Anatomy of synaptic circuits controlling the activity of sympathetic preganglionic neurons

Journal of Chemical Neuroanatomy 38 (2009) 231–239

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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu

Review

Anatomy of synaptic circuits controlling the activity of sympathetic preganglionic neurons Ida J. Llewellyn-Smith * Cardiovascular Medicine, Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 December 2008 Received in revised form 1 June 2009 Accepted 2 June 2009 Available online 11 June 2009

Sympathetic preganglionic neurons (SPN) are critical links in the sympathetic neural circuitry that controls every organ in the body. All sympathetic outflow to the periphery comes from SPN, which send their axons from thoracic and upper lumbar spinal segments to innervate post-ganglionic neurons in sympathetic ganglia and chromaffin cells in the adrenal medulla. Despite over 30 years of study, we still do not have a sufficiently detailed understanding of the synaptic circuits through which these important neurons receive information from other central sites. We know that there is direct synaptic input to SPN from both supraspinal and intraspinal neurons, but not sensory neurons. Ultrastructural studies support functional evidence that amino acids are the primary fast-acting transmitters controlling SPN activity and indicate that an amino acid transmitter occurs in every synaptic input to an SPN. In addition, axons that synapse on SPN contain neuropeptides and monoamines, which would co-exist with and be released with the amino acids. Receptors and transporters for transmitters have also been localized in SPN inputs. Light and electron microscopic observations suggest that there are qualitative and/or quantitative differences in the neurochemical types and origins of axons, which provide synaptic input to SPN that supply different targets or have different functions. However, more research is required before it can be confirmed that SPN receive projection- or function-specific patterns of innervation. This information is likely to be important if we are to understand how the central nervous system differentially regulates sympathetic outflow to different target tissues. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Amino acids Neuropeptides Monoamines Spinal cord Interneurons Immunocytochemistry Ultrastructure

Contents 1. 2. 3.

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Organization of SPN in the Lateral Horn. . . . . . . . . . . . Origin of synaptic inputs to SPN . . . . . . . . . . . . . . . . . . Neurotransmitters in synaptic inputs to SPN . . . . . . . . 3.1. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Neuropeptides. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Monoamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transporters and receptors in synaptic inputs to SPN . Rostrocaudal differences in synaptic input to SPN. . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Organization of SPN in the Lateral Horn Sympathetic preganglionic neurons (SPN) are the source of all sympathetic outflow to the periphery, providing central drive to

* Department of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Tel.: +61 8 8204 4456; fax: +61 8 8204 5268. E-mail address: Ida.Llewellyn-Smith@flinders.edu.au. 0891-0618/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2009.06.001

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post-ganglionic neurons in sympathetic ganglia as well as chromaffin cells in the adrenal medulla (Cabot, 1990). The cell bodies of the cholinergic SPN occur in the lateral horn of thoracic and upper lumbar segments of the spinal cord. The highest concentration of SPN cell bodies is found in the intermediolateral cell column (IML), where somata occur in closely spaced groups or ‘‘nests’’ that lie at the boundary between the grey and white matter (Fig. 1). Other SPN have their cell bodies in the central autonomic area (CAA) above the central canal, in the intercalated nucleus

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function and even on the autonomic subnucleus in which their cell bodies lie (Hinrichs and Llewellyn-Smith, 2009), it would be inadvisable to infer the neurochemistry of the input to SPN with cell bodies in the CAA, ICN and DLF on the basis of what is found for SPN with cell bodies in the IML. 2. Origin of synaptic inputs to SPN Fig. 1. Organization of SPN in the intermediolateral cell column (IML) in segment T4 from rat spinal cord. SPN supplying the stellate ganglion were retrogradely labelled with cholera toxin B subunit (CTB), which was detected with immunoperoxidase staining and a black reaction product. Horizontal section. The cell bodies of the stellate-projecting SPN in the IML are clumped together in ‘‘nests’’ that are connected by their rostrocaudally running dendrites. Bundles of labelled dendrites also travel medially (arrows) towards the central canal. The full trajectory of these dendrites is not apparent because they project ventrally as well as medially at this level of the cord. Other labelled dendrites (arrowheads) occur in the white matter (WM). Bar, 250 mm.

