Effects of spinal cord injury on synaptic inputs to sympathetic preganglionic neurons

L.C. Weaver and C. Polosa (Eds.) Progress in Brain Research, Vol. 152 ISSN 0079-6123 Copyright r 2006 Elsevier B.V. All rights reserved

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Effects of spinal cord injury on synaptic inputs to sympathetic preganglionic neurons Ida J. Llewellyn-Smith1,, Lynne C. Weaver2 and Janet R. Keast3 1 Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford Park, SA 5042, Australia Spinal Cord Injury Laboratory, BioTherapeutics Research Group, Robarts Research Institute, London, ON, Canada 3 Pain Management Research Institute, University of Sydney at Royal North Shore Hospital, St Leonards, NSW, Australia

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Abstract: Spinal cord injuries often lead to disorders in the control of autonomic function, including problems with blood pressure regulation, voiding, defecation and reproduction. The root cause of all these problems is the destruction of brain pathways that control spinal autonomic neurons lying caudal to the lesion. Changes induced by spinal cord injuries have been most extensively studied in sympathetic preganglionic neurons, cholinergic autonomic neurons with cell bodies in the lateral horn of thoracic and upper lumbar spinal cord that are the sources of sympathetic outflow. After an injury, sympathetic preganglionic neurons in mid-thoracic cord show plastic changes in their morphology. There is also extensive loss of synaptic input from the brain, leaving these neurons profoundly denervated in the acute phase of injury. Our recent studies on sympathetic preganglionic neurons in lower thoracic and upper lumbar cord that regulate the pelvic viscera suggest that these neurons are not so severely affected by spinal cord injury. Spinal interneurons appear to contribute most of the synaptic input to these neurons so that injury does not result in extensive denervation. Since intraspinal circuitry remains intact after injury, drug treatments targeting these neurons should help to normalize sympathetically mediated pelvic visceral reflexes. Furthermore, sympathetic pelvic visceral control may be more easily restored after an injury because it is less dependent on the re-establishment of direct synaptic input from regrowing brain axons.

and severity of these problems is dependent on the level and completeness of the injury. Two cardiovascular consequences of spinal cord injury are resting or postural hypotension and autonomic dysreflexia, a condition in which strokes or death can occur when noxious or innocuous sensory stimuli entering the cord below the injury reflexly induce episodes of hypertension. Spinal cord injury can also produce a variety of difficulties that impair the voiding of urine, including detrusor hyperreflexia, detrusor sphincter dyssynergia and detrusor areflexia. Fecal incontinence and constipation are also outcomes of spinal cord injury; and inability to achieve psychogenic and

Every year, tens of thousands of people worldwide suffer a spinal cord injury, often with devastating consequences. Injured people can lose mobility because they become partially or totally paralyzed and experience persistent pain and spasticity. As this volume documents, their quality of life can also be significantly damaged by injury-induced disorders in the control of autonomic function, including problems with blood pressure regulation, voiding, defecation and reproduction. The presence Corresponding author. Tel.: +61-8-8204-4456; Fax: +61-8-8204-5268; E-mail: Ida.Llewellyn-Smith@flinders.edu.au DOI: 10.1016/S0079-6123(05)52001-6

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reflexogenic erections and ejaculatory dysfunction in men and failure of vaginal lubrication in women have a significant impact on sexual life after injury. The root cause of all these autonomic sequelae of spinal cord injury is the disruption of the brain pathways that control spinal autonomic neurons. Subsequently, when axons conveying supraspinal drive are dying or dead, neuronal circuits caudal to the injury are able to reorganize. Primary afferent neurons sprout (Christensen and Hulsebosch, 1997; Krenz and Weaver, 1998b; Krenz et al., 1999; Wong et al., 2000; Weaver et al., 2001; Ondarza et al., 2003) and spinal autonomic interneurons can become key regulators of some of the information that is conveyed from the spinal cord to the periphery (see Schramm, this volume). Changes also occur in the synaptic circuitry controlling sympathetic preganglionic neurons that have axons providing central drive to all sympathetic postganglionic neuron in paravertebral and prevertebral ganglia and to chromaffin cells in the adrenal medulla.

dorsolateral funiculus in the white matter; sympathetic preganglionic neurons in the dorsolateral funiculus are more common in the rostral than the caudal thoracic cord (Strack et al., 1988). A small proportion of sympathetic preganglionic neurons are associated with the bundles of dendrites that course mediolaterally between the intermediolateral cell column and central canal. These sympathetic preganglionic neurons have spindle-shaped cell bodies and comprise the intercalated nucleus. The somata and dendrites of the sympathetic preganglionic neurons in the intercalated nucleus are oriented parallel to the dendritic bundles with which they are associated. A final concentration of sympathetic preganglionic cell bodies, which are usually fusiform in shape, occurs in the central autonomic area dorsal to the central canal. Somata occupying this position are most frequently encountered at the caudal end of the sympathetic preganglionic neuron distribution, i.e., in the lowest thoracic and upper lumbar segments.

Morphological changes after spinal cord injury Location and morphology of sympathetic preganglionic neurons Sympathetic preganglionic neurons are small- to medium-sized cholinergic neurons and their cell bodies are located in the thoracic and upper lumbar spinal cord in four distinct subnuclei within the lateral horn (Cabot, 1990). The majority of sympathetic preganglionic somata occur in the intermediolateral cell column, which lies at the border between the grey and white matter of the spinal cord. In the intermediolateral cell column, the cell bodies of sympathetic preganglionic neurons are spindle-shaped or fusiform and occur in groups or ‘‘nests’’ that are spaced at short intervals along the grey–white boundary. Most of the dendrites of the neurons in the intermediolateral cell column run rostrally or caudally and can be hundreds of micrometers long. In addition, some sympathetic preganglionic neurons in the intermediolateral cell column have dendrites that are oriented mediolaterally, traveling either into the dorsolateral funiculus or toward the central canal. Other sympathetic preganglionic somata are situated in the

