Dorsal Horn Plasticity Following Re-routeing of Peripheral Nerves: Evidence for Tissue-Specific Neurotrophic Influences from the Periphery

Some properties of primary sensory neurons change when they reinnervate new peripheral targets (McMahon et al., Neuroscience, 33, 67 - 75, 1989). We ask here if such influences can extend to the central connectivity of sensory neurons. In adult rats the nerve to the gastrocnemius muscle (GN) and the cutaneous sural nerve (SN) were self- and cross-anastomosed on left- and right-hand sides, respectively, so that they regenerated to either appropriate or inappropriate targets. Ten to 14 weeks later, the distribution and strength of spinal connections of the SN and GN were determined. The unmyelinated afferents in the GN innervating skin increased their connectivity to 286% of that seen for the GN innervating muscle (P < 0.005), and came to resemble normal cutaneous afferents. However, for the SN there was no significant difference between appropriately and inappropriately regenerated nerves by this measure. The ability of myelinated fibres to produce inhibitions and facilitations in dorsal horn cells was also assessed. The intact or self-anastomosed SN produced predominantly inhibitory effects, whilst the GN produced predominantly facilitatory effects. After the SN had regenerated to muscle its central effects became predominantly facilitatory, whilst those of the GN innervating skin became inhibitory. These changes were statistically significant. In conclusion, we have found that major changes in the physiology of central connections in the dorsal horn may occur following peripheral reinnervation of foreign targets. The changes that were seen were appropriate to the new target, and could not easily be explained by non-specific changes due to axotomy, or changes in A-fibre-mediated inhibitions. We suggest that these effects might arise because of trophic influences arising in and specific to different peripheral targets.     

Peripheral specification of sensory neurons transplanted to novel locations along the neuraxis

Thoracic dorsal root ganglia in bullfrogs contain sensory neurons that innervate the skin of the trunk and have synaptic connections in the dorsal horn of the spinal cord. The ganglion that innervates the forelimb contains, in addition to cutaneous afferents, many muscle afferents that project more ventrally in the spinal cord and make monosynaptic connections with motoneurons. In the present study, we have transplanted thoracic sensory neurons to the brachial level in tadpoles to discover whether they can innervate forelimb muscles and, if so, whether they form central connections characteristic of forelimb muscle afferents. The ganglion that normally supplies the forelimb was removed from tadpoles and replaced with 2 thoracic ganglia. After the tadpoles completed metamorphosis, the peripheral and central connections of the transplanted thoracic sensory neurons were examined with anatomical and electrophysiological techniques. When the ganglia were transplanted at stage XIV or earlier, transplanted sensory neurons innervated the forelimb and projected into the brachial spinal cord. Electrical stimulation of forelimb muscle nerves evoked impulses in the dorsal root, indicating that some centrally projecting sensory neurons were muscle afferents. Furthermore, muscle afferents were also activated by stretching muscles which suggest that they terminated on spindles. HRP labeling of the central projections revealed that transplanted sensory neurons terminated at sites characteristic of both cutaneous and muscle afferents. The pattern of synaptic connections was assessed by recording intracellularly from motoneurons. Stimulation of muscle afferents produced monosynaptic EPSPs in motoneurons. As in normal frogs, triceps muscle afferents projected more strongly to triceps motoneurons than to subscapularis and pectoralis motoneurons, while subscapularis afferents projected to all 3 types of motoneurons. Thus, the transplanted sensory neurons formed central connections appropriate to their novel peripheral targets. These observations suggest that interactions between sensory neurons and their targets may be important in determining their central connections.     