(ICN) between the IML and the CAA and in the dorsolateral funiculus (DLF) of the spinal white matter. The dendrites of SPN in the IML run mainly rostrocaudally, connecting the nests that contain the SPN cell bodies (Fig. 1). Mediolaterally oriented bundles of SPN dendrites also link the IML with the CAA. The SPN somata in the IML plus the rostrocaudal and mediolateral SPN dendrites form a ladder-like configuration. This organization is reflected in the immunohistochemical staining patterns of axons that heavily innervate the lateral horn, such as those containing catecholamine-synthesizing enzymes, serotonin (5-hydroxytryptamine, 5-HT) or enkephalin (Krukoff et al., 1985; Romagnano et al., 1987; Fuxe et al., 1989; Newton and Hamill, 1989; Hosoya et al., 1991). Intracellular dye-filling of individual neurons in the IML of cat and rat spinal cord has shown that the dendrites of SPN can travel for many hundreds of micrometers rostrocaudally (Dembowsky et al., 1985; Pilowsky et al., 1994). Because their dendritic arbours are so extensive, SPN receive most of their synaptic input on their dendrites. SPN show a topographical organization in the spinal cord of rats (Strack et al., 1988), the species in which SPN have been most frequently studied. SPN at the rostral end of the SPN distribution innervate post-ganglionic neurons in rostral sympathetic ganglia, regulating targets in the upper body, like the pupils, the salivary glands and the heart. SPN at the caudal end of the SPN distribution innervate sympathetic post-ganglionic neurons in the lower part of the body that regulate targets like the pelvic viscera. Despite this general rostrocaudal topography, there is overlap in the distributions of SPN that supply different targets, resulting in an intermixing of different functional classes of SPN within each spinal segment. For example, the IML of thoracic segment 13 in the rat contains SPN that innervate the adrenal medulla and major pelvic ganglion as well as SPN supplying the coeliac ganglion (Strack et al., 1988). There are also rostrocaudal differences in the neurochemistry of SPN. Many anatomical studies have now shown that mammalian SPN with different projections and/or functions vary in the neurochemicals that they contain and in the types of neurotransmitters and neuromodulators that they synthesize and release in addition to acetylcholine (e.g., Gibbins, 1992; Shafton et al., 1992; Grkovic and Anderson, 1995; Edwards et al., 1996; Grkovic and Anderson, 1997; Llewellyn-Smith et al., 1997b; Grkovic et al., 1999; Fenwick et al., 2006; Ito et al., 2007; Hinrichs and Llewellyn-Smith, 2009). Post-ganglionic neurons also appear to be chemically coded on the basis of innervation target and therefore function (reviewed by Gibbins, 1995). As noted above, mammalian SPN have their cell bodies in four spinal autonomic subnuclei. However, almost all of the ultrastructural and physiological data on synaptic input to SPN relates to neurons with cell bodies in the IML. Since the neurochemical phenotypes of SPN vary depending on their innervation target and

