation of sympathetic outflow. Different

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sympathetic outflows are regulated in a specific and differentiated manner. For example, subsets of sympathetic premotor neurons of the rostral ventrolateral medulla preferentially regulate sympathetic outflow to the heart or to blood vessels in specific vascular beds (75, 211, 232). Therefore, gap junction expression between SPNs with the same autonomic functions may play an important role in coordinating the activities of different neurons involved in the same sympathetic response. In this way gap junctions may coordinate populations of neurons to produce a synchronized response. Indeed, Logan et al. (210) observed that spikelets were not coincident with the action potentials seen in the recorded pair of neurons, spikelets were recorded even when neither of the recorded neurons reached the threshold for firing. This suggests that at least one additional neuron is present in the coupled network of neurons. Electrotonic coupling via gap junctions may allow SPNs to generate synchronized/rhythmic activity patterns which will influence the outflow of these preganglionic neurons onto postganglionic targets. Indeed if gap junctions, including those between SPNs but not exclusively at this site, are blocked, as performed in the working heart brainstem preparation with the anti-malarial drug mefloquine (which has a profound but relatively selective effect at connexin 36-containing gap junctions), the sympathetic nerve excitation to chemoreceptor stimulation is significantly attenuated (183). This effect may be mediated in part by the loss of synchronization of SPNs which normally enable precise and rapid activation to produce the appropriate response. In spinal cord slice preparations, only around a quarter of SPNs exhibit gap junction coupling in the form of spikelets (210) which would indicate some degree of functional specificity. In fact, recent evidence has clearly supported such a suggestion; in the working heart brainstem preparation, the regular spikelet firing was observed contributing to the activity of SPNs classified as CVC-like while MVC-like SPNs did not exhibit such spikelets (327). These gap junctions likely contributed to the lower input resistance and longer AHPs in the CVC-like SPNs compared with the MVC-like neurons. These CVC-like SPNs also exhibited strong inhibitory responses to peripheral chemoreceptor activation. Blockade of gap junctions was shown by Lall et al. to reduce the sympathetic responses to chemoreceptor stimulation (183). The precise mechanism of how disruption of the gap junctions between the CVC-like SPNs may contribute to overall sympathoinhibition is not fully understood—perhaps the site of action is not restricted to the sympathetic component of the pathway but it appears that gap junctions may play a role in coordinated and functionally specific sympathetic responses to occur. There is still some debate over the contribution of gap junctions to SPN coordinated activity and indeed some may argue that the presence of gap junctions in SPNs is merely a developmental phenomenon since the studies above showing spikelet activity were mainly carried out using slices or in the working heart brainstem preparation of younger animals. However, immunohistochemical data indicating an

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abundance of Cx36 immunoreactive SPNs were obtained in the adult rat (223) and the effects of blockade of Cx36 containing gap junctions on sympathetic variables were recorded in rats aged 4 to 6 weeks (183). Furthermore, gap junction coupling between SPNs in adult spinal cord slices was reported in a brief communication (187) while in the working heart brainstem preparation with increasing age from P5-P16, there is no reduction in the number of SPNs that exhibit spikelets (327). In fact, in the adult rat, the majority of SPN were immunopositive for Cx36 (223) but in only around a quarter of SPN were spikelets observed (210) suggesting that there is a redundancy in the system with respect to functional gap junction coupling. After nerve injury in the adult animal, dye coupling (an indication of functional gap junctions) between somatic motor neurons is upregulated, although connexin subunit expression did not change (56), suggesting that existing gap junction proteins can be modulated to become functionally active if required. Such a phenomenon may be worthy of investigation in the sympathetic circuits.

If SPNs exhibit synchronized firing through gap junctions, is there a specific order of recruitment? In other CNS regions such as the motor neurons of the spinal cord that are so critical for enabling an appropriate response to a stimulus, a coordinated and sequenced recruitment of motor units that results in this response is observed (252). SPNs are coupled through gap junctions that may contribute to a coordinated response, but is there an orderly recruitment of SPNs to produce an overall response? Early studies by Wallin and colleagues (214, 366) suggested that in human muscle sympathetic fibres such orderly recruitment does occur, with units with faster conduction velocities firing earlier in cardiac related bursts of sympathetic activity. However further work by McAllen and Trevaks (233) in the cat suggested that not only was the synchrony between “cardiac” SPNs rather imprecise but that it did not appear that SPNs were recruited in particular order. The significance of gap junctions and the recruitment and synchrony of SPNs should be further elucidated.

Extrinsic Influences on SPN Activity Understanding what extrinsic inputs influence SPNs has relied on a combination of tracing, electrophysiological and immunohistochemical studies. From extracellular recordings, it appears that in anesthetized preparations at least, a high proportion of SPNs do not display ongoing activity (68, 111, 112, 114, 123, 125, 222, 274, 292). Once again, it is critical to consider the functional specificity of these firing properties; those SPNs involved in sudomotor, pilomotor or vasodilator functions are often silent (145, 151, 152). However, if these previously silent neurons are made to fire by iontophoretic application of glutamate, then patterns of activity are similar to those observed in spontaneously firing SPNs


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and intracellular recordings confirm that most SPNs have subthreshold ongoing excitatory and inhibitory activity in the form of postsynaptic potentials which appear to be generated from three sources: supraspinal inputs, afferent innervation and local circuits and these shall be considered in the following subsections.

Afferent Inputs to the Spinal Cord Affect the Activity of SPNs If supraspinal inputs to SPNs are removed by spinalization, the level of ongoing activity in SPNs is severely reduced but still present; a proportion of this is further decreased by severing the dorsal roots, thus removing afferent inputs (222). Indeed stimulation of visceral or somatic afferents elicits up to 4 waves of reflex excitation in pre- or postganglionic nerves or SPNs themselves with the two waves of longer latency abolished by spinalization, suggesting that they are of supraspinal origin (67, 79, 184) while the shorter latency pathways may involve just spinal cord pathways. The extent of influence of afferent pathways may differ according to projection targets of SPNs since in cats with spinal cord transection (which removes descending influences), the activity of splanchnic sympathetic nerves is well maintained after dorsal rhizotomy to remove afferent inputs (242), but in spinal rats a similar rhizotomy reduces renal nerve activity by around 25% (352). This afferent influence on SPNs is not due to a direct input since there is no evidence of afferent axon terminals directly impinging onto SPN in degeneration studies (284). In fact, most afferent terminals are located in laminae I-V of the dorsal horn (132) and these sites correspond well with the locations of many of the local interneurons (see below), indicating that sensory innervation of SPN is polysynaptic and complex. There is also a direct sensory afferent input to lamina X of the spinal cord (132), an area where inhibitory interneurons innervating SPNs have been identified (87) and these may provide some of the inhibition in SPNs from afferent activation. Many small diameter Aδ and C fibres that transmit information regarding pain, itch, temperature and osmoregulation terminate in lamina I of the dorsal horn (72) and there is evidence of a direct pathway from this region to SPNs (71). These afferent spinal pathways may not necessarily involve just local interneurons; stimulation of the femoral nerve (whose afferents enter the spinal cord at the L6 and L7 level) excited the majority of SPNs recorded in the upper thoracic spinal cord (184), suggesting an involvement of propriospinal interneurons in at least some of the sensory evoked responses. This idea is supported by the fact that chemical stimulation of the cervical cord (which is likely to activate cervical neuronal cell bodies rather than fibres of passage) inhibited renal sympathetic nerve activity (309). So, sensory influences on sympathetic activity may involve long propriospinal pathways, thus adding to the complexity of this control.



To study how individual SPNs respond to sensory and other inputs, many researchers have utilized extracellular recordings in vivo [e.g., (69)]; however, intracellular recordings from SPNs or single fibre recordings from SPN axons in sympathetic trunk enabled extensive classification of these neurons. The majority of postsynaptic potentials in SPNs elicited by stimulation of somatic or visceral afferents were excitatory but inhibitory postsynaptic potentials were also observed. Based on the firing patterns and response patterns to activation of specific stimuli such as chemoreceptor or noxious activation, retinal stimulation or blood pressure rises, these researchers classified SPNs into at least four groups (Fig. 10; (32, 33, 151). The majority of group I neurons had activity related to the cardiac cycle and also coupled to the respiratory cycle (this was enhanced in hypercapnic conditions). These SPNs were excited by noxious, nasopharyngeal or chemoreceptor stimulation. The neurons had similar proportions of unmyelinated axons as groups II and IV and a broad distribution through the thoracic segments. Group II neurons on the other hand exhibited weak coupling to both the cardiac and respiratory cycles and were most likely of all the

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Classification of SPNs in the cat according to their responses to noxious stimulation and degree of cardiac modulation. Recordings are from SPNs in the anesthetized cat and various stimuli applied. (A) This group I SPN (middle trace is extracellular action potentials recorded) was excited by noxious stimulation of the ear (black line, Ai) and had strong cardiac rhythmicity, as illustrated in the pulse triggered histogram (Aii). (B) This group II SPN was inhibited by noxious stimulation and showed no cardiac rhythmicity. PHR: phrenic nerve activity; n(8 ms)-1: number of action potentials occurring in an 8 ms time period. Taken, with permission. from Boczek-Funcke et al. (32).