The central projection of masticatory afferent fibers to the trigeminal sensory nuclear complex and upper cervical spinal cord

Retrograde and anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) conjugate was used to study the organization of primary afferent neurons innervating the masticatory muscles. HRP applied to the nerves of jaw-closing muscles--the deep temporal (DT), masseter (Ma), and medial pterygoid (MP)--labeled cells in the trigeminal ganglion and the mesencephalic trigeminal nucleus (Vmes), whereas HRP applied to nerves of the jaw-opening muscles--anterior digastric (AD) and mylohyoid (My)--labeled cells only in the trigeminal ganglion. Cell bodies innervating the jaw-closing muscles were found with greater frequency in the intermediate region of the mandibular subdivision, while somata supplying the jaw-opening muscles were predominant posterolaterally. The distribution of their somatic sizes was unimodal and limited to a subpopulation of smaller cells. Projections of the muscle afferents of ganglionic origin to the trigeminal sensory nuclear complex (TSNC) were confined primarily to the caudal half of pars interpolaris (Vi), and the medullary and upper cervical dorsal horns. In the Vi, Ma, MP, AD, and My nerves terminated in the lateral-most part of the nucleus with an extensive overlap in projections, save for the DT nerve, which projected to the interstitial nucleus or paratrigeminal nucleus. In the medullary and upper cervical dorsal horns, the main terminal fields of individual branches were confined to laminae I/V, but the density of the terminals in lamina V was very sparse. The rostrocaudal extent of the terminal field in lamina I differed among the muscle afferents of origin, whereas in the mediolateral or dorsoventral axis, a remarkable overlap in projections was noted between or among muscle afferents. The terminals of DT afferents were most broadly extended from the rostral level of the pars caudalis to the C3 segment, whereas the MP nerve showed limited projection to the middle one-third of the pars caudalis. Terminal fields of the Ma, AD, and My nerves appeared in the caudal two-thirds of the pars caudalis including the first two cervical segments, the caudal half of the pars caudalis and the C1 segment, and in the caudal part of the pars caudalis including the rostral C1 segment, respectively. This rostrocaudal arrangement in the projections of muscle nerves, which corresponds to the anteroposterior length of the muscles and their positions, indicates that representation of the masticatory muscles in lamina I reflects an onion-skin organization. These results suggest that primary muscle afferent neurons of ganglionic origin primarily mediate muscle pain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of central versus peripheral nerve pathways to the guinea pig inferior mesenteric ganglion determined electrophysiologically after chronic nerve section

The contributions of central versus peripheral nerve pathways to neurons of the inferior mesenteric ganglion of guinea pigs were studied. Nerve trunks innervating neurons in the ganglion were surgically sectioned and intracellular electrical responses to nerve stimulation were measured 6-8 days after surgery. Guinea pigs were divided into two experimental groups: (1) those that had the lumbar sympathetic chain ganglia (LSG) L2 through L4 removed and (2) those that had the intermesenteric, lumbar colonic and hypogastric nerves sectioned leaving central connections intact. After 6-8 days fast excitatory postsynaptic potentials (EPSPs) and slow EPSPs were recorded intracellularly in randomly selected principal ganglionic neurons. The threshold stimulus voltage to elicit a fast EPSP, the amplitude of the slow EPSP and the number of neurons in which each type of synaptic potential occurred in response to stimulation of each of the nerve trunks was compared between surgically-sectioned animals and sham-operated controls. Neither section of preganglionic nerve trunks nor of postganglionic nerve trunks eliminated all synaptic input to neurons in the ganglion, indicating that neurons with cell bodies located central to the ganglion as well as in visceral target organs made synaptic connections in the ganglion. Both fast and slow synaptic potentials could be evoked by stimulation of postganglionic nerve trunks even after they were sectioned provided that preganglionic nerves were intact, indicating that axons of central origin which synapse in the ganglion may continue out into postganglionic nerve trunks. In like manner, evidence was obtained indicating that fibers from peripheral nerve trunks which initiate either fast or slow synaptic potentials in ganglionic neurons may continue out into the lumbar splanchnic nerves. These studies demonstrate that the synaptic potentials recorded in the inferior mesenteric ganglion arise not only from neurons with cell bodies central to the ganglion but also from neurons with cell bodies located in the visceral organs which this ganglion subserves.     

The organization of neuronal somata in the first sacral spinal ganglion of the cat.