Spinal cord injury can evoke significant changes in sympathetic preganglionic neurons, as well as depriving them of input from the brain. While neurons at the site of a cord injury can be killed, sympathetic preganglionic neurons that lie distant from the lesion site may also be affected, at least in the acute phase of injury. Within 3 days of a complete spinal cord transection at thoracic segment 4/5, the dendrites of sympathoadrenal preganglionic neurons in the intermediolateral cell column of mid-thoracic cord have retracted to about onethird of their original length and the diameter of their cell bodies has decreased to about 60% of that in intact cord (Llewellyn-Smith and Weaver, 2001). This reduction in soma size and dendrite length is less pronounced 7 days after transection; and, by 14 days post-operatively, the sympathetic preganglionic neurons and their dendrites are not significantly different in size from those in intact cord (Krassioukov and Weaver, 1996; Krenz and Weaver, 1998a). The shrinkage and regrowth of sympathetic preganglionic somata and dendrites correlate with the degeneration and clearance from

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the intermediolateral cell column of axons detached from their cell bodies by the transection. Many degenerating profiles of severed axons can be seen ultrastructurally at 3 days after injury, but virtually none are present at 14 days (Weaver et al., 1997). Although new synapses form on sympathoadrenal preganglionic neurons after a spinal cord injury (Weaver et al., 1997), reinnervation by axons of spinal neurons is unlikely to be the spur for regrowth of dendrites. Even at 14 days after a complete transection, sympathetic preganglionic neurons in the mid-thoracic cord are profoundly denervated. The density of synapses on their cell bodies has been cut to half of that in intact cord and their axodendritic input is reduced by 70% (Llewellyn-Smith and Weaver, 2001). Whether or not sympathetic preganglionic neurons continue to be reinnervated after 2 weeks of injury has not been studied ultrastructurally. However, anastomosing networks of axons immunoreactive for growthassociated protein 43, a marker for developing and regenerating axons, are present at least as long as 6 weeks after spinal cord injury (Cassam et al., 1999). Continuing reorganization of synaptic circuitry controlling the activity of sympathetic preganglionic neurons may underlie the increasing severity of attacks of autonomic dysreflexia (Maiorov et al., 1997a, b; Marsh and Weaver, 2004).

Innervation of sympathetic preganglionic neurons in intact and injured cord In intact cord, sympathetic preganglionic neurons are innervated by both supraspinal and intraspinal neurons. Virus tracing studies have been particularly useful for revealing the locations of presympathetic neurons, i.e., those that are directly antecedent to sympathetic preganglionic neurons and are likely to be involved in regulating their activity. Supraspinal inputs to sympathetic preganglionic neurons come from five main brain regions, including the rostral ventrolateral medulla, 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). Spinal neurons that project to sympathetic preganglionic neurons occur within

the intermediolateral cell column, around it in spinal cord laminae V and VII and in lamina X dorsal to the central canal (Cabot et al., 1994; Joshi et al., 1995; Clarke et al., 1998; Cano et al., 2001; Deuchars et al., 2001; Tang et al., 2004). The axons of presympathetic supraspinal and intraspinal neurons contain a diverse array of neurotransmitters, many of which have been shown to have direct effects on sympathetic preganglionic neurons. Neurons in the brain are probably the exclusive source of monoamine innervation, whereas both supraspinal and intraspinal neurons contribute axons containing amino acids and neuropeptides.

Amino acids Glutamate, g-aminobutyric acid (GABA) and glycine, all produce fast synaptic responses in sympathetic preganglionic neurons (e.g., Mo and Dun, 1987a, b; Inokuchi et al., 1992a, b; Krupp and Feltz, 1995; Krupp et al., 1997) and are considered the main fast-acting transmitters regulating their activity (Dampney, 1994). Axons immunoreactive for these amino acids synapse on sympathetic preganglionic neurons (Bacon and Smith, 1988; Bogan et al., 1989; Cabot et al., 1992) and quantitative ultrastructural studies have demonstrated that synaptic vesicles containing at least one type of amino acid are present in virtually all of the axons that provide input to these neurons (Llewellyn-Smith et al., 1992, 1995b, 1998). Brainstem neurons innervate sympathetic preganglionic neurons monosynaptically (Zagon and Smith, 1993; Deuchars et al., 1995, 1997); and these spinally projecting neurons contain markers for glutamate and GABA axons, including phosphate activated glutaminase, vesicular glutamate transporter 2 and glutamic acid decarboxylase (Minson et al., 1991; Stornetta et al., 2002, 2004). Furthermore, immunoreactivity for glutamate or GABA occurs in boutons in the intermediolateral cell column that have been anterogradely labeled from the medulla (Llewellyn-Smith et al., 1995b). Although neurons in the brain provide many of the glutamate- and GABA-immunoreactive axons in the intermediolateral cell column, spinal cord injury does not deprive sympathetic preganglionic neurons of amino acid-containing inputs. Ultrastructural

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studies at times when severed supraspinal axons are degenerating or have just been removed from below a lesion show that glutamate- and GABA-immunoreactive synaptic contacts persist on sympathetic preganglionic neurons caudal to either 3- or 7-day complete spinal cord transections (Llewellyn-Smith et al., 1997; Llewellyn-Smith and Weaver, 2001). Hence, intraspinal neurons as well as supraspinal neurons provide amino acid-containing inputs to sympathetic preganglionic neurons.

Monoamines Adrenaline and noradrenaline evoke both excitatory and inhibitory responses in sympathetic preganglionic neurons (Coote et al., 1981; Kadzielawa, 1983; Ma and Dun, 1985a; Miyazaki et al., 1989; Lewis and Coote, 1990a). At the light microscope level, sympathetic preganglionic neurons at all levels of intact cord are surrounded by networks of nerve fibers immunoreactive for enzymes of catecholamine synthesis, such as tyrosine hydroxylase (Fig. 1A) and phenylethanolamine N-methyltransferase (Fig. 1B). In intact cord, phenylethanolamine N-methyltransferase-immunoreactive axons have been confirmed to synapse on sympathetic preganglionic neurons at the electron microscope level (Milner et al., 1988; BernsteinGoral and Bohn, 1989) and we have found that sympathetic preganglionic neurons receive synapses from axons with immunoreactivity for tyrosine hydroxylase (Fig. 2A). All of this catecholamine input to sympathetic preganglionic neurons probably originates in the brain (see below). Major sources are the C1 adrenergic neurons of the rostral ventrolateral medulla and the noradrenergic neurons of the A5 group (Jansen et al., 1995). The supraspinal origin of catecholamine input is supported by the disappearance of immunoreactivity for catecholamine-synthesizing enzymes caudal to a complete spinal cord transection. Staining for tyrosine hydroxylase and phenylethanolamine N-methyltransferase is substantially reduced at 2 weeks post-operatively (Figs. 1C and D) and is absent by 11 weeks (Figs. 1E and F). Interestingly, after spinal cord injury, some neurons in the intermediate grey of the spinal cord become