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groups to be inhibited by noxious, chemoreceptor or nasopharyngeal stimulation. Group III neurons had a restricted location in T2 and T3 and had significantly faster conduction velocities than groups I and II with only 8% of axons that were unmyelinated. Their activity was coupled to the respiratory but not the cardiac cycles and they were mainly excited by noxious, chemoreceptor and nasopharyngeal stimulation. Group IV SPNs were not influenced by the cardiac or respiratory rhythms but were excited by noxious inputs. Considering these group classifications together with information gathered over many years on the postganglionic neuronal classifications, the authors assigned potential functional roles to these four classes (32,33,151), such that group I neurons likely control the skeletal muscle resistance vessels (MVCs), while most group II SPNs may regulate blood vessels in the skin (CVCs). However firm functional roles could not be assigned to the other groups and there was some duplication of function in groups such that some of the group II and group IV SPNs may be involved in pupil or pineal gland control. In the working heart-brainstem preparation, the intrinsic properties of functional groups of SPNs (such as CVCs and MVCs), such as inward rectification were correlated with their response profile to baroreceptor or chemoreceptor stimulation or simulation of the diving reflex (327). This provides evidence of how different cellular mechanisms may enable adaptation of the barrage of synaptic inputs to SPNs to produce the appropriate output to the end organ. So there are distinct groups of SPN with regards to their response to afferent and other inputs and these groupings may be related to their function, a phenomenon reviewed by Janig (146, 149, 150). One issue with recording from SPNs is an inability to gather all the relevant information from one single experiment to gain a complete picture of how an SPN with a specific function is influenced by distinct synaptic inputs or displays a certain neurochemical profile. SPNs which had excitatory responses to 5-HT showed only decreased discharge or no response to noxious stimulation of the ipsilateral hindlimb (114). Conversely, SPNs which had their discharge rates decreased by 5-HT had primarily excitatory responses to noxious inputs. To gain a complete picture of the characteristics of SPNs, their location, their responses to specific stimuli or perturbations in their environment and their responses to receptor activation, never mind filling the neurons and looking at their anatomical profile, then revealing their target organ would require something of a miracle in recording. Perhaps the answer may come with use of combinations of optogenetic and tracing tools similar to that used in recent studies into the role of C1 neurons in homeostasis (126).

Descending Inputs have Significant Influence on SPNs For many years it has been known that if you transect the spinal cord at the cervical level, the level of sympathetic activity below the lesion is profoundly decreased and blood pressure

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drops considerably. Spinalization decreases the numbers of SPNs which exhibit ongoing activity and of those that still show spontaneous firing, the frequency of this firing is diminished (79, 222, 243, 293). Furthermore, the respiratory and cardiac related activity is also lost with lesioning of the spinal cord, suggesting that this input also arises from supraspinal regions (222). However, our recent understanding shows that some level of activity is generated within the spinal cord and this is also important to consider. Often just by increasing the tonic levels of excitability of the spinal cord circuits, rhythms can reemerge (4),which suggests that these spinal cord circuits are complex and could be critical in situations where supraspinal control is compromised, such as in spinal cord injury. Early studies injecting retrograde tracers such as horseradish peroxidise (6) or fluorescent tracers (358) resulted in labeling of those regions now established as critical suppliers of supraspinal inputs: the rostral ventrolateral medulla (RVLM), rostral ventromedial medulla (RVMM), A5 noradrenergic cell group in the pontine region, raphe nuclei, the lateral hypothalamus, and the paraventricular nucleus of the hypothalamus (PVN). Our understanding of the location of supraspinal regions critical in controlling SPN activity has really benefitted from the use of transneuronal tracing studies which clearly delineated the entirety of the pathways from the supraspinal region to the SPNs themselves. RVLM, A5, raphe, and hypothalamic neurons were some of the first infected after injections of pseudorabies virus into various sympathetic ganglia (49, 104, 115, 156, 228, 268, 270, 310, 330, 335, 337,347,365) (Fig. 11). Other regions also provide less prominent but direct inputs; these include Barrington’s nucleus, the Kolliker-Fuse nucleus, the nucleus subcoeruleus, the arcuate nucleus and the infralimbic cortex. Transneuronal tracing studies focusing on specific end organs such as hind limb muscle blood vessels (168,185), kidney (48,137,310), spleen (50), adrenal gland (337), the bladder or external urethral sphincter (78, 269, 282), BAT (49, 219, 270, 275), and stellate ganglia (104,156) have shown that many of these supraspinal pathways provide input to SPNs influencing different functions with the level of contribution varying to some degree depending on final output. However, few studies have investigated whether there are specific groups of descending neurons underlying control of different end organs since it is hard to use double labeling with different strains of pseudorabies virus. In the RVLM, A5 region of the pons, raphe nuclei, periaqueductal gray region and the paraventricular and lateral hypothalamic nuclei, a small proportion of neurons became infected with two strains of pseudorabies virus injected into the stellate ganglion and adrenal gland (104, 155) leading to the intriguing thought that some of these neurons act as central command neurons to enable global sympathetic responses during situations such as arousal. It must be noted that in these studies, there is a likelihood of undercounting due to a low rate of coinfection such that a theory of sparse and specialized central command neurons should be treated with caution. Inoculation of the left and right kidney with different


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Transneuronal labeling of neurons in the brain after injections of PRV into the right stellate ganglion of the rat. A. Line drawings of brain sections where dots denote labeled cells in the different regions. Bilateral labeling is observed. (B) Organization of neuropeptide and monoamine neurons projecting to the stellate SPNs. The width of the line denotes quantitative differences in this projection system but does not necessarily reflect the potency of the pathway Important Abbreviations Rpa: raphe pallidus; R Ob: raphe obscurus; RMg: raphe magnus; RVLM: rostral ventrolateral medulla; A5: noradrenergic cell group; LC: locus coerulus; PVN: paraventricular nucleus; AHN: anterior hypothalamic nucleus. Taken and adapted, with permission, from Jansen et al. (156).

transneuronal tracers resulted in high percentages (up to 58%) of neurons that were infected with both viruses (48) in brain regions, including the RVLM and A5 regions (but to a lesser extent the raphe nuclei and Barrington’s nucleus). Those areas with high proportions of dual infections may be major contributors to enabling the coordinated activation of both kidneys.


Recently an area known as the medullo-cervical pressor area due to its specific location in the caudal medulla/cervical spinal cord region was identified that has projections to the IML and chemical stimulation of this region caused sympathoexcitation suggesting a direct new caudal pathway to SPNs (315).

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The neurochemistry of the different pathways innervating SPNs has been studied by combining transneuronal tracing with immunohistochemistry for specific markers (156, 337), (Fig. 11). In the RVMM, neurons were identified as adrenergic, serotonergic (5-HT), thyrotropin releasing hormone (TRH), substance P, somatostatin, enkephalin, and vasoactive intestinal peptide (VIP) immunoreactive while in the RVLM, neurons were mainly adrenergic with colocalization of neuropeptide Y (NPY); other neurons also contained enkephalin, somatostatin and VIP. Raphe neurons were 5HT immunopositive but also labeled for substance P and/or TRH. In the A5 region, noradrenergic neurons were common but neurons infected after adrenal gland inoculations were sometimes somatostatin immunopositive. Stellate ganglion inoculations infected neurons in the PVN that were mainly oxytocin-immunopositive, while those PVN neurons from adrenal gland infections were mainly immunoreactive for tyrosine hydroxylase (an enzyme involved in the synthesis of noradrenaline; TH) and substance P- with fewer oxytocin immunopositive neurons. Thus, target-specific supraspinal pathways with distinct neurochemical profiles may enable precise activation of SPNs for appropriate responses to a single perturbation. The idea that different descending pathways subserve distinct functional roles is clear from many studies; one such for example, used autocorrelation, power spectral analysis of postganglionic sympathetic activity, and spike triggered averaging with single cell recordings from raphe and RVLM neurons to demonstrate that these regions differentially influence the renal, cardiac and external carotid sympathetic outflows (24). We will consider each region in turn but try and give a flavor of this selective control of sympathetic spinal cord outflow.