Neurons that are in synaptic contact with SPN and therefore likely to be directly involved in regulating their activity (‘‘presympathetic neurons’’) have their cell bodies in both the brain and the spinal cord. The locations of brain presympathetic neurons in small laboratory mammals have been revealed by injecting sympathetic ganglia or sympathetically innervated target tissues with attenuated strains of neurotropic viruses. These viruses multiply and cross synapses, ultimately labelling chains of neurons that are synaptically connected. Viral tracing studies have shown that SPN receive inputs from five main brain regions. These are the rostral ventrolateral medulla (RVLM), the rostral ventromedial medulla, the caudal raphe´ nuclei, the A5 region and the paraventricular nucleus of the hypothalamus (Strack et al., 1989; Sved et al., 2001). The presence of monosynaptic connections between the RVLM and SPN has been confirmed electrophysiologically (Deuchars et al., 1995, 1997). Other supraspinal neurons, including orexin neurons in the lateral hypothalamus (Llewellyn-Smith et al., 2003; Geerling et al., 2003), have been shown to contribute synapses to SPN. Autonomic nuclei in the thoracic and upper lumbar cord also receive projections from a range of other supraspinal regions, including the locus coeruleus, arcuate nucleus and cortex (Fritschy et al., 1987; Holets et al., 1988; Hurley et al., 1991; Bacon and Smith, 1993; Elias et al., 1998); but whether or not these projections synapse directly on SPN is not known. Spinal neurons that innervate SPN have also been primarily detected by trans-synaptic viral tracing and electrophysiological studies have contributed to their identification. The cell bodies of the intraspinal presympathetic neurons lie within the IML, around the IML in spinal cord laminae V and VII and also in lamina X above the central canal (Cabot et al., 1994; Joshi et al., 1995; Clarke et al., 1998; Deuchars et al., 2001, 2005; Cano et al., 2001; Brooke et al., 2002; Tang et al., 2004). Studies on rats with spinal cord transections have also yielded valuable information about the origin of synaptic inputs to SPN. For more than a week after a transection, the cell bodies and dendrites of SPN undergo substantial morphological changes (Krassioukov and Weaver, 1996; Krenz and Weaver, 1998; Llewellyn-Smith and Weaver, 2001). However, by 2 weeks after transection, the SPN have returned to their original shape; all severed axons arising from neurons above the lesion have disappeared from the lateral horn and the SPN have acquired only a very small number of new synapses (Weaver et al., 1997; Llewellyn-Smith et al., 2006). Comparing observations made on intact cords and cords at 2 weeks posttransection can therefore provide insights into which synapses are likely to come from supraspinal neurons (i.e., those synapses that have disappeared after injury) and which synapses are of intraspinal origin (i.e., those synapses that persist). Sensory neurons cannot be the source of the surviving synapses on SPN in 2-week transected cord because trans-synaptic tracing indicates that interneurons, rather than dorsal horn neurons, are the first spinal population to become virus-labelled after SPN are labelled (see below). 3. Neurotransmitters in synaptic inputs to SPN The axons of supraspinal and intraspinal neurons that innervate SPN contain a diverse array of neurotransmitters; and there is pharmacological, physiological and electrophysiological evidence

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that many of these transmitters directly affect the activity of SPN. Amino acids and neuropeptides occur in synaptic inputs arising from both supraspinal and intraspinal neurons whereas the monoamine innervation of SPN originates exclusively from supraspinal neurons. 3.1. Amino acids The most important neurotransmitters in synaptic inputs to SPN are the amino acids, glutamate and g-aminobutyric acid (GABA); and some SPN synapses also contain glycine (Dampney, 1994). All three of these amino acids produce fast responses, with glutamate being excitatory whereas GABA and glycine are

Fig. 2. Amino acid immunoreactivity in axon terminals that synapse on ChATimmunoreactive SPN in the IML of segment T8 from rat spinal cord. A, An axon terminal showing glutamate (GLU)-immunoreactivity forms a synapse with multiple small asymmetrical active zones (arrowheads) on an SPN dendrite (D) that contains ChAT-immunoreactivity (asterisk). Immunogold staining for GABA in a serial section showed that this terminal was negative for GABA. B, An axon terminal showing GABA-immunoreactivity directly contacts a spine (star) that arises from an SPN dendrite (D) that contains ChAT-immunoreactivity (asterisk). This GABA-positive terminal formed a synapse on the spine in another ultrathin section. Immunogold staining for glutamate in a serial section showed that this terminal was negative for glutamate. Bars, 500 nm. Reproduced from LlewellynSmith and Weaver (2001).

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inhibitory (e.g., Mo and Dun, 1987a,b; Inokuchi et al., 1992a,b; Krupp and Feltz, 1995; Krupp et al., 1997). SPN receive synapses from axons that contain immunoreactivity for glutamate (Fig. 2A), GABA (Fig. 2B) and glycine (Bacon and Smith, 1988; Bogan et al., 1989; Cabot et al., 1992); and quantitative ultrastructural studies have demonstrated that around 95% of the axons that provide synaptic input to SPN contain either glutamate or GABA (Llewellyn-Smith et al., 1992, 1995, 1998). Since the proportion of synapses on identified SPN that contain glycine has not been determined (Cabot et al., 1992), glycine could occur in SPN inputs that do not contain glutamate or GABA. Alternatively, since glycine and GABA are transported into synaptic vesicles by the same transporter protein (Chaudhry et al., 1998; Dumoulin et al., 1999), glycine could co-exist with GABA. Co-existence of GABA and glycine has been demonstrated in synaptic inputs to other types of spinal neurons (Ornung et al., 1994; Maxwell et al., 1995; Todd et al., 1996) and in dorsal horn and commissural spinal interneurons (Todd and Sullivan, 1990; Weber et al., 2007).