immunoreactive for dopamine b-hydroxylase (Cassam et al., 1997), the enzyme that produces noradrenaline from dopamine; and we have described axons caudal to a 2-week transection that contain tyrosine hydroxylase and form synapses in the intermediolateral cell column (Fig. 2B; Llewellyn-Smith et al., 1995a). Hence, the catecholamine enzyme-immunoreactive fibers present at 2 weeks may arise from these neurons. Some immunoreactivity may also be present in the nonterminal portions of severed axons since, at 2 weeks, degenerating terminals cannot be found in the intermediolateral cell column at the ultrastructural level (Weaver et al., 1997). Determining whether the spinal neurons that express catecholamine enzymes at 2 weeks after transection synthesize dopamine, adrenaline or noradrenaline will require different experimental strategies, such as fluorescence histochemistry, or multiple-label immunofluorescence for investigating coexistence of relevant synthetic enzymes or amine transporters. A more detailed anatomical analysis of 11-week transected cord will also be needed to ascertain whether there are any enzyme-immunoreactive fibers present at the chronic stage of injury. Pharmacological and physiological studies indicate that, in general, serotonin (5-hydroxytryptamine (5-HT)) has a sympathoexcitatory action on sympathetic nerve activity and directly on sympathetic preganglionic neurons (e.g., Ma and Dun, 1986; Yusof and Coote, 1988; Lewis and Coote, 1990b; Pickering et al., 1994). Serotonergic axons, marked by either immunoreactivity for 5-HT or the serotonin transporter, also form a dense plexus of axons around sympathetic preganglionic neurons (Figs. 3A and C) and synapses by 5-HTimmunoreactive axons have been demonstrated on sympathetic preganglionic neurons that project to the superior cervical ganglion and adrenal medulla (Bacon and Smith, 1988; Vera et al., 1990; Jensen et al., 1995). Retrograde and viral tracing studies indicate that the serotonergic axons surrounding sympathetic preganglionic neurons arise from raphe neurons, mainly those in the medullary nuclei (Loewy and McKellar, 1981; Bowker et al., 1982; Jansen et al., 1995). Very rare 5-HT-immunoreactive neurons have been detected in the spinal cord (Newton et al., 1986). However, these are unlikely

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Fig. 1. Axons containing immunoreactivity for the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), disappear from the intermediolateral cell column caudal to a complete spinal cord transection (TX). (A, B) Intact cord. (C, D) 2-week transected cord. (E, F) 11-week transected cord. (A, C, E) Stained for tyrosine hydroxylase and (B, D, F) stained for phenylethanolamine N-methyltransferase (PNMT). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. All micrographs show the intermediolateral cell column in thoracic segment 6. Bars, 100 mm.

to nnervate sympathetic preganglionic neurons because 5-HT-immunoreactive and serotonin transporter-immunoreactive axons are absent from autonomic areas caudal to a 2-week transection (Figs. 3B and D). Hence, severed serotonergic axons disappear from the cord before axons that are immunoreactive for catecholamine enzymes.

Neuropeptides Axons containing a substantial array of neuropeptides have been demonstrated by light microscopy in regions of the cord where the cell bodies and dendrites of sympathetic preganglionic neurons are located (Table 1) and a number of these

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Fig. 2. Axons immunoreactive for tyrosine hydroxylase form synapses in intact and transected cord. (A) In the intermediolateral cell column of intact cord, a tyrosine hydroxylase-immunoreactive varicosity (TH) forms a synapse (arrowhead) on a dendrite that contains cholera toxin B subunit (CTB) retrogradely transported from the adrenal medulla. An adjacent non-immunoreactive varicosity (asterisk) synapses (arrowheads) on the same dendrite. (B) In the intermediolateral cell column of thoracic segment 8, a tyrosine hydroxylase-immunoreactive varicosity (TH) forms a synapse (arrowheads) on a dendrite 2 weeks after a complete spinal cord transection (TX) in caudal thoracic segment 4/rostral thoracic segment 5. An adjacent non-immunoreactive varicosity (asterisk) synapses (arrowhead) on the same dendrite. Bars, 500 nm.

Fig. 3. Axons containing the serotonergic markers, 5-HT or the serotonin transporter, disappear from the intermediolateral cell column caudal to a complete spinal cord transection (TX). (A, C) Intact cord. (B, D) 2-week transected cord. (A, B) Stained for 5-HT and (C, D) stained for the serotonin transporter (SERT). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. All micrographs show the intermediolateral cell column in thoracic segment 6. Bar in (D) applies to (A—D), 100 mm.

17 Table 1. Neuropeptide immunoreactivity identified in axons in autonomic regions of the thoracic and upper lumbar spinal cord Angiotensin II Avian pancreatic polypeptide (APP) Calcitonin gene-related peptide (CGRP) Cholecystokinin (CCK) Cocaine and amphetamine regulated transcript (CART)a Corticotropin releasing factor (CRF) Enkephalina Galanin Neuropeptide Y (NPY)a Neurophysin

Neurotensin Nociceptin Orexina Oxytocin Pituitary adenylate cyclase activating polypeptide (PACAP)a Somatostatin Substance Pa Thyrotropin releasing hormone Vasoactive intestinal peptide (VIP) Vasopressin

a Axons containing these neuropeptides have been shown to form synapses either in the intermediolateral cell column or on identified sympathetic preganglionic neurons.