The basis of organization of neuronal somata within a spinal ganglion has been studied using histological techniques. The positions of the largest neuronal cell bodies, which are thought to innervate muscle receptors, were plotted for unoperated and sham operated animals. These ganglion cells were found in all sectors of the ganglion and their distribution generally reflected that of the total ganglion cell population in the respective sectors. The positions of neuronal somata undergoing the axonal reaction following various nerve lesions were plotted. Altered neurons were not preferentially accumulated in any sector of the ganglion after lesions of cutaneous nerves, nerves to muscle, or nerves innervating the distal part of the hind limb, but they often occurred in clusters which were found in all parts of the ganglion. Following a lesion of either the pudendal nerve, the S1 dorsal primary ramus, or the S1 ventral root, altered neurons tended to be grouped in one or more sectors of the ganglion. These data suggest that ganglion cells innervating skin or muscle often occur in small groups distributed throughout the ganglion, while processes of afferent neurons which branch from the spinal nerve and do not travel through a limb plexus tend to have their cell bodies restricted to a particular region of the ganglion.     

Axotomy alters putative neurotransmitters in visceral sensory neurons of the nodose and petrosal ganglia.

Acute peripheral axotomy of the visceral sensory neurons of the vagus and glossopharyngeal nerves removes peripheral depolarizing and trophic influences to their sensory ganglia. To study axotomy-induced changes in the putative neurotransmitters of visceral sensory neurons, rats were sacrificed 1, 3, 7 or 14 days after transection of either the cervical vagus and superior laryngeal nerves (to affect peripheral axotomy of the nodose ganglion) or the glossopharyngeal and carotid sinus nerves (to affect peripheral axotomy of the petrosal ganglion). The numbers of tyrosine hydroxylase (TH)-immunoreactive (ir), vasoactive intestinal peptide (VIP)-ir, calcitonin-gene-related peptide (CGRP)-ir, and substance P (SP)-ir neurons in the respective ganglia were analyzed in axotomized and control ganglia. In the nodose ganglion, axotomy of the cervical vagus resulted in a rapid (by 1 day) reduction in the number of TH-ir cells, whereas VIP-ir neurons were dramatically increased in number by 3 days. CGRP- and SP-ir cells in the nodose ganglion were relatively unaffected by axotomy. In the petrosal ganglion, axotomy of the glossopharyngeal and carotid sinus nerves greatly reduced the number of TH-ir cells but did not alter the number VIP-ir neurons. CGRP- and SP-ir neurons in the petrosal ganglion were reduced in number by axotomy. Thus, axotomy of visceral sensory neurons differentially changed the content and perhaps the expression of putative transmitters. Differential changes were seen among transmitters in a single ganglia and between ganglia. These data demonstrate the plasticity of putative neurotransmitter systems in visceral afferent systems of adult rats.     

Regulation of afferent connectivity in the adult spinal cord by nerve growth factor

During development, nerve growth factor (NGF) regulates the density and character of peripheral target innervation (Barde, Neuron , 2 , 1525–1534, 1989; Ritter et al., Soc. Neurosci. Abstr. , 17 , 546.2, 1991); its role in adult animals is less well defined. Here we have asked if the availability of growth factors such as NGF in peripheral tissues can influence the pattern of primary afferent connections in the CNS. Using osmotic minipumps, we raised the levels of NGF in rat skeletal muscle in vivo , a tissue where the levels of this factor are normally very low (Korsching and Thoenen, Proc. Natl. Acad. Sci. USA , 80 , 3513–3516, 1983; Shelton and Reichardt, Proc. Natl. Acad. Sci. USA , 81 , 7951–7955, 1984; Goedert et al., Mol. Brain Res. , 1 , 85–92, 1986). After 2 weeks of treatment we asked if the sensory neurons innervating this tissue showed an altered strength and distribution of connections with dorsal horn neurons. The contralateral (vehicle-treated) muscle, and totally untreated animals, served as controls. In normal and vehicle-treated animals, electrical stimulation of muscle afferents excited relatively few neurons in the dorsal horn, and these generally showed only weak responses. In contrast, on the NGF-treated side many more dorsal horn neurons in the lumbar enlargement of the spinal cord were excited by muscle afferents. The increased responsiveness could not be explained by a generalized increase in dorsal horn excitability, since spontaneous activity was not enhanced, nor by a change in A-fibre-mediated inhibitions from the treated afferents. Thus, these afferents appeared to establish new synaptic connections or strengthened previously weak ones as a result of increased neurotrophic factor availability. The data suggest that, in the adult rat, the levels of growth factors in peripheral targets may be used to regulate an appropriate degree of afferent connectivity within the central nervous system.     