Fig. 3. Double immunogold labelling of axon terminals that synapse on rat SPN retrogradely labelled by injection of cholera toxin B subunit into the superior cervical ganglion (SCG). Glutamate-immunoreactivity was localized with 10 nm gold particles; and GABA-immunoreactivity, with 15 nm gold particles. A, A terminal forms a synapse (arrowheads) on a CTB-immunoreactive SPN dendrite (D). Many 10 nm gold particles but few 15 nm gold particles overlie the terminal, indicating that it is immunoreactive for glutamate but not GABA. The deposits of peroxidase reaction product marking the dendrite as CTB-immunoreactive lay outside the area shown in the micrograph. B, A terminal forms a synapse (arrowheads) on an SPN dendrite (D) containing CTB-immunoreactivity (asterisk). Many 15 nm gold particles but few 10 nm gold particles overlie the terminal, indicating that it is immunoreactive for GABA but not glutamate. An adjacent varicosity (star) is immunoreactive for glutamate but not GABA. Bars, 500 nm. Reproduced from Llewellyn-Smith et al. (1998).

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In general, terminals that synapse on SPN contain either glutamate or GABA (Fig. 3). However, after a spinal cord injury, some terminals show immunoreactivity for both of these amino acids (Llewellyn-Smith and Weaver, 2001). The terminals that contain both GABA and glutamate could be GABAergic since glutamate is a precursor for GABA synthesis. Alternatively, these terminals could actually release both amino acids because GABA can be co-stored and co-released with glutamate from synaptic vesicles (Takamori et al., 2000a,b). SPN receive monosynaptic input from neurons in brainstem regions, including the RVLM and caudal raphe´ nuclei (i.e., raphe´ pallidus, raphe´ magnus and raphe´ obscurus) (Bacon et al., 1990; Zagon and Smith, 1993; Deuchars et al., 1995, 1997). Neurochemical markers indicative of amino acid neurotransmission (e.g., immunoreactivity for phosphate activated glutaminase, vesicular glutamate transporter (VGLUT) 2 mRNA and glutamic acid decarboxylase mRNA) occur in spinally projecting brainstem neurons in these areas (Minson et al., 1991; Stornetta et al., 2002, 2004). Furthermore, glutamate- or GABA-immunoreactivity can be found in axons in the IML that have been anterogradely labelled by injections of horseradish peroxidase that fill the ventral medulla (Fig. 4; Llewellyn-Smith et al., 1995). While brainstem neurons provide a considerable proportion of the amino acid input to the IML (at least in mid-thoracic cord, see below), SPN also receive synapses from intraspinal neurons that contain amino acids. Ultrastructural studies on rats with complete spinal cord transections have shown that glutamate- and GABAimmunoreactive axons still synapse on SPN caudal to 7- or 14-day injuries. These are time points at which severed, degenerating supraspinal axons have already been removed from the IML below the lesion and few if any new synapses have formed (LlewellynSmith et al., 1997a; Llewellyn-Smith and Weaver, 2001). Hence, the persisting amino acid input must come from intraspinal neurons. Consistent with this suggestion, SPN in slice preparations, where input from the brain is absent, show ongoing inhibitory and excitatory synaptic activity that is blocked by amino acid antagonists (e.g., Dun and Mo, 1989; Spanswick et al., 1994; Krupp and Feltz, 1995; Spanswick et al., 1998); and the spinal cord contains interneurons with activity that is positively and negatively correlated with sympathetic nerve activity (Chau et al., 2000; Miller et al., 2001; Tang et al., 2003). Glutamic acid decarboxylase has been shown to occur in spinal interneurons