have been shown to synapse on sympathetic preganglionic neurons (Bacon and Smith, 1988; Vera et al., 1990; Llewellyn-Smith et al., 1991; Pilowsky et al., 1992). When applied to sympathetic preganglionic neurons, many of these neuropeptides evoke synaptic responses (e.g., Ma and Dun, 1985b; Dun and Mo, 1988; Kolaj et al., 1997; Lai et al., 1997; Antunes et al., 2001; van den Top et al., 2003). Some of the neuropeptide-immunoreactive axons supplying sympathetic preganglionic neurons arise exclusively from neurons in the brain, including those containing oxytocin, vasopressin and orexin. However, other neuropeptides, such as substance P, enkephalin and neuropeptide Y, occur in autonomic areas of intact and transected cord (Figs. 4–6), suggesting that both supraspinal and intraspinal neurons supply the lateral horn. Substance P is co-localized with serotonin in brainstem neurons that innervate the spinal cord (Sasek et al., 1990). A large subset of spinally projecting cardiovascular neurons in the medulla contain preproenkephalin mRNA (Stornetta et al., 1999) and immunoreactivity for neuropeptide Y and mRNA for preproneuropeptide Y have been identified in medullospinal neurons (Minson et al., 1994; Stornetta et al., 1999). Nevertheless, complete spinal cord transection does not destroy all varicose axons in the intermediolateral cell column immunoreactive for substance P, enkephalin or neuropeptide Y (Davis and Cabot, 1984; Romagnano et al., 1987; Cassam et al., 1997) and we have shown that, caudal to a 7day complete transection, axons containing each of these neuropeptides form synapses on choline

acetyltransferase-immunoreactive (i.e., cholinergic) neurons in the intermediolateral cell column (Llewellyn-Smith and Weaver, 2004). Hence, some of the axons containing these neuropeptides come from spinal interneurons to innervate the cholinergic sympathetic preganglionic neurons. The source of the neuropeptide Y-immunoreactive axons that persist in the lateral horn may be the neurons in laminae V and VII in intact cord that express neuropeptide Y mRNA (Minson et al., 2001). The intraspinal enkephalin innervation of autonomic regions may arise from small enkephalin-immunoreactive neurons in lamina X that we have detected in sections from intact cord fixed with high concentrations of glutaraldehyde (Llewellyn-Smith and Keast, unpublished observations), as neurons in this location are known to communicate with sympathetic preganglionic neurons (Cano et al., 2001; Tang et al., 2004). The cell bodies of origin of the intraspinal substance P input have yet to be defined.

Rostrocaudal differences in sympathetic preganglionic neurons and their innervation The sympathetic nervous system was originally thought to act in an undifferentiated way to allow an animal to respond appropriately to lifethreatening situations. However, over the past two decades, it has become increasingly clear that central control of sympathetic outflow is differential, permitting specific functional groups of sympathetic preganglionic neurons to respond in different ways

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Fig. 4. Caudal to a complete spinal cord transection (TX), axons containing the neuropeptides, substance P, enkephalin and neuropeptide Y, are still present in the intermediolateral cell column of mid-thoracic spinal cord segments. (A, C, E) Intact cord. (B, D, F) 2-week transected cord. (A, B) Stained for substance P (SP), (C, D) stained for enkephalin (ENK) and (E, F) stained for neuropeptide Y (NPY). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. Micrographs show the intermediolateral cell column from thoracic segments 7, 8 or 9. Bars, 100 mm.

to the same homeostatic challenge (reviewed by Morrison, 2001). Differences in the spatial arrangement of sympathetic preganglionic neurons and in their innervation are likely to be the anatomical basis for these differentiated physiological responses. The differences in the innervation of sympathetic preganglionic neurons suggest that

the outcomes of spinal cord injury will vary depending on the functional group of sympathetic preganglionic neurons that are deprived of their supraspinal input. Sympathetic preganglionic neurons are topographically organized along the rostrocaudal axis of the spinal cord (Strack et al., 1988). Preganglionic

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Fig. 5. Caudal to a complete spinal cord transection (TX), axons containing the neuropeptides, substance P, enkephalin and neuropeptide Y, are still present in the intermediolateral cell column of lower thoracic and upper lumbar segments. (A, C, E) Intact cord. (B, D, F) 2-week transected cord. (A, B) Stained for substance P (SP), (C, D) stained for enkephalin (ENK) and (E, F) stained for neuropeptide Y (NPY). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. Micrographs show the intermediolateral cell column from thoracic segment 13 or lumbar segment 1. Bars, 100 mm.

neurons in the rostral thoracic cord supply rostral sympathetic ganglia (e.g., the superior cervical ganglion and the stellate ganglion) and participate in the regulation of targets in the upper body, like pupils, salivary glands and the heart. The midthoracic cord contains sympathetic preganglionic neurons that project to the celiac ganglion as part of the circuitry controlling mesenteric vasculature, gut motility and gut secretion as well as sympathetic preganglionic neurons projecting to the adrenal medulla to regulate release of noradrenaline

and adrenaline from chromaffin cells. The caudal end of the range includes sympathetic preganglionic neurons involved in regulating the activity of organs in the lower body, like the urinary bladder, lower bowel and reproductive organs. Despite this general topographical organization, the distributions of sympathetic preganglionic neurons projecting to different target ganglia or adrenal chromaffin cells overlap so that sympathetic preganglionic neurons of different functions are intermixed within each spinal segment. For example,

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Fig. 6. Caudal to a complete spinal cord transection (TX), axons containing the neuropeptides, substance P, enkephalin and neuropeptide Y, are still present in the central autonomic area of upper lumbar segments. (A, C, E) Intact cord. (B, D, F) 2-week transected cord. (A, B) Stained for substance P (SP), (C, D) stained for enkephalin (ENK) and (F) stained for neuropeptide Y (NPY). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. All micrographs show the central autonomic area in lumbar segment 2. cc, central canal. Bars, 100 mm.

in the rat, thoracic segment 6 contains a mixture of sympathetic preganglionic neurons that send axons to the superior cervical ganglion, stellate ganglion, celiac ganglion or adrenal medulla. Although not very well studied, rostrocaudal differences in the innervation of sympathetic preganglionic neurons parallel the target-based

rostrocaudal arrangement of their cell bodies. In autonomic areas of cat thoracolumbar cord, nerve fibers immunoreactive for 5-HT, substance P, somatostatin, oxytocin, neurotensin or neurophysin show a non-uniform rostrocaudal distribution (Krukoff et al., 1985) as do 5-HT-immunoreactive axons in rabbit intermediolateral cell column