Neurochemical and morphological phenotypes of vagal afferent neurons innervating the adult mouse jejunum

Abstract  Whilst much is known about the function and influence of vagal afferents on the mammalian upper gastrointestinal tract, the phenotypes of the different types of vagal afferent neurons innervating the jejunum is unknown. We have previously shown that spinal afferents supplying the jejunum are predominantly medium-sized sensory neurons that express specific combinations of transient receptor potential vanilloid type 1 (TRPV1), neuronal nitric oxide synthase (NOS) and calcitonin-gene related peptide (CGRP) and that they lack binding for isolectin B4 (IB4). This study aimed to identify the chemical phenotypes and somal sizes of jejunal afferent neurons in the mouse vagal ganglion. Jejunal vagal afferents were identified by retrograde labelling with sub-serosal injections of cholera toxin B (CTB) into the jejunal wall and assessed for IB4-binding, TRPV1-, NOS- and CGRP-immunoreactivities using fluorescent microscopy. Almost all (99%) of CTB-labelled vagal afferent neurons were small- and medium-sized sensory cells. Most (81%) jejunal vagal afferents bound IB4 but fewer (32%) expressed TRPV1. A quarter (25%) of those that bound IB4 co-expressed TRPV1-immunoreactivity whilst 77% of TRPV1-expressing jejunal vagal afferent neurons bound IB4. NOS (0%) and CGRP (0%) expression was absent from all CTB-labelled cells examined. In conclusion, vagal afferents innervating the jejunum differ in their expression of IB4, TRPV1, CGRP and NOS from their spinal counterparts, suggesting that the peripheral endings for extrinsic sensory neurons terminating within the enteric nervous system can be identified selectively.     

Brain stem projections of the aortic nerve in the cat: A study using tetramethyl benzidine as the substrate for horseradish peroxidase

The intra-axonal transport of horseradish peroxidase (HRP) has been used to trace the nodose ganglion and brain stem projections of a physiologically distinct nerve - the aortic depressor nerve - following electrophysiological identification. Tetramethyl benzidine (TMB) has been used as the substrate for demonstrating the centrally transported HRP15, 16. This sensitive method for horseradish peroxidase histochemistry has permitted the visualization of the central projections of aortic nerve afferents and has also provided information regarding the anatomical localization of cell bodies of these sensory nerve fibers within the nodose ganglion. This study demonstrates the usefulness of using TMB as a substrate for HRP histochemistry in anatomical studies where the detection of anterogradely transported HRP is an essential prerequisite. The uptake of HRP from the cut central ends of sensory nerve fibers and the transport of this enzyme to the sensory ganglion and subsequently into the central processes of these sensory neurons have made possible this study of the central projections of a functionally distinct peripheral nerve. Information has been provided by this study that cell bodies of aortic nerve afferent fibers are localized in the rostrolateral pole of the nodose ganglion. Dense central projections of sensory terminals of aortic afferents have been found in the dorsolateral and medial subdivisions of the nucleus of the tractus solitarius. These central projections of aortic afferents extend for 6 mm rostrocaudally in the medulla with the densest projection being found at the level of the obex. These projections are bilateral at all rostrocaudal levels. This anatomical demonstration of the dorsolateral and medial subdivisions of the nucleus of the tractus solitarius confirms earlier reports based on electrophysiological studies. Of particular interest in this study is the new observation that there exists a dense projection of aortic nerve afferents to the area postrema. The possible physiological implications of a direct input of peripheral chemoreceptor afferents to a region of central chemosensitivity are discussed. The complete absence of any retrogradely labeled cell body in the brain stem from exposure of the aortic nerve to horseradish peroxidase is noteworthy. This indicates that the aortic nerve is purely afferent in function and that reflex control of afferent activity in the aortic nerve is not mediated by brain stem neurons projecting down the same nerve.     