transneuronally labelled from the adrenal medulla and stimulation of spinal regions containing presympathetic neurons evokes inhibitory post-synaptic potentials in SPN that are antagonized by bicuculline (Deuchars et al., 2005). These data confirm that GABAergic interneurons are part of the spinal circuitry regulating the activity of SPN. Excitatory spinal interneurons that release glutamate onto SPN have not yet been localized anatomically. VGLUT2, which packages glutamate into synaptic vesicles, occurs in neurons in spinal regions known to contain sympathetic interneurons (Llewellyn-Smith et al., 2007); but whether these neurons are presynaptic to SPN is not known. The central processes of sensory neurons are another potential source of amino acid-containing synapses caudal to a complete transection. However, there is no evidence for a monosynaptic connection between primary afferents and SPN. Primary afferent terminals target dorsal horn laminae rather than the IML (e.g., Neuhuber, 1982; Grant, 1995) and no degenerating terminals are found in the IML when dorsal roots are cut (Petras and Cummings, 1972). Furthermore, the time course of infection with transneuronal viral tracers indicates that spinal interneurons are immediately antecedent to SPN (Joshi et al., 1995; Clarke et al., 1998; Cano et al., 2001; Tang et al., 2003). These observations imply that sensory neurons do not provide any of the amino acid input to SPN and that sensory information must be conveyed to SPN via spinal interneurons. The finding that virtually all of the axons that synapse on SPN contain at least one fast-acting amino acid transmitter has important implications for understanding how the activity of SPN is regulated. If every synaptic terminal on an SPN contains an amino acid, then any other transmitter in those terminals must coexist with, and is likely to be co-released with, an amino acid. If the appropriate receptors are present post-synaptically, then the SPN is likely to be driven by the amino acid transmitter and the colocalized transmitter(s) will modulate its effects. 3.2. Neuropeptides Axons containing various types of neuropeptide-immunoreactivity have been found in the lateral horn, where the cell bodies and dendrites of SPN are located (Table 1). Ultrastructural studies have demonstrated that terminals immunoreactive for some of these neuropeptides, including substance P, enkephalin, neuropeptide Y,

Fig. 4. Amino acid immunoreactivity in terminals in the IML that were anterogradely labelled from bilateral injections of horseradish peroxidase (HRP) that filled the ventral medulla (see Llewellyn-Smith et al., 1995, for sizes and locations of injection sites). A, A terminal in the IML contains a tetramethylbenzidine (TMB)-tungstate crystal (arrow) as a result of anterograde transport of HRP from the ventral medulla. The terminal is immunoreactive for glutamate and forms a synapse (arrowheads) on a dendrite (D). B, A terminal in the IML contains TMB-tungstate crystals (arrow) as a result of anterograde transport of HRP from the ventral medulla. The terminal is immunoreactive for GABA. Bars, 500 nm. Reproduced from Llewellyn-Smith et al. (1995).

I.J. Llewellyn-Smith / Journal of Chemical Neuroanatomy 38 (2009) 231–239 Table 1 Neuropeptide-immunoreactive axons in spinal regions where SPN are located. Angiotensin II Avian pancreatic polypeptide (APP) Calcitonin gene-related peptide (CGRP) Cholecystokinin (CCK) Cocaine and amphetamine regulated transcript (CART) Corticotropin-releasing factor (CRF) Enkephalin Galanin Neuropeptide Y (NPY) Neurophysin

Neurotensin Nociceptin Orexin Oxytocin Pituitary adenylate cyclase activating polypeptide (PACAP) Somatostatin Substance P Thyrotropin-releasing hormone Vasoactive intestinal peptide (VIP) Vasopressin