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(Jensen et al., 1995). In rats, oxytocin-immunoreactive axons closely appose sympathetic preganglionic neurons retrogradely labeled from the cervical sympathetic trunk; but sympathoadrenal preganglionic neurons are not innervated by oxytocin fibers (Holets and Elde, 1982; Appel and Elde, 1988). Inputs to choline acetyltransferaseimmunoreactive sympathetic preganglionic neurons that express Fos in response to hypotension also show a heterogenous pattern of innervation by some types of axons (Minson et al., 2002). Although apposing almost all Fos-positive sympathetic preganglionic neurons in upper and middle thoracic cord, neuropeptide Y or phenylethanolamine N-methyltransferase-immunoreactive axons avoid significant proportions of these neurons in lower thoracic segments. More than half of the hypotension-sensitive sympathetic preganglionic neurons in the middle and lower thoracic cord lacked appositions from galaninimmunoreactive axons, whereas some choline acetyltransferase-positive, Fos-negative neurons in the lumbar cord lay in dense baskets of galaninpositive fibers. Recently, we have examined enkephalinimmunoreactive inputs to sympathetic preganglionic neurons retrogradely labeled with cholera toxin B subunit from the major pelvic ganglion (Llewellyn-Smith et al., 2005), which contains sympathetic and parasympathetic preganglionic neurons innervating the urinary bladder, lower bowel and reproductive organs. This work has revealed a striking difference between the reaction to spinal cord injury of these neurons, which have somata in thoracic segment 12 to lumbar segment 2, and more rostral sympathetic preganglionic neurons. In contrast to choline acetyltransferaseimmunoreactive sympathetic preganglionic neurons in thoracic segment 8 (Llewellyn-Smith and Weaver, 2001), sympathetic preganglionic neurons that are in circuits controlling pelvic viscera appear to retain most of their innervation after a complete spinal cord transection. In intact cord, we found that sympathetic preganglionic neurons projecting to the major pelvic ganglion from the intermediolateral cell column, the intercalated nucleus and central autonomic area were surrounded by very dense baskets of enkephalin-immunoreactive

axons at the light microscopic level (Figs. 5C, and 6C). Similarly, dense baskets were present around retrogradely labeled neurons in cords at 2 and 11 weeks after transection (e.g., Figs. 7 and 8). These observations imply that most of the enkephalin input to pelvic visceral sympathetic preganglionic neurons comes from spinal neurons below the transection. Furthermore, the density of the enkephalin innervation of neurons projecting to the major pelvic ganglion suggests that interneurons are likely to provide the predominant input to sympathetic preganglionic neurons that control the pelvic viscera. At the electron microscope level, we observed that the density of synapses on sympathetic preganglionic neurons projecting to the major pelvic ganglion did not appear to differ in intact and transected cord, although this observation was not quantified. This conclusion was supported by quantification of enkephalin-immunoreactive input to these neurons. In intact cord, sympathetic preganglionic neurons that projected to the major pelvic ganglion received many synapses from enkephalin-immunoreactive axon terminals. In the intermediolateral cell column, 52% of the synaptic input to retrogradely labeled cell bodies was enkephalin-immunoreactive. Furthermore, this enkephalin innervation was targeted to cell bodies in preference to dendrites since only 29% of the input to retrogradely labeled dendrites was enkephalin positive. In the 2-week transected cord, enkephalin occurred in 65% of the varicosities that synapsed on sympathetic preganglionic somata that projected to the major pelvic ganglion from the intermediolateral cell column. The proportional change in input between cell bodies in intact and transected cord was not statistically significant. However, the increase in enkephalin input from 52% to 65% suggests a small loss of synapses due to transection. This loss might have been revealed if data had been collected from a larger number of rats. These data indicate that the enkephalin input to pelvic visceral sympathetic preganglionic neurons is not significantly affected by transection, due to the fact that it is predominantly intraspinal. Since pelvic visceral sympathetic preganglionic neurons are not substantially denervated after spinal cord injury, their somata and dendrites may

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Fig. 7. In transected cord, enkephalin-immunoreactive axons closely appose sympathetic preganglionic neurons projecting to the major pelvic ganglion. Transections (TX) were located in caudal thoracic segment 4/rostral thoracic segment 5. (A) A sympathetic preganglionic neuron (asterisk) that has retrogradely transported cholera toxin B subunit (CTB) from the major pelvic ganglion (MPG) lies at the lateral edge of the intermediolateral cell column (IML) in 11-week transected cord. A host of enkephalin (ENK)immunoreactive varicosities form close appositions on the sympathetic preganglionic neuron. A retrogradely labeled dendrite in the white matter (WM) also receives many close appositions from enkephalin-containing terminals. Some appositions are indicated by arrowheads. Bar, 20 mm. (B) Retrogradely labeled sympathetic preganglionic neurons (asterisks) in the central autonomic area lie within a very dense network of enkephalin (ENK)-immunoreactive axons. Bar, 50 mm.

not undergo the shrinkage and regrowth that we have previously documented in more rostral sympathetic preganglionic neurons immediately after injury. However, further studies are needed to explore this possibility. The dominance of intraspinal pathways in the control of sympathetic preganglionic neurons supplying the major pelvic ganglion has important implications for the restoration of pelvic visceral function after spinal cord injury. Since intraspinal circuits controlling pelvic visceral sympathetic preganglionic neurons are relatively unaffected by spinal cord injury, drug treatments that target this persistent circuitry should help to normalize sympathetically mediated pelvic visceral reflexes. Furthermore, since sympathetic components are less

affected, they should be more easily restored after an injury because there will be less dependence on the re-establishment of direct synaptic input from regrowing supraspinal axons. It will be interesting to see whether spinal interneurons are equally important in the regulation of parasympathetic preganglionic neurons, which project to the major pelvic ganglion from the lower lumbar and upper sacral cord and are also critical for pelvic visceral function. Acknowledgments Project Grants (#229907 to ILS and #000044 to JRK) and Research Fellowships (#229921 to ILS and #358709 to JRK) from the National Health and Medical Research Council of Australia, grants

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Fig. 8. In transected cord, enkephalin-immunoreactive axons synapse on sympathetic preganglionic neurons projecting to the major pelvic ganglion. In the central autonomic area of 2-week transected cord, an enkephalin-immunoreactive varicosity (ENK) forms a synapse (arrowheads) on a dendrite that contains a crystal due to retrograde transport of cholera toxin B subunit (CTB) from the major pelvic ganglion (MPG). The transection was located in caudal thoracic segment 4/rostral thoracic segment 5. Bar, 500 nm.

from the National Heart Foundation of Australia (#G98A0097 and #G00A0512 to ILS), a Visiting Scientist Award from the Heart and Stroke Foundation of Canada (ILS), Ontario Heart and Stroke Foundation (LCW) and the Canadian Institutes of Health Research (LCW) supported this work. Carolyn Martin, Natalie Fenwick and Lee Travis provided expert technical assistance.