Segmental distribution and central projectionsof renal afferent fibers in the cat studied by transganglionic transport of horseradish peroxidase

The segmental and central distributions of renal nerve afferents in adult cats and kittens were studied by using retrograde and transganglionic transport of horseradish peroxidase (HRP). Transport of HRP from the central cut ends of the left renal nerves labeled afferent axons in the ipsilateral minor splanchnic nerves and sensory perikarya in the dorsal root ganglia from T12 to L4. The majority of labeled cells (85%) were located between L1 and L3. A few neurons in the contralateral dorsal root ganglia were also labeled. Labeled cells were not confined to any particular region within a dorsal root ganglion. Some examples of bifurcation of the peripheral and central processes within the ganglion were noted. A small number of preganglionic neurons, concentrated in the intermediolateral nucleus, were also identified in some experiments. In addition, many sympathetic postganglionic neurons were labeled in the renal nerve ganglia, the superior mesenteric ganglion, and the ipsilateral paravertebral ganglia from T12 to L3 Transganglionic transport of HRP labeled renal afferent projections to the spinal cord of kittens from T1 1 to L6, with the greatest concentrations between Ll and L3. These afferents extended rostrocaudally in Lissauer's tract and sent collaterals into lamina I. In the transverse plane, a major lateral projection and a minor medial projection were observed along the outer and inner margins of the dorsal horn, respectively. From the lateral projection many fibers extended medially in laminae V and VI forming dorsal and ventral bundles around Clarke's nucleus. The dorsal bundle was joined by collaterals from the medial afferent projection and crossed to the contralateral side. The ventral bundle extended into lamina VII along the lateroventral border of Clarke's nucleus. Some afferents in the lateral projection could be followed ventrally into the dorsolateral portion of lamina VII in the vicinity of the intermediolateral nucleus. In the contralateral spinal cord, labeled afferent fibers were mainly seen in laminae V and VI These results provide the first anatomical evidence for sites of central termination of renal afferent axons. Renal inputs to regions (laminae I, V, and VI) containing spinoreticular and spinothajamic tract neurons may be important in the mediation of supraspinal cardiovascular reflexes as well as in the transmission of activity from nociceptors in the kidney. In addition, the identification of a bilateral renal afferent projection in close proximity to the thoracolumbar autonomic nuclei is consistent with the demonstration in physiological experiments of a spinal pathway for the renorenal sympathetic reflexes.     

Plasticity of primary afferent acid phosphatase expression following rerouting of afferents from muscle to skin in the adult rat

We have examined the possibility that reinnervation of a new peripheral target by primary afferent neurones can alter the histochemical properties of those afferents in the adult rat. The hindlimb sural and gastrocnemius nerves largely supply skin and muscle, respectively. In adult animals these nerves were cut and rejoined to either their own distal stumps (self-anastomosis) or that of the other nerve (cross-anastomosis) and allowed to regenerate for 12-16 weeks to reinnervate an appropriate or inappropriate target. Fluoride-resistant acid phosphatase (FRAP) is a chemical marker found in many unmyelinated afferents. We have determined the FRAP expression in normal and regrown nerves and examined its distribution in the dorsal horn of animals with self- and cross-anastomosed nerves. While normal and self-anastomosed sural nerves stained heavily for FRAP, gastrocnemius nerves showed either no staining or only the occasional fibre. Cross-anastomosed gastrocnemius nerves, now innervating the skin, showed a significant increase in staining, in some cases approaching the levels normally seen in sural nerves. Conversely, cross-anastomosed sural nerves (innervating muscle) showed decreased FRAP staining. In the normal dorsal horn the terminals of FRAP containing afferents form a thin band extending throughout the mediolateral extent of lamina II (Devor and Claman: Brain Res. 190:17-28, '80). One week after axotomy of the sural nerve, FRAP is depleted from its terminals and a gap appears in the normal FRAP staining pattern in the lumbar enlargement of the spinal cord. The new expression of FRAP in cross-anastomosed nerves was also seen in their terminals in the dorsal horn.(ABSTRACT TRUNCATED AT 250 WORDS)     