thyrotropin-releasing hormone, orexin, cocaine and amphetamine regulated transcript (CART) and pituitary adenylate cyclase activating polypeptide (PACAP), occur in synapses in the IML or on identified SPN (Chiba and Masuko, 1987; Bacon and Smith, 1988; Vera et al., 1990; Llewellyn-Smith et al., 1991; Poulat et al., 1992; Pilowsky et al., 1992; Chiba et al., 1996; Dun et al., 2002; Llewellyn-Smith et al., 2003). Only some of the neuropeptides that occur in synapses on mammalian SPN have been tested for their effects on the activity of these neurons. These include substance P, thyrotropin-releasing hormone, oxytocin and vasopressin, PACAP and orexin (Backman and Henry, 1984; Ma and Dun, 1985; Dun and Mo, 1988; Cammack and Logan, 1996; Kolaj et al., 1997; Lai et al., 1997; Antunes et al., 2001; van den Top et al., 2003). When applied to slice preparations of mammalian spinal cord, all of these neuropeptides excite SPN, either depolarizing them, causing them to fire or increasing their firing rate. Neurons in the brain are the exclusive source of some of the neuropeptide-containing inputs to spinal sympathetic regions, including the oxytocin-, vasopressin- and orexin-immunoreactive innervation of areas where SPN are located. Other neuropeptides, such as substance P, enkephalin and neuropeptide Y, occur not only in supraspinal but also in intraspinal neurons that supply the lateral horn. Substance P is present in brainstem serotonin neurons that innervate the spinal cord (Sasek et al., 1990); preproenkephalin mRNA occurs in a significant fraction of spinally projecting neurons in the RVLM (Stornetta et al., 1999) and immunoreactivity for neuropeptide Y or mRNA for preproneuropeptide Y have been identified in medullospinal neurons (Minson et al., 1994; Stornetta et al., 1999). Nevertheless, axons immunoreactive for substance P, enkephalin or neuropeptide Y persist in the IML caudal to a complete transection and form synapses on SPN (Davis and Cabot, 1984; Romagnano et al., 1987; Cassam et al., 1997; LlewellynSmith and Weaver, 2004). The survival of synaptic terminals immunoreactive for substance P, enkephalin or neuropeptide Y caudal to a complete transection indicates that spinal interneurons must be the source of a proportion of these neuropeptidecontaining inputs to SPN. The neuropeptide Y-immunoreactive axons that are present after transection are likely to originate from neurons in laminae V and VII that express neuropeptide Y mRNA (Minson et al., 2001). The intraspinal enkephalin innervation may come from neurons in lamina X above the central canal that can be detected after high glutaraldehyde fixation (Llewellyn-Smith et al., 2005), a suggestion that is consistent with lamina X neurons being labelled by trans-synaptic viral tracers immediately after SPN are labelled (Cano et al., 2001; Tang et al., 2004). The location of the neurons that supply the intraspinal substance P-immunoreactive input to SPN has yet to be identified. 3.3. Monoamines Application of noradrenaline and adrenaline to mammalian spinal cord slices causes both excitatory and inhibitory responses in SPN (Kadzielawa, 1983; Yoshimura et al., 1986; Miyazaki et al.,