References Antunes, V.R., Brailoiu, G.C., Kwok, E.H., Scruggs, P. and Dun, N.J. (2001) Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro. Am. J. Physiol. Regul. Integr. Comp. Physiol., 281: R1801–R1807. Appel, N.M. and Elde, R.P. (1988) The intermediolateral cell column of the thoracic spinal cord is comprised of targetspecific subnuclei: evidence from retrograde transport studies and immunohistochemistry. J. Neurosci., 8: 1767–1775. Bacon, S.J. and Smith, A.D. (1988) Preganglionic sympathetic neurones innervating the rat adrenal medulla: immunocytochemical evidence of synaptic input from nerve terminals containing substance P, GABA or 5-hydroxytryptamine. J. Auton. Nerv. Syst., 24: 97–122. Bernstein-Goral, H. and Bohn, M.C. (1989) Phenylethanolamine N-methyltransferase-immunoreactive terminals synapse on adrenal preganglionic neurons in the rat spinal cord. Neuroscience, 32: 521–537. Bogan, N., Mennone, A. and Cabot, J.B. (1989) Light microscopic and ultrastructural localization of GABA-like immunoreactive input to retrogradely labeled sympathetic preganglionic neurons. Brain Res., 505: 257–270.

Bowker, R.M., Westlund, K.N., Sullivan, M.C. and Coulter, J.D. (1982) Organization of descending serotonergic projections to the spinal cord. Prog. Brain Res., 57: 239–265. Cabot, J.B. (1990) Sympathetic preganglionic neurons: Cytoarchitecture, ultrastructure and biophysical properties. In: Loewy A.D. and Spyer K.M. (Eds.), Central Regulation of Autonomic Functions. Oxford University Press, pp. 44–67. Cabot, J.B., Alessi, V. and Bushnell, A. (1992) Glycine-like immunoreactive input to sympathetic preganglionic neurons. Brain Res., 571: 1–18. Cabot, J.B., Alessi, V., Carroll, J. and Ligorio, M. (1994) Spinal cord lamina V and lamina VII interneuronal projections to sympathetic preganglionic neurons. J. Comp. Neurol., 347: 515–530. Cano, G., Sved, A.F., Rinaman, L., Rabin, B.S. and Card, J.P. (2001) Characterization of the central nervous system innervation of the rat spleen using viral transneuronal tracing. J. Comp. Neurol., 439: 1–18. Cassam, A.K., Llewellyn-Smith, I.J. and Weaver, L.C. (1997) Catecholamine enzymes and neuropeptides are expressed in fibres and somata in the intermediate gray matter in chronic spinal rats. Neuroscience, 78: 829–841. Cassam, A.K., Rogers, K.A. and Weaver, L.C. (1999) Co-localization of substance P and dopamine beta-hydroxylase with growth-associated protein-43 is lost caudal to a spinal cord transection. Neuroscience, 88: 1275–1288. Christensen, M.D. and Hulsebosch, C.E. (1997) Spinal cord injury and anti-NGF treatment results in changes in CGRP density and distribution in the dorsal horn in the rat. Exp. Neurol., 147: 463–475. Clarke, H.A., Dekaban, G.A. and Weaver, L.C. (1998) Identification of lamina V and VII interneurons presynaptic to adrenal sympathetic preganglionic neurons in rats using a recombinant herpes simplex virus type 1. Neuroscience, 85: 863–872.

24 Coote, J.H., Macleod, V.H., Fleetwood-Walker, S.M. and Gilbey, M.P. (1981) The response of individual sympathetic preganglionic neurons to microiontophoretically applied endogenous monoamines. Brain Res., 215: 135–145. Dampney, R.A.L. (1994) Functional organization of central pathways regulating the cardiovascular system. Physiol. Rev., 74: 323–364. Davis, B.M. and Cabot, J.B. (1984) Substance P-containing pathways to avian sympathetic preganglionic neurons: evidence for major spinalspinal circuitry. J. Neurosci., 4: 2145–2159. Deuchars, S.A., Brooke, R.E., Frater, B. and Deuchars, J. (2001) Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1. Neuroscience, 106: 433–446. Deuchars, S.A., Morrison, S.F. and Gilbey, M.P. (1995) Medullary-evoked EPSPs in neonatal rat sympathetic preganglionic neurones in vitro. J. Physiol., 487(Part 2): 453–463. Deuchars, S.A., Spyer, K.M. and Gilbey, M.P. (1997) Stimulation within the rostral ventrolateral medulla can evoke monosynaptic GABAergic IPSPs in sympathetic preganglionic neurons in vitro. J. Neurophysiol., 77: 229–235. Dun, N.J. and Mo, N. (1988) In vitro effects of substance P on neonatal rat sympathetic preganglionic neurones. J. Physiol., 399: 321–333. Holets, V. and Elde, R. (1982) The differential distribution and relationship of serotonergic and peptidergic fibers to sympathoadrenal neurons in the intermediolateral cell column of the rat: a combined retrograde axonal transport and immunofluorescence study. Neuroscience, 7: 1155–1174. Inokuchi, H., Yoshimura, M., Trzebski, A., Polosa, C. and Nishi, S. (1992a) Fast inhibitory postsynaptic potentials and responses to inhibitory amino acids of sympathetic preganglionic neurons in the adult cat. J. Auton. Nerv. Syst., 41: 53–60. Inokuchi, H., Yoshimura, M., Yamada, S., Polosa, C. and Nishi, S. (1992b) Fast excitatory postsynaptic potentials and the responses to excitant amino acids of sympathetic preganglionic neurons in the slice of the cat spinal cord. Neuroscience, 46: 657–667. Jansen, A.S.P., Wessendorf, M.W. and Loewy, A.D. (1995) Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion. Brain Res., 683: 1–24. Jensen, I., Llewellyn-Smith, I.J., Minson, J.B., Pilowsky, P.M. and Chalmers, J.P. (1995) Serotonin inputs to rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion or adrenal medulla. J. Comp. Neurol., 353: 427–438. Joshi, S., LeVatte, M.A., Dekaban, G.A. and Weaver, L.C. (1995) Identification of spinal interneurons antecedent to adrenal sympathetic preganglionic neurons using trans-synaptic transport of herpes simplex virus type 1. Neuroscience, 65: 893–903. Kadzielawa, K. (1983) Inhibition of the activity of sympathetic preganglionic neurones and neurones activated by visceral