The rostral ventrolateral medulla The RVLM is implicated in providing one of the major inputs to cardiovascular SPNs but may play a less prominent role in control of sympathetic outflow to noncardiovascular tissues. Injections of a variety of retrograde tracers such as HRP (30, 54, 303), fluorescent tracers (302, 359), Fluorogold (127, 158) or cholera toxin B/colloidal gold (203, 247) into the spinal cord or more precisely into the IML (although such injections, although not often totally restricted to the IML, will not result in labeling of other areas containing SPNs, such as the intercalated nucleus or central autonomic area; see above groupings) led to labeling in the RVLM. If anterograde tracers are injected into the RVLM, then very dense terminal labeling is observed in IML (208, 303). Retrograde labeling of SPNs from the adrenal gland combined with anterograde tracing from the RVLM revealed close appositions between the labeled terminals and SPNs which at the electron microscopic level were identified as monosynaptic connections from RVLM neurons onto SPNs in the IML (385). Both symmetrical and asymmetrical synaptic contacts of RVLM neurons onto SPNs were reported, with specific subregions in the RVLM differing such that inputs from the

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rostroventrolateral reticular nucleus of the RVLM synapse mainly onto the dendrites while those from the lateral paragigantocellular nucleus region of the RVLM predominantly innervate the somata. Thus researchers concluded that there may be more than one RVLM input. This monosynaptic pathway was supported by studies in the brainstem spinal cord preparation using either direct stimulation of RVLM and analysis of the resulting excitatory postsynaptic potentials (EPSPs) (88) or spike triggered averaging (279). The major neurotransmitters in this pathway from the RVLM, based on early immunohistochemical studies, may be considered to be glutamate and adrenaline; the glutamate synthesising hormone phosphate activated glutaminase is extensively co-localized with phenylethanolamineN-methyltransferase (an enzyme involved in the synthesis of adrenaline from noradrenaline; PNMT) in bulbospinal RVLM neurons (248). The PNMT-positive neurons form the C1 adrenergic cell group. Combining in situ hybridization and immunohistochemical studies determined that around 70% of C1 neurons coexpressed vGlut2 (a vesicular glutamate transporter) (332) and identification of those C1 neurons considered as bulbospinal through retrograde labeling from the IML showed that an even higher proportion of these cells was also glutamatergic (79%) (333). Both glutamate (257, 258) and PNMT containing terminals (246) synapse directly on IML neurons. Indeed, the terminals are directly onto identified SPNs and there are both small round vesicles (likely glutamate containing) and large dense core vesicles (likely PNMTimmunopositive) (246), which is consistent with the colocalization of these neurotransmitters. Many studies have shown that a high proportion of RVLM neurons retrogradely labeled after IML injections are PNMT-immunoreactive (158,359). In one study, it was suggested that bulbospinal RVLM neurons that were baroreceptor sensitive were also mainly TH positive; indeed, all neurons with slower conducting c fibres were from the C1 cell group of the RVLM (311). More recent evidence may dispute this somewhat: Stornetta used cfos labeling of RVLM neurons that were activated by decreases in blood pressure to show that only 61% of glutamatergic RVLM cells that were activated by hypotension were C1 cells. Furthermore, of 16 RVLM neurons recorded in vivo and antidromically identified as bulbospinal and with strong baroreceptor modulation, only 6 were identified as C1 neurons although the majority were glutamatergic (333). In fact, quantitative electron microscopic studies demonstrated that TH positive synapses comprised only 5% of total synapses onto SPNs in the second and third thoracic segments while PNMT and NPY positive synapses accounted for only 1% to 2% each, which suggests that C1 neurons may not provide a major synaptic input onto SPNs (202). Indeed using sensitive assays for adrenaline in the spinal cord, no adrenaline was detected in the IML (345). Thus, although the enzymes for synthesis of adrenaline are present in terminals onto SPNs, they may not actually release adrenaline. What is also not known is whether some C1 neurons form synaptic contacts onto SPNs containing glutamate but not PNMT; this could explain the fact that the C1


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neurons do contribute to sympathetic control but the influence of adrenaline may be minimal or non-existent. In the most sophisticated studies into the role of the C1 neurons, Guyenet and co-workers used a combination of approaches, including optogenetics. Firstly RVLM neurons were photostimulated to cause an increase in blood pressure that was attenuated by partial lesioning of the C1 cells (1). Then, using a lentivirus that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuronpreferring promoter PRSx8, they were able to selectively and robustly activate the C1 barosensitive RVLM neurons which caused profound increases in sympathetic activity and blood pressure [Fig. 12; (2)]. Indeed, the complexity of control by these neurons has been beautifully uncovered by these researchers who show how they fit into the circuitry involved not only in cardiorespiratory control but also in, for example, glucoprivic responses and responses to pain. Furthermore, their skilled use of Cre-driver mouse strains in combination with floxed-AAV2 has led to a revolution in our ability to follow the axonal projections of neuronal cell groups expressing specific neurochemicals and this technology will hopefully continue to inform us most precisely about the projections and functions of descending neurons (126, 329). The non-adrenergic RVLM neurons that project to the IML must also be considered; these display high degrees of barosensitivity. Moreover selective destruction of C1 cells using a conjugate of the toxin saporin and an antidopaminebeta-hydroxylase antibody (124, 217, 218, 312, 313) did not dramatically affect resting blood pressure or sympathetic nerve activity. If the GABA agonist muscimol was microinjected into the RVLM of rats treated with this toxin to reduce the levels of activity in the non-C1 neurons, this profoundly reduced blood pressure and sympathetic activity. This suggests that the non-C1 neurons are capable of maintaining resting sympathetic tone and blood pressure. In these same studies, C1 neuronal destruction still had a significant effect on sympathoexcitatory responses to specific perturbations such as baroreceptor unloading, sciatic nerve stimulation, glucoprivation or chemoreceptor stimulation, suggesting that the C1 neurons are critical in enabling the appropriate response to specific perturbations. The non-C1 neurons are also reported as glutamatergic (333) but may use different neuromodulators; most barosensitive bulbospinal noncatecholaminergic RVLM neurons are enkephalinergic (331) while both catecholaminergic and non-catecholaminergic RVLM bulbospinal neurons that are activated by decreases in blood pressure (and thus considered vasomotor) are CART+ve (41). NPY, somatostatin, VIP, PACAP and substance P are also found in RVLM descending neurons (95, 156). The critical role of glutamate as a neurotransmitter in the RVLM inputs onto SPNs is clear since in extracellular recordings from SPNs in vivo, stimulation of the RVLM elicited short and long latency excitation of these neurons, effects which were blocked by iontophoretic application of excitatory amino acid antagonists (260). Stimulation of the RVLM in a


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brainstem-spinal cord preparation (which enables whole cell patch clamp recording of SPNs and retains the long pathway from RVLM to the IML) elicits fast EPSPs in SPNs which are mediated via both N-methyl-D-aspartate (NMDA) and non-NMDA receptors (88).

Significance of the RVLM input to SPNs Many researchers have explored the nature and significance of the RVLM input to SPNs. It is clear that the RVLM is the major descending influence in controlling sympathetic outflow to cardiac and vasomotor targets. In fact, when spinal cord injury occurs in the form of a hemisection, baroreflex control of SPNs recovers (386) due to improved efficacy of the remaining contralateral pathways to retain this baroreceptor control (53). One study recording from muscle vasoconstrictor, visceral vasoconstrictor and renal sympathetic fibres showed that in all likelihood, there are subsets of RVLM neurons dedicated to driving sympathetic outflow to specific targets (231). This concept of differential control of sympathetic targets is supported by others (211, 262); what is more, the RVLM can change the level of excitability of adrenal, renal or lumbar sympathetic nerve activity either concurrently, differentially or selectively (265). The latencies of the responses of specific groups of SPNs to RVLM stimulation may also reflect their different functions: adrenal SPNs that were strongly activated by the glucopenic agent 2 deoxyglucose (suggesting that they were adrenaline secreting SPNs) but not affected by baroreceptor activation responded to RVLM stimulation with a long latency response (129 ms) while those SPNs activated by RVLM stimulation at shorter latencies (29 ms) were likely the noradrenaline secreting SPNs (259). Regions close to the RVLM may also be critical in providing direct descending inhibition of SPNs—injections of retrograde tracers into the IML led to labeling of RVLM neurons, some of which were GABAergic (250) and stimulation of the RVLM region in the presence of excitatory amino acid antagonists elicits monosynaptic IPSPs which are blocked by bicuculline (89). These effects may be due to activation of regions just medial to the RVLM and are unlikely to be due to activation of the C1 neurons. In considering the role of the RVLM, it is important to be aware of the complexity of control that SPNs may receive via the RVLM and other medullary regions. One recent study highlights this very well. Using a neonatal rat brainstem spinal cord preparation, Kasumacic and colleagues (165) electrically stimulated the vestibular afferents of the VIII cranial nerve and optically recorded the synaptic response of SPNs. They showed, by lesioning the medulla, that these vestibulospinal inputs may be indirectly activating the SPNs through a pathway in the RVLM. Therefore the heterogeneity of this region, the complexity of neurochemical content and the specific inputs onto SPNs may be the beautiful way in which we have developed functionally specific pathways to enable appropriate responses to a particular perturbation without resulting in global changes to autonomic outflow.