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1989; Lewis and Coote, 1990a). The excitatory responses are mediated by alpha 1 receptors while alpha 2 receptors mediate the inhibitory responses (Inokuchi and Yoshimura, 1992). Light microscopic studies have shown that dense networks of axons immunoreactive for catecholamine synthetic enzymes (i.e., tyrosine hydroxylase and phenylethanolamine N-methyltransferase) surround SPN at all levels of their distribution (e.g., Minson et al., 2002; Llewellyn-Smith et al., 2006); and synapses from axons immunoreactive for these enzymes have been identified on SPN (Milner et al., 1988; Bernstein-Goral and Bohn, 1989; LlewellynSmith et al., 2006). All of this catecholamine input to SPN originates supraspinally, a conclusion that is confirmed by the disappearance of axons with immunoreactivity for catecholamine synthetic enzymes caudal to a transection (Llewellyn-Smith et al., 2006). Major sources of catecholamine input to SPN are the C1 adrenergic neurons of the rostral ventrolateral medulla and the noradrenergic neurons of the A5 group (Jansen et al., 1995). Neurons that use serotonin as a neurotransmitter also directly innervate SPN. Serotonin increases sympathetic nerve activity and excites SPN (Ma and Dun, 1986; Lewis and Coote, 1990b; Lewis et al., 1993; Pickering et al., 1994; Madden and Morrison, 2006, 2008). However, when 5-HT is injected over the central canal in spinal cord slices, a few SPN hyperpolarize before they depolarize (Pickering et al., 1994). Light microscopic studies have shown that SPN are surrounded by dense plexuses of serotonergic axons (revealed by either immunoreactivity for 5-HT or the serotonin transporter) and SPN retrogradely labelled from the superior cervical ganglion or adrenal medulla receive synapses from terminals that contain 5-HTimmunoreactivity (Bacon and Smith, 1988; Vera et al., 1990; Jensen et al., 1995; Llewellyn-Smith et al., 2006). The main source of serotonergic input to SPN is the medullary raphe´ nuclei (Loewy and McKeller, 1981; Bowker et al., 1982; Jansen et al., 1995). Although occasional 5-HT-immunoreactive neurons have been detected in the spinal cord (Newton et al., 1986), studies on completely transected cord confirm that the serotonergic input to SPN arises only from supraspinal neurons (Llewellyn-Smith et al., 2006). 4. Transporters and receptors in synaptic inputs to SPN Transporters that carry neurotransmitters into vesicles and receptors that bind neurotransmitters also occur in terminals that synapse in the IML and/or on SPN. Consistent with the extensive glutamate-immunoreactive innervation of SPN, axons containing all three vesicular glutamate transporters occur in spinal autonomic regions. VGLUT1-immunoreactive innervation is sparse whereas VGLUT2-immunoreactive axons are abundant. The VGLUT2-containing terminals closely appose SPN retrogradely labelled from a variety of peripheral targets and form synapses on SPN containing Fluorogold as a result of transport of this tracer from intraperitoneal injections (Llewellyn-Smith et al., 2007). VGLUT3 has been identified in axons and in synaptic terminals in the lateral horn (Nakamura et al., 2004; Stornetta et al., 2005). However, the precise complement of amino acids that occur in this VGLUT3-immunoreactive input remains unresolved. Consistent with observations that VGLUT3 expression is not restricted to glutamatergic neurons (Fremeau, Jr. et al., 2002; Somogyi et al., 2004; Herzog et al., 2004; Takamori, 2006), spinally projecting VGLUT3 neurons in medullary raphe´ nuclei express the GABAergic marker, glutamic acid decarboxylase 67; and VGLUT3-immunoreactive terminals in the IML also contain GABA-immunoreactivity (Stornetta et al., 2005). Post-embedding immunogold double labelling would be a useful for determining whether the VGLUT3/GABA terminals also contain glutamate. In addition to axons containing VGLUTs, axons immunoreactive for vesicular monoamine transporter 2 occur in the IML and

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originate from the adrenergic C1 neurons of the RVLM (Sevigny et al., 2008). These axons closely appose SPN at the light microscope level and presumably would be in synaptic contact with them. SPN are known to receive synapses from terminals that contain the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase (Milner et al., 1988). Immunoreactivity for the adenosine 2 A receptor occurs in a dense network of axons around SPN and immuno-positive axons form synapses in the IML (Brooke et al., 2004). Although the postsynaptic targets of these synapses were not conclusively identified, at least some are likely to occur on SPN. Activation of pre-synaptic adenosine 2 A receptors increased the amplitude of inhibitory post-synaptic potentials in SPN and this effect was blocked by a specific antagonist for this receptor (Brooke et al., 2004). The neurokinin 1 receptor also occurs in inputs to SPN. Terminals containing immunoreactivity for this receptor synapse on dendrites in the IML that contain the receptor. The postsynaptic neurons must be SPN because they are the only IML neurons showing receptor immunoreactivity (Llewellyn-Smith et al., 1997b). Spinal neurokinin 1 receptors have been implicated in the control of vasomotor sympathetic outflow (e.g., Hassessian et al., 1988; Solomon et al., 1999; Cloutier et al., 2006) and these receptors have been localized on hypotension-sensitive SPN (Burman et al., 2001). 5. Rostrocaudal differences in synaptic input to SPN Over the past 30 years, evidence has been accumulating that the central control of different sympathetic outflows is not uniform. Consequently, different functional groups of SPN respond in different ways to the same physiological challenge (reviewed by Morrison, 2001). These differentiated physiological responses are likely to reflect differences in the innervation of different functional classes of SPN by the various groups of neurochemically identified axons discussed above. The rostrocaudal differences in the locations of different functional populations of SPN (see above) probably dictate the rostrocaudal variations that have been observed in the innervation of the IML. Light microscopic studies have shown that nerve fibers immunoreactive for 5-HT, substance P, somatostatin, oxytocin, neurotensin or neurophysin show a non-uniform rostrocaudal distribution in autonomic regions of cat thoracolumbar cord, as do serotonergic axons in the rabbit IML (Krukoff et al., 1985; Jensen et al., 1995). Hypotension-sensitive SPN also show rostrocaudal heterogeneity in their innervation by some types of neurochemically defined axons, including those containing immunoreactivity for neuropeptide Y, phenylethanolamine N-methyltransferase or galanin (Minson et al., 2002). Furthermore, in preference to other neurons in the IML, somatostatin-immunoreactive axons target sympathoadrenal SPN (Holets and Elde, 1982). There are only a few ultrastructural studies that have quantified immunocytochemically defined synaptic inputs to SPN. These studies support the implication of the light microscopic data that different groups of SPN vary with respect to the proportion of synaptic input that they receive from individual classes of neurotransmitter-defined axons. Glutamate-immunoreactive terminals provide about 70% of the synaptic input to sympathoadrenal SPN whereas only half of the input to SPN supplying the superior cervical ganglion is glutamatergic (Llewellyn-Smith et al., 1992, 1998). The proportion of input to these two groups of SPN that is GABA-immunoreactive is about 30% and about 50%, respectively (Bacon and Smith, 1988; Llewellyn-Smith et al., 1998), as would be expected if all the terminals that synapse on SPN contain an amino acid (see above). SPN that project to the major pelvic ganglion and are involved in regulating the pelvic viscera receive more than half of their synaptic input from