afferents, by alpha-methylnoradrenaline and endogenous catecholamines. Neuropharmacology, 22: 3–17. Kolaj, M., Shefchyk, S.J. and Renaud, L.P. (1997) Two conductances mediate thyrotropin-releasing-hormone-induced depolarization of neonatal rat spinal preganglionic and lateral horn neurons. J. Neurophysiol., 78: 1726–1729. Krassioukov, A.V. and Weaver, L.C. (1996) Morphological changes in sympathetic preganglionic neurons after spinal cord injury in rats. Neuroscience, 70: 211–225. Krenz, N.R., Meakin, S.O., Krassioukov, A.V. and Weaver, L.C. (1999) Neutralizing intraspinal nerve growth factor blocks autonomic dysreflexia caused by spinal cord injury. J. Neurosci., 19: 7405–7414. Krenz, N.R. and Weaver, L.C. (1998a) Changes in the morphology of sympathetic preganglionic neurons parallel the development of autonomic dysreflexia after spinal cord injury in rats. Neurosci. Lett., 243: 61–64. Krenz, N.R. and Weaver, L.C. (1998b) Sprouting of primary afferent fibers after spinal cord transection in the rat. Neuroscience, 85: 443–458. Krukoff, T.L., Ciriello, J. and Calaresu, F.R. (1985) Segmental distribution of peptide- and 5HT-like immunoreactivity in nerve terminals and fibres of the thoracolumbar sympathetic nuclei of the cat. J. Comp. Neurol., 240: 103–116. Krupp, J., Bordey, A. and Feltz, P. (1997) Electrophysiological evidence for multiple glycinergic inputs to neonatal rat sympathetic preganglionic neurons in vitro. Eur. J. Neurosci., 9: 1711–1719. Krupp, J. and Feltz, P. (1995) Excitatory postsynaptic currents and glutamate receptors in neonatal rat sympathetic preganglionic neurons in vitro. J. Neurophysiol., 72(4): 1503–1512. Lai, C.C., Wu, S.Y., Lin, H.H. and Dun, N.J. (1997) Excitatory action of pituitary adenylate cyclase activating polypeptide on rat sympathetic preganglionic neurons in vivo and in vitro. Brain Res., 748: 189–194. Lewis, D.I. and Coote, J.H. (1990a) Excitation and inhibition of rat sympathetic preganglionic neurons by catecholamines. Brain Res., 530: 229–234. Lewis, D.I. and Coote, J.H. (1990b) The influence of 5-hydroxytryptamine agonists and antagonists on identified sympathetic preganglionic neurons in the rat, in vivo. Br. J. Pharmacol., 99: 667–672. Llewellyn-Smith, I.J., Arnolda, L.F., Pilowsky, P.M., Chalmers, J.P. and Minson, J.B. (1998) GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion. J. Auton. Nerv. Syst., 71: 96–110. Llewellyn-Smith, I.J., Cassam, A.K., Krenz, N.R., Krassioukov, A.V., Minson, J.B., Pilowsky, P.M., Arnolda, L.F., Chalmers, J.P. and Weaver, L.C. (1995a) Do sympathetic preganglionic neurons receive synapses from intraspinal catecholamine neurons? Soc. Neurosci. Abstr., 21: 1401. Llewellyn-Smith, I.J., Cassam, A.K., Krenz, N.R., Krassioukov, A.V. and Weaver, L.C. (1997) Glutamate- and GABAimmunoreactive synapses on sympathetic preganglionic neurons caudal to a spinal cord transection in rats. Neuroscience, 80: 1225–1235.

25 Llewellyn-Smith, I.J., DiCarlo, S.E., Collins, H.L. and Keast, J.R. (2005) Enkephalin-immunoreactive interneurons extensively innervate sympathetic preganglionic neurons regulating the pelvic viscera. J. Comp. Neurol., 488: 278–289. Llewellyn-Smith, I.J., Minson, J.B., Pilowsky, P.M., Arnolda, L.F. and Chalmers, J.P. (1995b) The one hundred percent hypothesis: glutamate or GABA in synapses on sympathetic preganglionic neurons. Clin. Exp. Hypertens., 17: 323–334. Llewellyn-Smith, I.J., Minson, J.B., Pilowsky, P.M. and Chalmers, J.P. (1991) There are few catecholamine- or neuropeptide Y-containing synapses in the intermediolateral cell column of rat thoracic spinal cord. Clin. Exp. Pharmacol. Physiol., 18: 111–115. Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M. and Chalmers, J.P. (1992) Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res., 581: 67–80. Llewellyn-Smith, I.J. and Weaver, L.C. (2001) Changes in synaptic inputs to sympathetic preganglionic neurons after spinal cord injury. J. Comp. Neurol., 435: 226–240. Llewellyn-Smith, I.J. and Weaver, L.C. (2004) Interneuronal inputs to sympathetic preganglionic neurons: evidence from transected spinal cord. In: Dun N.J., Machado B.H. and Pilowsky P.M. (Eds.), Neural Mechanisms of Cardiovascular Regulation. Kluwer, pp. 265–283. Loewy, A.D. and McKellar, S. (1981) Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat. Brain Res., 211: 146–152. Ma, R.C. and Dun, N.J. (1985a) Norepinephrine depolarizes lateral horn cells of neonatal rat spinal cord in vitro. Neurosci. Lett., 60: 163–168. Ma, R.C. and Dun, N.J. (1985b) Vasopressin depolarizes lateral horn cells of the neonatal rat spinal cord in vitro. Brain Res., 348: 36–43. Ma, R.C. and Dun, N.J. (1986) Excitation of lateral horn neurons of the neonatal rat spinal cord by 5-hydroxytryptamine. Dev. Brain Res., 24: 89–98. Maiorov, D.N., Krenz, N.R., Krassioukov, A.V. and Weaver, L.C. (1997a) Role of spinal NMDA and AMPA receptors in episodic hypertension in conscious spinal rats. Am. J. Physiol., 273: H1266–H1274. Maiorov, D.N., Weaver, L.C. and Krassioukov, A.V. (1997b) Relationship between sympathetic activity and arterial pressure in conscious spinal rats. Am. J. Physiol., 272: H625–H631. Marsh, D.R. and Weaver, L.C. (2004) Autonomic dysreflexia, induced by noxious or innocuous stimulation, does not depend on changes in dorsal horn substance P. J. Neurotrauma, 21: 817–828. Milner, T.A., Morrison, S.F., Abate, C. and Reis, D.J. (1988) Phenylethanolamine N-methyltransferase-containing terminals synapse directly on sympathetic preganglionic neurons in the rat. Brain Res., 448: 205–222. Minson, J.B., Arnolda, L.F. and Llewellyn-Smith, I.J. (2002) Neurochemistry of nerve fibers apposing sympathetic preganglionic neurons activated by sustained hypotension. J. Comp. Neurol., 449: 307–318.