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Photostimulation of channelrhodopsin-expressing RVLM neurons in the rat activates blood pressure, heart rate, and sympathetic nerve discharge. (A) The cardiovascular and sympathetic effects produced by photostimulation of the RVLM with pulsed laser light (20 Hz, 10 ms, and 9 mW). Traces from top to bottom represent arterial pressure (AP), heart rate (HR), integrated sympathetic nerve discharge (SND; rectified and integrated with 2 s time constant and expressed relative to resting discharge, 0% representing the value observed at saturation of the baroreflex and 100% the resting level), and raw SND. (B) A waveform average of rectified SND triggered by laser pulse onset. (C) The effects of photostimulation on neurons in the RVLM. Cells were grouped based on their sensitivity to laser light (LL) stimulation, those activated were silenced by elevated arterial pressure while those that were insensitive to LL were not. The grouped data from event-triggered histograms (1 ms bin) from LL activated neurons demonstrated that during 20 Hz laser stimulation, virtually all action potentials (AP on y-axis) occur within 10 ms of the laser pulse onset with a large peak occurring 5 ms after the onset of the laser pulse. A subsidiary peak occurred between 9 and 10 ms representing cases when cells fired in couplets. For comparison, the same averaging of LL-insensitive cells shows no relationship with the timing of the laser pulse. The right panel shows that LL sensitive neurons (filled with biotinamide during recording) expressed channelrhodopsin while the insensitive cells did not. Taken, with permission, from Abbott et al. (2).

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Raphe nuclei and hypothalamic inputs Raphe-spinal neurons have been identified by many researchers using various combinations of retrograde and anterograde tracers (46, 128). Once again definitive experiments were carried out using electron microscopy (19) to confirm monosynaptic connections between anterogradely labeled raphe-spinal terminals and retrogradely labeled SPNs. Neurons in the PVN and to a lesser degree the lateral hypothalamus also project directly to SPNs in a variety of species such as rat, cat, monkey and baboon (156, 337). Close appositions between axons of PVN neurons and SPNs projecting to a variety of targets such as the SCG, stellate ganglion and adrenal medulla (135, 264, 301) suggest monosynaptic inputs onto these SPNs, which may in part release vasopressin (264). Dahlstrom and Fuxe (74) first identified the raphespinal pathway as 5-HT positive but many neuromodulators and neurotransmitters are colocalized with 5-HT, including Substance P, thyrotropin releasing hormone (307), neuropeptide Y (31), enkephalin (186), GABA (28) and glutamate (164, 271). Indeed, glutamate and 5-HT may act in tandem in the regulation of cutaneous sympathetic discharge (277) or in the control of sympathetic outflow to BAT (215). These data suggest that all raphe-spinal neurons are serotonergic; however, this is likely not to be the case. In studying how the raphe nuclei may influence SPN activity, it is important to take into account the fact that the raphe region comprises nuclei with diverse outputs and functions so responses obtained vary according to exactly which of the subdivisions are targeted by the different electrodes. In early studies, using direct recordings from SPNs in the pigeon, raphe stimulation strongly inhibited SPN discharge (46) and lesioning the raphe nuclei elicited hypertension and increases in sympathetic nerve discharge but this sympathoinhibition is likely due to a tonic nonbaroreceptor mediated effect since activation of the baroreceptor reflex after raphe lesioning still inhibited sympathetic nerve discharge (236). More specific stimulation of the raphe pallidus nucleus excited SPNs— furthermore, this study considered the response profiles of different groups of SPNs to enable classification of the SPNs that were activated (253). There is a unique class of splanchnic SPNs that is excited by raphe stimulation via an unmyelinated pathway and does not respond to RVLM stimulation with short latency responses but only with longer latency responses that are likely due to activation of an axon collateral originating in the raphe pallidus, suggesting that this one class of SPNs does not have direct input from the RVLM (253). This study also showed that other classes of splanchnic SPNs are excited at short latency by RVLM stimulation but unlikely to also be excited by raphe stimulation, while yet others exhibit both short and long latency responses to RVLM stimulation that imply inputs from both RVLM and raphe pallidus. Such studies clearly support the potential functional segregation of SPNs in terms of their inputs. So it is worthwhile considering what that functional specification could be. Transneuronal tracing studies demonstrate that, although raphe spinal pathways are commonly labeled after injections


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into many end organs (see above), a pattern seems to emerge suggesting a specialised role for some of the raphe-spinal and paraventricular neurons in temperature control. Raphe and PVN neurons target SPNs influencing the tail, an important source of heat loss in rats (321, 357), and BAT (49), which is critical for thermogenesis during cold exposure. Importantly, exposure to cold led to activation of these two groups of spinally projecting neurons (49). Thus these pathways are likely to be the source of the direct influence on SPNs involved in thermoregulation. Morrison and colleagues provide insight into the particular roles that some of these PVN- and raphespinal neurons play (261). Disinhibition of the rostral raphe pallidus nuclei (which itself is activated by cold, as shown by c-fos labeling of neurons in this region after this perturbation) caused significant increases in the activity of sympathetic nerves innervating BAT (254, 263) (Fig. 13) and these were significantly more increased (up to 2000% of the control activity) than the increases in the activity of sympathetic nerves supplying the splanchnic nerve (which only increased by 25% of the control activity) (256), giving some of the first evidence that the raphe nuclei play a particular role in thermoregulatory and metabolic function. Indeed, it is now clear that the raphe pallidus is a major conduit for the sympathetic components of regulatory mechanisms that are triggered by stressful situations such as an immune insult (51), psychological stress (166), or those involving release of corticotrophin releasing factor (55). The hypothalamus has many diverse roles in homeostasis and this is reflected in the complexity of the inputs to the SPNs and the neurochemistry. SPNs are inhibited by PVN stimulation, although it is not known if this is a direct effect, and the targets of these SPNs were not identified (110). PVN stimulation also inhibits BAT sympathetic nerve activity (216), which suggests that BAT-SPNs are specifically inhibited. Orexin positive lateral hypothalamic neurons project to SPNs and these may be important in sympathetic responses to arousal; in fact since a single orexinergic neuron projects to different SPNs that in turn innervate separate targets, it is feasible that these are like central command neurons, orchestrating appropriate responses to arousal, feeding or stress (104, 167). Similar to the raphe nuclei, it seems that there are target specific pathways from the hypothalamic inputs, indicated by oxytocin immunopositive fibres which are found in close apposition to SPNs labeled from the cervical sympathetic trunk but which seem to avoid sympathoadrenal preganglionic neurons (11). This difference was not observed with somatostatin or serotonin. Melanocortin-concentrating hormone positive spinally projecting neurons are also located in the lateral hypothalamus and the hypothalamic arcuate nucleus (167) and activation of melanocortin receptors in the IML by microinjections of alpha-melanocyte stimulating hormone induces tachycardia while similar tachycardic responses due to chemical activation of the arcuate nucleus were blocked by intermediolateral cell column applications of melanocortin receptor 4 antagonists (144). The authors conclude that this pathway may mediate some of the cardiovascular responses to stress.

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Figure 13 Effect of hypothermia and bicuculline microinjection into raphe pallidus (RPa) on sympathetic nerve activity to brown adipose tissue in anesthetized rat. Panels A and B are from same experiment. (A) AP, BAT SNA, and BAT SNA PWR during control conditions (colonic temperature: 37.5◦ C) and acute hypothermia (34.7◦ C). It is clear that BAT SNA is increased during hypothermia. (B) AP, BAT SNA, and HR responses to microinjection (arrow; 60 nL) of bicuculline (500 μmol/L) into RPa. (C) Immunocytochemical staining for Fos (used as an indicator of activated neurons) in neurons of midline brain stem after 4-h exposure to environmental temperature of 4◦ C (cold) or 22◦ C (control). Note large increase in Fos expression in RPa at level of bregma −10.30 in animal exposed to acute hypothermia. Abbreviations: BAT SNA: sympathetic nerve activity to brown adipose tissue; BAT SNA PWR: autospectrum of BAT SNA; AP: arterial pressure; HR: heart rate; RPa: raphe pallidus. Adapted, with permission, from Morrison et al. (263).