BDNF is a target-derived survival factor for arterial baroreceptor and chemoafferent primary sensory neurons.

Brain-derived neurotrophic factor (BDNF) supports survival of 50% of visceral afferent neurons in the nodose/petrosal sensory ganglion complex (NPG; Ernfors et al., 1994a; Jones et al., 1994; Conover et al., 1995; Liu et al., 1995; Erickson et al., 1996), including arterial chemoafferents that innervate the carotid body and are required for development of normal breathing (Erickson et al., 1996). However, the relationship between BDNF dependence of visceral afferents and the location and timing of BDNF expression in visceral tissues is unknown. The present study demonstrates that BDNF mRNA and protein are transiently expressed in NPG targets in the fetal cardiac outflow tract, including baroreceptor regions in the aortic arch, carotid sinus, and right subclavian artery, as well as in the carotid body. The period of BDNF expression corresponds to the onset of sensory innervation and to the time at which fetal NPG neurons are BDNF-dependent in vitro. Moreover, baroreceptor innervation is absent in newborn mice lacking BDNF. In addition to vascular targets, vascular afferents themselves express high levels of BDNF, both during and after the time they are BDNF-dependent. However, endogenous BDNF supports survival of fetal NPG neurons in vitro only under depolarizing conditions. Together, these data indicate two roles for BDNF during vascular afferent pathway development; initially, as a target-derived survival factor, and subsequently, as a signaling molecule produced by the afferents themselves. Furthermore, the fact that BDNF is required for survival of functionally distinct populations of vascular afferents demonstrates that trophic requirements of NPG neurons are not modality-specific but may instead be associated with innervation of particular organ systems.     

Organization of the primary projections of the lateral line nerves in the lamprey Lampetra japonica

The lateral line sensory system of Lampetra japonica is innervated by the anterior and posterior lateral line nerves. The anterior lateral line nerve innervates all electroreceptors throughout the body and mechanoreceptors of the head. The posterior lateral line nerve innervates trunk mechanoreceptors. The anterior lateral line nerve consists of two ganglia (anterior lateral line and intracapsular) and four major peripheral branches (superficial ophthalmic, buccal, hyomandibular, and recurrent nerves). The posterior lateral line nerve has one posterior lateral line ganglion and one peripheral branch. The location and central projection patterns of the primary sensory neurons of these branches of the lateral line nerves were studied with the aid of horseradish peroxidase labeling. The ganglion cells of the buccal nerve were found in the rostral half, and those of the hyomandibular nerve were found in the caudal half of the medial part of the anterior lateral line ganglion. The lateral part of the anterior lateral line ganglion contains ganglion cells of the recurrent nerve and the superficial ophthalmic nerve. The rostral half of the intracapsular ganglion contains ganglion cells of the recurrent, hyomandibular, and buccal nerves. The ganglion cells of the posterior lateral line nerve were found in the posterior lateral line ganglion. The buccal nerve afferents terminated mainly in the lateral part of the ipsilateral mechanoreceptive medial nucleus. The peripheral part of the electroreceptive dorsal nucleus also received several afferents. The hyomandibular afferents terminated ipsilaterally in the central part of the medial nucleus and in the dorsolateral part of the dorsal nucleus. Some afferents of the hyomandibular nerve ascended and descended in the descending nucleus of the trigeminal nerve near its dorsal margin. The ventral nucleus, the primary nucleus of the VIIIth nerve, received a few fibers of the buccal and hyomandibular nerves. In the recurrent nerve, the fibers of the lateral part of the anterior lateral line ganglion terminated throughout the entire dorsal nucleus, and the fibers of the intracapsular ganglion projected to the dorsolateral part of the nucleus. The afferents of the posterior lateral line nerve terminated in the medial part of the ipsilateral medial nucleus and in the lateral part of the contralateral medial nucleus. In the cerebellar area, afferents of the anterior lateral line nerve were located laterally to those of the posterior lateral line nerve. Several fibers terminated in some branchiomotor nuclei, the cerebellar crest, and the dorsal gray near the obex level. No efferent cell bodies were found in the place where efferent neurons of the VIIIth nerve have been previously reported.     