Minson, J.B., Llewellyn-Smith, I.J. and Arnolda, L.F. (2001) Neuropeptide Y mRNA expression in interneurons in rat spinal cord. Auton. Neurosci., 93: 14–20. Minson, J.B., Llewellyn-Smith, I.J., Pilowsky, P.M. and Chalmers, J.P. (1994) Bulbospinal neuropeptide Y-immunoreactive neurons in the rat: comparison with adrenaline-synthesising neurons. J. Auton. Nerv. Syst., 47: 233–243. Minson, J., Pilowsky, P., Llewellyn-Smith, I., Kaneko, T., Kapoor, V. and Chalmers, J. (1991) Glutamate in spinally projecting neurons of the rostral ventral medulla. Brain Res., 555: 326–331. Miyazaki, T., Coote, J.H. and Dun, N.J. (1989) Excitatory and inhibitory effects of epinephrine on neonatal rat sympathetic preganglionic neurons in vitro. Brain Res., 497: 108–116. Mo, N. and Dun, N.J. (1987a) Excitatory postsynaptic potentials in neonatal rat sympathetic preganglionic neurons: possible mediation by NMDA receptors. Neurosci. Lett., 77: 327–332. Mo, N. and Dun, N.J. (1987b) Is glycine an inhibitory transmitter in rat lateral horn cells? Brain Res., 400: 139–144. Morrison, S.F. (2001) Differential control of sympathetic outflow. Am. J. Physiol. Regul. Integr. Comp. Physiol., 281: R683–R698. Newton, B.W., Maley, B.E. and Hamill, R.W. (1986) Immunohistochemical demonstration of serotonin neurons in autonomic regions of the rat spinal cord. Brain Res., 376: 155–163. Ondarza, A.B., Ye, Z. and Hulsebosch, C.E. (2003) Direct evidence of primary afferent sprouting in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and CGRP. Exp. Neurol., 184: 373–380. Pickering, A.E., Spanswick, D. and Logan, S.D. (1994) 5-Hydoxytryptamine evokes depolarizations and membrane potential oscillations in rat sympathetic preganglionic neurones. J. Physiol., 480: 109–121. Pilowsky, P., Llewellyn-Smith, I.J., Lipski, J. and Chalmers, J. (1992) Substance P immunoreactive boutons form synapses with feline sympathetic preganglionic neurons. J. Comp. Neurol., 320: 121–135. Romagnano, M.A., Braiman, J., Loomis, M. and Hamill, R.W. (1987) Enkephalin fibers in autonomic nuclear regions: intraspinal vs. supraspinal origin. J. Comp. Neurol., 266: 319–331. Sasek, C.A., Wessendorf, M.W. and Helke, C.J. (1990) Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neuroscience, 35: 105–119. Stornetta, R.L., Akey, P.J. and Guyenet, P.G. (1999) Location and electrophysiological characterization of rostral medullary adrenergic neurons that contain neuropeptide Y mRNA in rat medulla. J. Comp. Neurol., 415: 482–500. Stornetta, R.L., McQuiston, T.J. and Guyenet, P.G. (2004) GABAergic and glycinergic presympathetic neurons of rat medulla oblongata identified by retrograde transport of pseudorabies virus and in situ hybridization. J. Comp Neurol., 479: 257–270.

26 Stornetta, R.L., Sevigny, C.P., Schreihofer, A.M., Rosin, D.L. and Guyenet, P.G. (2002) Vesicular glutamate transporter DNPI/VGLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla. J. Comp. Neurol., 444: 207–220. Strack, A.M., Sawyer, W.B., Hughes, J.H., Platt, K.B. and Loewy, A.D. (1989) A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res., 491: 156–162. Strack, A.M., Sawyer, W.B., Marubio, L.M. and Loewy, A.D. (1988) Spinal origin of sympathetic preganglionic neurons in the rat. Brain Res., 455: 187–191. Sved, A.F., Cano, G. and Card, J.P. (2001) Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clin. Exp. Pharmacol. Physiol., 28: 115–119. Tang, X., Neckel, N.D. and Schramm, L.P. (2004) Spinal interneurons infected by renal injection of pseudorabies virus in the rat. Brain Res., 1004: 1–7. van den Top, M., Nolan, M.F., Lee, K., Richardson, P.J., Buijs, R.M., Davies, C.H. and Spanswick, D. (2003) Orexins induce increased excitability and synchronisation of rat sympathetic preganglionic neurones. J. Physiol., 549: 809–821. Vera, P.L., Holets, V.R. and Miller, K.E. (1990) Ultrastructural evidence of synaptic contacts between substance P-, enke-

phalin-, and serotonin-immunoreactive terminals and retrogradely labeled sympathetic preganglionic neurons in the rat: a study using a double-peroxidase procedure. Synapse, 6: 221–229. Weaver, L.C., Cassam, A.K., Krassioukov, A.V. and Llewellyn-Smith, I.J. (1997) Changes in immunoreactivity for growth associated protein-43 suggest reorganization of synapses on spinal sympathetic neurons after cord transection. Neuroscience, 81: 535–551. Weaver, L.C., Verghese, P., Bruce, J.C., Fehlings, M.G., Krenz, N.R. and Marsh, D.R. (2001) Autonomic dysreflexia and primary afferent sprouting after clip-compression injury of the rat spinal cord. J. Neurotrauma, 18: 1107–1119. Wong, S.T., Atkinson, B.A. and Weaver, L.C. (2000) Confocal microscopic analysis reveals sprouting of primary afferent fibres in rat dorsal horn after spinal cord injury. Neurosci. Lett., 296: 65–68. Yusof, A.P.M. and Coote, J.H. (1988) Excitatory and inhibitory actions of intrathecally administered 5-hydroxytryptamine on sympathetic nerve activity in the rat. J. Auton. Nerv. Syst., 22: 229–236. Zagon, A. and Smith, A.D. (1993) Monosynaptic projections from the rostral ventrolateral medulla oblongata to identified sympathetic preganglionic neurons. Neuroscience, 54: 729–743.