The A5 region The A5 region of the pons is perhaps the least studied of the pathways directly impinging on SPNs but this may be associated with the relative importance of this region. Early studies suggested a dense projection of A5 noradrenergic neurons to the rat spinal cord, with injections of anterograde tracers such as tritiated amino acids (209) or HRP-wheatgerm agglutinin (42) labeling fibres in the IML, intercalated nucleus and other autonomic regions. To show that these descending neurons were noradrenergic, retrograde labeling of HRP was combined with catecholamine fluorescence labeling which resulted in double labeling of these cells while injections of the catecholamine neurotoxin 6-OHDA destroyed both the

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retrogradely labeled neurons in A5 and anterogradely labeled terminals in the IML (209). Similar projections were also shown in the rabbit (30). With the advent of neurochemically select tracers such as an adeno-associated viral (AAV) vector encoding green fluorescent protein (GFP) driven by the artificial dopamine-β-hydroxylase promoter, PRSx8, it is possible to determine the exact termination patterns of the only 3 noradrenergic spinally projecting CNS regions, A5, A6, and A7 (40). Combining select injections into each region with immunohistochemistry for ChAT to label the SPNs, it was clear that the major noradrenergic descending pathway to the sympathetic areas arose from the A5 region and that the densest labeling was in the upper thoracic region. Spinally


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projecting A5 neurons demonstrated robust barosensitivity, with the majority being inhibited by increases in blood pressure, suggesting that the A5 projection is excitatory (121,122,138). Indeed using spike triggered averaging, a clear correlation was observed between splanchnic sympathetic discharge and firing of A5 neurons (138). Selective activation of noradrenergic A5 neurons using optogenetic tools elicited a fivefold greater activation of the renal nerve than the lumbar sympathetic chain (163), suggesting functionally specific activation of sympathetic outflow. This study also recorded from bulbospinal A5 neurons to show that they were powerfully activated by peripheral chemoreceptor stimulation, suggesting that the A5 neurons play a role in producing the rapid sympathetic responses to hypoxia. Although the RVLM appears to play a critical role in sympathetic responses to blood pressure changes, an idea supported by such observations that bilateral injections of GABA receptor antagonists abolish the sympathetic response to baroreceptor activation (341), this dominance has been disputed with studies in conscious rabbits implicating the A5 region in this control. Induced hypotension caused an increase in activated A5 neurons (shown with c-fos staining) (76, 191) and at least a proportion of those cells that were activated were shown to be spinally projecting (295). This was not the case for PVN neurons, suggesting that at least in rabbits, the PVN spinally projecting neurons do not contribute significantly to the sympathetic response arm of the baroreceptor reflex. Together these data regarding supraspinal inputs onto SPNs serve to emphasize the complexity of descending control of sympathetic outflow. The major direct inputs to the SPNs from brainstem regions have distinct roles in controlling the levels of activity in SPNs, which is dependent on the function and appropriate response to a particular perturbation. It is also important to remember that other regions such as the medial prefrontal cortex and other corticospinal pathways project to autonomic regions but may not always directly innervate SPNs; these are discussed in the next section on interneurons.

Spinal Interneurons Involved in Sympathetic Control Within the spinal cord, interneurons are important components of the circuits controlling sympathetic outflow, participating in some of the descending and all of the afferent circuits influencing SPN activity (83,84). Interneurons may be of particular importance in situations such as spinal cord injury when spinal circuits are modified after severance or damage to the descending pathways, thus losing the important supraspinal control of sympathetic outflow. In this section, we will consider the evidence for the location and properties of these interneurons. Some of the first clues about the existence of these interneurons were evident with in vitro spinal cord slices when ongoing synaptic activity was regularly recorded from SPNs


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(87, 92, 176, 190, 323); here authors concluded that the source of these synaptic inputs must be from interneurons still intact and indeed spontaneously active in this reduced preparation. Thus, within very small spinal cord compartments, interneuronal circuitry exists to enable local control of SPN activity.

Location and properties of interneurons Strack et al. (335, 337) first demonstrated the presence of pseudorabies virally labeled interneurons that were involved in sympathetic spinal circuits and located in spinal cord laminae VII and X. They injected into a number of sympathetic ganglia and into the adrenal gland which all led to similar degrees of infection of these interneurons. Injections of a different transneuronal tracer (wheatgerm agglutinin) into the SCG led to a higher extent of retrograde labeling with interneurons located in lamina V in high numbers and also in the IML itself (44). This was of particular interest since previously, it was considered that this autonomic nucleus was almost exclusively packed with SPNs. However such an idea was corroborated by extracellular recordings from neurons in the dorsal horn and IML region that were not considered SPNs due to their electrophysiological characteristics (57, 244, 350); these non-SPNs fired action potentials correlating with the sympathetic activity recorded simultaneously from sympathetic renal nerves (Fig. 14). In fact these presumptive interneurons were both positively and negatively correlated with this firing, potentially enabling subclassification of these interneurons into excitatory and inhibitory groups. Their action potential firing preceded the peak of the sympathetic renal activity, suggesting they were driving the SPNs. These interneurons were located not just in the IML region but also in laminae I-V and around the central canal, in lamina X. The contributions of these interneurons in the intact spinal cord may be limited since there is a lower degree of correlation with the renal sympathetic nerve activity in intact compared to spinalized rats (244). Interneurons may be involved in many sympathetic circuits controlling different end organs since they are labeled after injections into the adrenal gland (64, 159), BAT (49), spleen (50), kidney (351), colon (364), bladder or urethral sphincter (340, 362, 365), and stellate ganglion (156). Perhaps critically, when injections are made simultaneously into two end organs such as the bladder and colon (304), doublelabeled interneurons are rare, suggesting that interneurons are involved in coordinating very specific functions, as would be expected to enable appropriate and exclusive responses. By combining transneuronal tracing with in situ hybridization with probes against the enzyme glutamic acid decarboxylase (GAD), a group of GABAergic interneurons in the central autonomic area was identified (87) and these formed monosynaptic inhibitory contacts onto SPNs in the IML. These interneurons could be the ones reported by Schramm’s group above that exhibited renal-related activity (57, 244, 350). Interneurons are themselves contacted by supraspinal and afferent inputs that thus exert some of their effects on SPN

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Figure 14

Sympathetic interneurons in the rat spinal cord. (A) Top panel: low magnification photograph of a juxtacellularly labeled, sympathetically correlated neuron histologically located in lamina IV of the dorsal horn. Bottom panel: dark trace: cross-correlation between the incidence of discharge of a sympathetically correlated dorsal horn interneuron and simultaneously recorded RSNA in the anaesthetized rat. Light traces: dummy cross correlations between RSNA and discharges of a simulated neuron. (B) Action potentials recorded from an SPN and an IML interneuron in rat spinal cord slices showing the faster repolarization phase of the interneuronal action potential. On the right, immunohistochemical analysis of Kv3.1b (top) combined with fluorogold labeling of SPNs (bottom) show that interneurons, not SPNs, express Kv3.1b. C. Filled IML interneuron (inset) and line drawing of the axonal and dendritic arborization. Adapted, with permission, from Tang et al. (350) (A) and Deuchars et al. (86) (B and C).

activity through spinal interneurons rather than directly. This would enable amplification of a signal or coordination of specific sympathetic outflows in a more efficient way. Many afferent terminals are in dorsal horn regions containing the interneurons that in turn send projections to autonomic regions such as the IML (see section above), indeed some afferent axons terminate in lamina X which suggest the involvement of interneurons around the central canal (87). Injections of anterograde tracer into the corticospinal tract and RVLM resulted in terminal labeling surrounding both SPNs and interneurons, although the extent of labeling from the RVLM was higher onto SPNs than interneurons (280). The opposite is true of the corticospinal tract since although the extent of labeling was quite low, perhaps due to difficulties in anterograde labeling of this descending pathway, the majority of labeled axons closely apposed interneurons rather than SPNs (280). The medial prefrontal cortical input

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is also likely to be onto interneurons rather than SPNs since anterograde tracing from this region terminated in the central autonomic area but away from the locations of SPNs (18). Indeed, the targets of these cortical projections may be the GABAergic interneurons directly innervating SPNs that are located in the vicinity of the terminal labeling (87). This would explain the decrease in blood pressure recorded when this cortical region is stimulated (18). Some of the descending inputs from the PVN may predominately act through activation of interneurons; after pseudorabies viral infections of the adrenal gland, few oxytocin or vasopressin neurons are infected in the PVN, suggesting that at least one further neuron is involved in these pathways compared to other descending pathways that were infected (370). This contrasts clearly with the heavy and rapid labeling of the PVN after inoculation of BAT, indicating fewer neurons involved in this PVN-BAT pathway (49, 275).