Fig. 5. Enkephalin (ENK)-containing synaptic input to SPN. An ENK-immunoreactive terminal forms a synapse on the dendrite (D) of an SPN that has been retrogradely labelled with CTB from the major pelvic ganglion (MPG). Heavy deposits of amorphous diaminobenzidine reaction product are associated with large granular vesicles (asterisks) in the ENK-positive terminal. CTB-immunoreactivity in the dendrite was visualized with a TMB-tungstate reaction, which produces crystals (star). Bar, 500 nm.

terminals that are immunoreactive for enkephalin (Fig. 5; Llewellyn-Smith et al., 2005). However, whether an equivalently large proportion of the axons that synapse on other groups of SPN contains enkephalin remains to be determined. In addition to rostrocaudal differences in the types of neurotransmitter-identified axons that innervate SPN, it is likely that SPN at different rostrocaudal levels of the cord differ in the proportion of input that they receive from supraspinal versus intraspinal sources. In thoracic segment 8, rat SPN identified by immunoreactivity for choline acetyltransferase, the enzyme that synthesizes acetylcholine, were found to be profoundly denervated at 14 days after a complete spinal cord transection. About half of the synapses on the cell bodies of these neurons and 70% of the synapses on their dendrites had degenerated and disappeared (Llewellyn-Smith and Weaver, 2001). Since most synapses occur on the dendrites of SPN, these data indicate that significantly more than half of the synaptic input to mid-thoracic SPN comes from neurons above the lesion, likely supraspinal neurons. In contrast, in segments T12-L2, a complete transection appeared to leave almost untouched the innervation of SPN that projected to the major pelvic ganglion, which contains post-ganglionic neurons innervating the pelvic viscera (Llewellyn-Smith et al., 2005). In intact cord, 52% of the terminals that synapsed on the cell bodies of retrogradely labelled SPN in the IML contained enkephalinimmunoreactivity whereas the figure was 65% for cell bodies of SPN in the IML in 2-week transected cord. Hence, in contrast to SPN in T8, there was not a substantial loss of somatic synapses on pelvic visceral SPN as a result of the transection. These quantitative data on intact and transected cord imply that spinal interneurons are the source of most of the synapses on SPN that project to the major pelvic ganglion and therefore that only a small proportion of their input comes from supraspinal neurons. At least some of the interneuronal synapses on pelvic visceral SPN are likely to convey the sensory information that is important for regulating the function of pelvic organs. These interneurons may also integrate sensory and supraspinal information since both types of terminals occur in lamina X, where sympathetic interneurons are known to occur. Similar ultrastructural studies after retrograde labelling from other peripheral targets are needed to clarify just how variable are the proportions of supraspinal and intraspinal input to different groups of SPN.

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