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Stimulation of the dorsolateral funiculus [which contains many of the descending inputs to the autonomic regions (79)] evoked both IPSPs and EPSPs in interneurons (38, 85). There is less direct evidence of afferent inputs eliciting responses in autonomic interneurons although afferent influences on SPNs are known to be indirect (see above) and persist in spinalized animals (376) which indicates that local interneurons are involved. In this study, some presumptive interneurons were excited or inhibited by ulnar or sciatic nerve stimulation. The time course of the inhibitory effects on interneurons was similar to that of the inhibition of the SPNs that were also recorded in this study, suggesting that the mechanism of action of afferent induced SPN inhibition may be disfacilitation through inhibition of excitatory interneurons. Interneurons are also subject to cardiac modulation since single extracellular recordings from interneurons show activity correlated with the cardiac cycle (22, 102). The peak of this activity is at a shorter latency than that of SPNs, signifying that they are antecedent to these neurons. Furthermore some interneurons were inhibited by increased blood pressure and activated by stimulation of the RVLM, implying a sympathoexcitatory role. Still other cardiac-related interneurons are likely sympathoinhibitory based on their response pattern, since their activity is related to the R-wave of the electrocardiogram but they are activated by stimulation of the vasodepressor region of the NTS (235). Propriospinal circuits using interneurons located within the spinal cord but distant from the SPNs also play a role in sympathetic control. Transneuronal tracing studies identified two intraspinal circuits arising from the lateral funiculus and lateral spinal nucleus at cervical levels (154). Electrical or chemical stimulation of the cervical spinal cord inhibits renal sympathetic activity (309); effects only observed in spinalized rats so the authors suggested the presence of a pathway from the cervical interneurons that somehow interacts with the RVLM to control sympathetic activity (296). IML interneurons have shorter action potentials than those recorded from SPNs in the same study, due to a more rapid repolarization phase, rather than a difference in the depolarization phase of the action potential (Fig. 14, (86)). This faster repolarization is due in part to the presence of the potassium channel Kv3.1b (important in other CNS regions in enabling fast firing properties) since blocking this channel prolonged the action potential and decreased the firing frequencies of the interneurons. There was little electrophysiological evidence of gap junctions in interneurons—this is also hard to determine with other techniques since no study has combined the necessary transneuronal tracing with immunohistochemistry for different gap junction proteins. In the same study, the recorded interneurons were filled with neurobiotin and a fluorescent dye and some of the spinal cord sections were then processed for immunohistochemistry for the Kv3.1b subunit [Fig. 14, (86)] which revealed that these fast firing interneurons were indeed Kv3.1b immunopositive. The interneurons directly influence SPNs since morphological analysis of filled cells at the electron microscopic level combined with



immunohistochemistry for ChAT to label SPNs show direct interneuronal synaptic contacts onto SPNs (83), while other studies using transneuronal labeling of interneurons innervating SPNs combined with immunohistochemistry for Kv3.1b further confirm the data (39). If these interneurons form part of the circuits within the spinal cord then is it possible to generate any rhythmic sympathetic activity from spinal cord slices that may involve the SPNs and interneurons firing in appropriate synchrony? A recent study (288) recorded with extracellular electrodes from the IML region of the spinal cord and reported that sympathetic neuronal network oscillatory activity could be generated either spontaneously or by application of 5-HT. They proposed that small local circuits involving interneurons (since the GABA A receptor antagonists bicuculline reduced the power of this oscillation) and SPNs that were communicating by gap junctions (since gap junction blockers reduced or abolished the oscillation) were capable of generating these rhythms. Our understanding of these circuits may enable us to harness the potential of the spinal cord circuits when damage to supraspinal pathways has occurred.

Neurochemistry of interneurons Not surprisingly, given the evidence above of the diversity of inputs onto SPNs, some of this diversity is due to populations of interneurons with differing neurochemical coding. Once again many of these findings are the results of the combination of transneuronal tracing and in situ hybridization or immunohistochemistry. Virally infected local interneurons express substance P, enkephalin and somatostatin but do not contain cholecystokinin-8, corticotrophin releasing factor or vasoactive intestinal peptide (156). In other studies, many interneurons in the vicinity of autonomic regions that are likely to innervate SPNs contain a variety of neurotransmitters and neuromodulators. For example, CARTimmunopositive, ChAT-immunonegative neurons (and thus presumptive interneurons) are located in the IML (97) while dorsal horn axon terminals that are not primary afferents contain both glutamate and substance P and these may impinge on dendrites of SPNs as a local interneuronal input. Transsynaptic tracing combined with immunohistochemistry revealed GABAergic interneurons in lamina X directly innervating SPNs (87). In addition, small enkephalin-containing interneurons are present in lamina X (197). Since enkephalin often coexists with GABA in the dorsal horn (355), enkephalin may be present in these GABAergic neurons. Neuropeptide Y-containing interneurons are found in the vicinity of spinal autonomic regions such as laminae V and VII, in close proximity to SPNs (249). Because most neuropeptide Y-containing axons in the dorsal horn also express GABA (291), these interneurons may also have an inhibitory role. Cholinergic influences on SPNs are also likely to be local since cholinergic inputs persist after spinalization (319). We do not understand the role of all interneurons in sympathetic control; obviously many form part of the afferent

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or descending pathways that shape sympathetic outflow from the spinal cord but still others may help to form local network oscillators that can contribute to sympathetic outflow in the absence of other inputs. After spinal cord transection, the number of active local interneurons increases dramatically as little as two and a half hours after the injury; this increase may reflect increases in their activity due to loss of inhibitory supraspinal inputs (306). Moreover, the proportion of interneurons with firing patterns that correlate with renal sympathetic nerve activity is higher after acute transection than in the intact cord (244). This immediate change in activity may be the cause of exaggerated reflexes observed shortly after spinal cord injury. We need to understand the role of interneurons better to know whether it is possible to manipulate them where appropriate to restore sympathetic homeostasis after any perturbations.

Determination of Inputs onto SPNs—Neurochemical Studies Thus far, this review has considered the intrinsic properties of SPNs, the gap junctions that enable synchronized activity to occur between SPNs and the sources of the various inputs onto SPNs which include afferent pathways, supraspinal influences

and local interneurons. However, many neurotransmitters and neuromodulators have significant effects on SPNs and these neurochemicals are not restricted to specific descending or local pathways so it is hard to assign specific effects of activating receptors on SPNs to particular pathways or input. Therefore this section considers as a whole, how SPNs are targeted by inputs containing specific neurochemicals, rather than how they are affected by activation of a specific pathway. This section extends the review on the anatomy of synaptic circuitry onto SPNs by Llewellyn-Smith (193). A summary of key inputs onto SPNs and their source is given in Table 1.

Glutamate Glutamate plays a critical role in the control of SPN activity, being the major excitatory neurotransmitter in afferent, descending and local inputs onto these neurons. Since we have touched on the specific pathways and their use of glutamate above, this part will focus on the actual effects at the level of the spinal cord. The enormity of the influence of glutamate in SPN control is shown in a series of experiments investigating the extent of glutamatergic inputs (192, 196, 201, 204). They make a bold statement that nearly one hundred percent of the synapses onto SPNs contained either glutamate or GABA, such that all inhibition and excitation likely involves

Table 1

Overview of Effects of Different Neurotransmitters on SPNs and Their Potential Source—for Details of Papers, Refer to the Specific Sections in the Review

Neurotransmitter/ neuromodulator

Effect on SPNs

Receptors involved

Used in which inputs onto SPNs?

Glutamate

Excitation

NMDA, non-NMDA, mGluRs

RVLM, A5, raphe, PVN, local interneurons

GABA

Inhibition

GABAA , GABAB extrasynaptic

RVLM, A5, raphe, PVN, local interneurons

Glycine

Inhibition

Glycine receptors (subunits not described)

RVLM, local interneurons

5-HT

Excitation and inhibition

5-HT2 (excitation), 5-HT1A (inhibition

Raphe

Substance P

Excitation

NK1

RVLM, raphe, PVN, local interneurons

Noradrenaline


α1 (excitation), α2 (inhibition)

PVN, A5

Adrenaline


α1 (excitation), α2 (inhibition)

RVLM (?)

Orexin

Excitation

OX1 and OX2

PVN

Oxytocin

Excitation

Oxytocin and V1 receptors

PVN

Vasopressin

Excitation

V1a

PVN

Enkephalin

Inhibition

Mu opioid

RVLM, raphe, local interneurons

Nociceptin

Inhibition

Opioid receptor like orphan receptor

Local interneurons?

Neuropeptide Y

Not known

Not described

RVLM, raphe, local interneurons

Acetylcholine


Nicotinic (excitation), muscarinic (inhibition)

Local interneurons

Angiotensin II

Excitation

Ang I

RVLM?

Adenosine

Overall inhibition

A1, A2A

Not known

PACAP

Excitation

PAC1, VPAC I, II

RVLM

N.B. Excitation refers to either depolarizations recorded with intracellular or patch clamp electrodes or increases in firing rate of extracellular action potentials. Inhibition refers to either hyperpolarizations recorded with intracellular or patch clamp electrodes or decreases in firing rate of extracellular action potentials.

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a release of one of these transmitters (192, 196, 201, 204). They combined retrograde labeling of SPN from the SCG or the adrenal gland with post embedding immunohistochemistry for glutamate and GABA to show that for adrenal SPNs, around two thirds of the synaptic boutons onto their cell bodies and dendrites were glutamatergic while the other third were GABAergic, while for SCG SPNs, the numbers for each neurotransmitter were more evenly split (48.9% GABA, 51.7% glutamate; Fig. 15). As discussed above, much of this input comes from descending pathways. However, after a complete spinal cord transection, which removes influence of these descending pathways, there is still a high degree of glutamatergic (and GABAergic) immunoreactive terminals onto SPNs. This infers that local intraspinal glutamatergic neurons are a strong influence on SPNs; these may be those involved in the local circuits responsible for afferent innervation or others that may be part of descending pathways but may also be involved in local networks that generate sympathetic rhythmic activity (see below). Since these widespread glutamatergic inputs do not discriminate between specific pathways, it is perhaps more informative to examine the extent of expression of the vesicular glutamate transporters that are not ubiquitously expressed by all glutamatergic cells. Vesicular glutamate transporter 1 is not widely expressed in terminals onto SPNs but vesicular glutamate transporter 2 is widespread and these terminals innervate SPNs retrogradely labeled from many different end organs or ganglia (198). Indeed vesicular glutamate 3 immunoreactive terminals are also distributed in the IML and these are an independent population from the vesicular glutamate transporter 2-immunopositive terminals (271). This study also reported that a population of the vesicular glutamate transporter 2-immunopositive terminals contains PNMT while some of the vesicular glutamate transporter 3 expressing terminals were serotonergic. Thus different descending glutamatergic pathways utilise specific transporters that aids in their identification. Bath application of selective amino acid agonists NMDA, kainate and quisqualate all induced membrane depolarizations associated with a decrease in input resistance, reflective of opening of nonselective cation channels (143, 177, 322) while ongoing EPSPs were glutamatergic in nature (143,177). The majority of ongoing activity in individual SPNs recorded in vivo can be antagonized by application of kynurenic acid so excitatory amino acid receptors are providing the main excitatory drive to SPNs (257). Furthermore, stimulation of the dorsal horn, ipsilateral or contralateral lateral funiculus or contralateral IML elicits EPSPs through effects at both NMDA and non-NMDA receptors (85, 325), although earlier studies (317) indicated that excitatory responses due to dorsal root stimulation were primarily mediated through NMDA receptor activation. Medullary evoked EPSPs are also due to activation of both NMDA and non-NMDA receptors, indicating that both major inotropic receptors are critical in mediating these responses (88). Metabotropic receptors can play a role in sympathetic control with differential effects mediated by the different receptors such that activation of group I


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Figure 15 GABA and glutamate terminals influence SPNs in the rat. (A) Immunohistochemical localization of glutamate and GABA in terminals onto SPNs retrogradely labeled after injection into the superior cervical ganglion (reaction product arrowed). Small dots (10-nm gold particles) are in glutamatergic terminals and larger dots (15-nm gold particles) are in GABAergic terminals. It is clear that the two neurotransmitters do not colocalise-in the top panel, only small particles are present in the terminal (glutamatergic) while in the bottom panel, only larger gold particles are located in the terminal (GABAergic). (B) Stimulation of the lateral funiculus elicits fast excitatory postsynaptic potentials which are blocked by the antagonists for excitatory amino acids (CNQX and AP-5). (C) Ongoing inhibitory postsynaptic potentials in SPNs recorded in spinal cord slices. These are GABAergic since they are inhibited by bicuculline. Taken, with permission, from LlewellynSmith et al. (195) (A), Deuchars et al. (85) (B), and Wang et al. (369) (C).

metabotropic receptors causes depolarizations while group II receptors, which are sparsely expressed in comparison, hyperpolarises SPN (272,324,374). Furthermore, presynaptic metabotropic receptors depressed both EPSPs and IPSPs in SPNs, which enables specific dampening of inputs onto these neurons (91, 374).

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GABA Many studies have examined the extent of GABAergic innervation of SPNs, using immunohistochemistry, tract tracing, electrophysiology and combinations of techniques. Elucidating the source of these GABAergic inputs has often relied on tract tracing with immunohistochemistry for GAD or GABA and many groups suggested that at least a component of this GABAergic input is from descending pathways. For example, GABAergic neurons labeled from IML retrograde tracing were located either in the RVLM or CVLM, suggesting direct descending projections from those regions onto SPNs (229, 250). The use of in situ hybridization combined with retrograde tracing after injections of Fast Blue into the spinal cord (which in fact covered more than just the IML region, (328) or more precisely, transynaptic labeling of bulbospinal neurons after adrenal gland infections with PRV (330) suggested that some GABAergic spinally projecting neurons were located medial to the C1 and A1 neurons of the RVLM and CVLM respectively. None of these were TH positive, suggesting clear segregation of inhibitory pathways from the catecholaminergic inputs to SPNs. Furthermore, spinally projecting GABAergic neurons in the raphe nuclei were also 5-HT positive (328, 330) which confirmed earlier similar work (245). Importantly, a proportion of those descending GABAergic neurons in the ventral gigantocellular nucleus and medial reticular formation were found to be also glycinergic. Electrophysiological studies show the huge influence of GABA on the SPN activity. Application of GABA iontophoretically onto SPNs in cat rapidly inhibited extracellular spontaneous action potential firing (12) while bath application of GABA caused hyperpolarizations in the majority of SPNs recorded in cat (142) or rat spinal cord slices (91, 176, 373). Activation of the lateral funiculus in cat spinal cord slices elicited IPSPS (sometimes only unmasked after blocking excitatory synaptic transmission) that were mediated in part (about 30% of the time) solely by GABA receptors or sometimes through activation of both GABAergic and glycinergic receptors (142). This was in contrast to observations from the rat spinal cord slice where bicuculline, the GABA A antagonist only weakly affected lateral funiculus evoked IPSPs and had no effect on spontaneous IPSPs (176) but more recent reports showed clear antagonism by bicuculline of the lateral funiculus evoked IPSPs and spontaneous IPSPs in rat spinal cord slices (87, 369, 371). These studies focus on the synaptic transmission of GABA but there is a further role for extrasynaptic GABA receptors; application of bicuculline alone, but not gabazine depolarized SPNs and the pharmacological profile of these slow responses together with PCR analysis of the receptor subunits located in the IML indicated that the novel receptor contains α5 subunits, commonly found in such extrasynaptic locations (369). This dampening down of SPN excitability may be a novel site to target for therapies involving overactivity in SPNs. The effects above are all due to activation of ionotropic receptors but metabotropic

856


GABAB receptors also influence SPN activity with activation of postsynaptic receptors hyperpolarising SPNs (58,368,371) or reducing spontaneous activity in vivo (238). Moreover activation of presynaptic receptors reduced both EPSPs (58, 371) and IPSPs (368) and could also inhibit the response of SPNs to dorsal root stimulation (238). The source of these GABAergic inputs has been examined in a number of ways; electrical stimulation of the RVLM region in the neonatal rat brainstem spinal cord preparation in the presence of excitatory amino acid antagonists to block fast EPSPs revealed fast IPSPs that were blocked by the GABA antagonist bicuculline (89). This may be due at least in part to activation of regions just medial to the C1 and A1 groups of neurons since there was no overlap between GABAergic bulbospinal neurons and those that were TH-positive (328). Local GABAergic interneurons also provide a high degree of input since ongoing GABAergic IPSPs in spinal cord slice preparations are routinely observed at the upper thoracic level (369) and combining transneuronal tracing from the adrenal gland with in situ hybridization revealed GABAergic interneurons located in the central autonomic area of the spinal cord. These interneurons may be the crucial link in the conversion of excitatory spinal input from the medial prefrontal cortex to inhibition in SPNs (18).

Glycine Glycine receptors are located on both IML neuronal somata and dendrites (although these were not positively identified as SPN) and some of these are postsynaptic to GABAergic terminals suggesting that GABA and glycine are colocalized (59, 356). There is dense glycinergic input onto retrogradely identified SPNs and there are glycinergic positive interneurons located in laminae V and VII (43). Iontophoretic application of glycine inhibits SPN (12). At least a component of the glycinergic input onto SPNs may be supraspinal since transneuronal labeling of medullary neurons was combined with in situ hybridization for glycine and GABA to reveal distinct groups of inhibitory descending pathways: those in the raphe nuclei were GABAergic but never glycinergic while those in the other medial medullary regions often expressed both GABA and glycine (330)—this fits well with electrophysiological findings. There is discrepancy in the findings of some laboratories regarding the chemical nature of ongoing inhibitory synaptic potentials in spinal cord slices since in thoracic and lumbar spinal cord slices spontaneous IPSPs were glycinergic (175, 176) whilst others found that in similar preparations, they were commonly GABAergic in nature [e.g., (369)]. These differences may be due to the fact that both GABA and glycine are released from many terminals as shown above anatomically and the amount released in any experiment may be due to the anatomical level of slices recorded, SPNs targeted or preparation of slices. One noteworthy observation, based on event kinetics and pattern, is that there may be at least two sources of glycinergic input


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onto SPNs (175). Stimulation of the dorsal roots also elicits IPSPs that are glycinergic in nature and must therefore involve a local glycinergic interneuron (92). IPSPs from activation of descending inputs by stimulation of the lateral funiculus are occasionally blocked by either a combination of bicuculline and strychnine or strychnine alone which further confirms that some of the descending pathways express both glycine and GABA (87, 91).

5-HT 5-HT (i.e., serotonin) plays one of the most profound and diverse neuromodulatory roles of all the descending pathways to the spinal cord. Besides its role in autonomic control, there are major innervations of motor pathways and those dorsal horn regions involved in sensory processing. The raphe nuclei are the major sources of this input and these are lost following spinal cord injury, thus it is not surprising that therapeutic avenues involving 5-HT replacement are producing such encouraging results for combating some of the many symptoms of this condition, which include many autonomic dysfunctions (136). Immunohistochemical studies have shown dense innervations of the IML and other autonomic regions containing SPNs; the staining pattern resembles the ladder like location pattern of SPNs themselves, thus supporting this concept of global and prominent influences of this monoamine. There are dense plexi of 5-HT positive axons surrounding cell bodies and dendrites of SPNs (157, 361) and the innervation pattern is similar for adrenally projecting SPNs (17). Iontophoretic application of 5-HT increases the firing rate of cat SPNs with a slow onset and prolonged duration, an effect blocked by application of the nonselective 5-HT1/2 antagonist methysergide and indicative of a neuromodulatory effect (16). Effects were similar in adrenal and nonadrenal SPN. Interestingly a carefully conducted in vivo study (114) showed that SPNs which had excitatory responses to 5-HT showed only decreased action potential firing or no response to noxious stimulation of the ipsilateral hindlimb. Conversely, SPNs with action potential firing decreased by 5-HT had primarily excitatory responses to noxious inputs. This implies that 5-HT can either excite or inhibit SPNs, correlated with their reponses to other stimuli. When 5-HT is bath applied to the spinal cord slice, the responses of SPNs are mainly depolarizing, mediated by 5HT2 receptors (188,190,286), causing slow changes in membrane potential accompanied by an increase in input resistance due to closure of potassium channels. The responses of SPNs to 5-HT are influenced by the site of application since if 5-HT was applied in a concentrated manner over the central canal region (where the long medially projecting dendrites are located and where it is known that the 5-HT terminals are also found), a biphasic response is observed with initial depolarization followed by the long-lasting hyperpolarization, mediated by 5-HT1A receptors (286). This fits well with the finding that 5-HT is colocalized with both GABA and glutamate in



synaptic terminals (see above), so it may be that when 5-HT is released with GABA, it activates inhibitory 5-HT1A receptors while release of 5-HT from synapses that also contain glutamate will activate the excitatory 5-HT2 receptors. The degree of coupled activity is also increased with 5-HT application which may be due either to a change in the conductance of gap junctions or an increase in the firing rate of SPNs that are coupled to the recorded cell or a combination of both mechanisms (287). An indirect effect of 5-HT on SPNs that may involve depolarization of antecedent inhibitory interneurons is suggested from an increase in ongoing IPSPs in SPNs after 5-HT application (190). 5-HT may be also critical in the rhythmic activity of SPNs. Intrathecal application of 5-HT increased rhythmic activity with a long latency to onset (215) and similar rhythmic sympathetic activity was maintained after spinal cord transection (224), suggesting that the spinal cord itself was capable of generating this complex activity. Indeed in a reduced spinal cord slice preparation of just 500 μm thickness, 5-HT can initiate or increase network rhythmic activity and this rhythm involves the contributions of both SPNs and interneurons [Fig. 16 (288)]. This leads to the intriguing proposition that small groups of neurons involved in autonomic control can be induced to coordinate their activity just with the application of 5-HT to produce a sympathetic rhythm of a functionally relevant frequency, something that could be exploited in future to help combat autonomic dysfunction associated with spinal cord injury.

Substance P Substance P is another neuromodulator found in the terminals of a number of descending inputs onto SPNs and there is plenty of anatomical evidence of synapses onto SPNs containing this neuropeptide (17, 290, 361). The source of this neuromodulatory influence on SPN activity is also partly from within the spinal cord (77), although the identity of the neurons expressing substance P is not yet clear. Substance P depolarises SPNs through an action at Neurokinin 1 receptors (47) to depress a calcium-activated potassium channel and thus increase the excitability of the neurons. This is observed as an increase in firing rate of SPNs after iontophoretic application of substance P characterized by a slow onset and prolonged effect, indicative of a neuromodulatory role that enables heightened responses to other neurotransmitters in certain conditions (13, 16).

Catecholamines Enzymes involved in the synthesis of catecholamines are expressed in descending terminals onto SPNs however the levels of adrenaline in the IML are too low to be detected by high performance liquid chromatography (345). Since monoamine oxidase inhibition increased catecholamine levels in the spinal cord, these neurotransmitters may be synthesized but not stored in vesicles (346). Thus, the evidence of actual release of

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10 μM 5-HT

Control

(A)

(D) Power (μV2)

20 μV 0.2s 30

20

20

10

10 25

50

0

25

50

0

0

12

–1

–1 –0.2

0

0.2

–0.2

Fre

0

0.2

Time lag (s)

n=5

n=5

(F) Power (NAP)

1 Frequency (Hz)

Autocorrelation coefficient

(E) 1

e qu

0

Frequency (Hz)

(C)

8 4

z)

H

y( nc

) in (m T H 5-

0 0

25

e

μM

0

0 60 m Ti

30

10

Power μV2

(B)

10

0.7 0.5

n=8

n=8

0.3 0.1

0 Control

5-HT

Control

5-HT

Figure 16

5-HT induces oscillatory activity in the IML. (A) Low-pass filtered (

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

Synaptic mechanisms in sympathetic preganglionic neurons.

Development and migration of avian sympathetic preganglionic neurons.

Glycine-like immunoreactive input to sympathetic preganglionic neurons.

Spinally projecting preproglucagon axons preferentially innervate sympathetic preganglionic neurons.

Developmental reorientation of sympathetic preganglionic neurons in the rat.

Activation of α1-adrenoceptors facilitates excitatory inputs to medullary airway vagal preganglionic neurons.

The relation between end-tidal CO2 and discharge patterns of sympathetic preganglionic neurons.

Sympathetic preganglionic neurons projecting to the adrenal medulla and aorticorenal ganglion in the rabbit.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

Light and electron microscopic analyses of intraspinal axon collaterals of sympathetic preganglionic neurons.

Renal and adrenal sympathetic preganglionic neurons in rabbit spinal cord: tracing with herpes simplex virus.

Adrenergic receptors (alpha 1 and alpha 2) modulate different potassium conductances in sympathetic preganglionic neurons.

Effect of post-impulse depression on background firing of sympathetic preganglionic neurons.

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

Intracellular recordings from sympathetic preganglionic neurones.

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord.

Presynaptic GABAB receptor activation attenuates synaptic transmission to rat sympathetic preganglionic neurons in vitro.

Bulbo-spinal neurons activated by baroreceptor afferents and their possible role in inhibition of preganglionic sympathetic neurons.

Whole-cell recordings from sympathetic preganglionic neurons in rat spinal cord slices.

Morphology of sympathetic preganglionic neurons in the neonatal rat spinal cord: an intracellular horseradish peroxidase study.

Temporal patterns of antidromic invasion latencies of sympathetic preganglionic neurons related to central inspiratory activity and pulmonary stretch receptor reflex.

Oedema associated with the interruption of preganglionic sympathetic tract.

Fast inhibitory postsynaptic potentials and responses to inhibitory amino acids of sympathetic preganglionic neurons in the adult cat.