Effect of lumbar 5 ventral root transection on pain behaviors: a novel rat model for neuropathic pain without axotomy of primary sensory neurons

A peripheral nerve injury often causes neuropathic pain but the underlying mechanisms remain obscure. Several established animal models of peripheral neuropathic pain have greatly advanced our understanding of the diverse mechanisms of neuropathic pain. A common feature of these models is primary sensory neuron injury and the commingle of intact axons with degenerating axons in the sciatic nerve. Here we investigated whether neuropathic pain could be induced without sensory neuron injury following exposure of their peripheral axons to the milieu of Wallerian degeneration. We developed a unilateral lumbar 5 ventral root transection (L5 VRT) model in adult rats, in which L5 ventral root fibers entering the sciatic nerve were sectioned in the spinal canal. This model differs from previous ones in that DRG neurons and their afferents are kept uninjured and intact afferents expose to products of degenerating efferent ventral root fibers in the sciatic nerve and the denervated muscles. We found that the L5 VRT produced rapid (24 h after transection), robust and prolonged (56 days) bilateral mechanical allodynia, to a similar extent to that in rats with L5 spinal nerve transection (L5 SNT), cold allodynia and short-term thermal hyperalgesia (14 days). Furthermore, L5 VRT led to significant inflammation as demonstrated by infiltration of ED-1-positive monocytes/macrophages in the DRG, sciatic nerve and muscle fibers. These findings demonstrated that L5 VRT produced behavioral signs of neuropathic pain with high mechanical sensitivity and thermal responsiveness, and suggested that neuropathic pain can be induced without damage to sensory neurons. We propose that neuropathic pain in this model may be mediated by primed intact sensory neurons, which run through the milieu of Wallerian degeneration and inflammation after nerve injury. The L5 VRT model manifests the complex regional pain syndrome in some human patients, and it may provide an additional dimension to dissect out the mechanisms underlying neuropathic pain.     

Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice

Spinal afferent neurons are responsible for the transduction and transmission of noxious (painful) stimuli and innocuous stimuli that do not reach conscious sensations from visceral organs to the central nervous system. Although the location of the nerve cell bodies of spinal afferents is well known to reside in dorsal root ganglia (DRG), the morphology and location of peripheral nerve endings of spinal afferents that transduce sensory stimuli into action potentials is poorly understood. The individual nerve endings of spinal afferents that innervate the urinary bladder have never been unequivocally identified in any species. We used an anterograde tracing technique developed in our laboratory to selectively label only spinal afferents. Mice were anesthetized and unilateral injections of dextran‐amine made into lumbosacral DRGs (L5‐S2). Seven to nine days postsurgery, mice were euthanized, the urinary bladder removed, then fresh‐fixed and stained for immunoreactivity to calcitonin‐gene‐related‐peptide (CGRP). Four distinct morphological types of spinal afferent ending in the bladder were identified. Three types existed in the detrusor muscle and one major type in the sub‐urothelium and urothelium. Most nerve endings were located in detrusor muscle where the three types could be identified as having: “branching”, “simple”, or “complex” morphology. The majority of spinal afferent nerve endings were CGRP‐immunoreactive. Single spinal afferent axons bifurcated many times upon entering the bladder and developed varicosities along their axon terminal endings. We present the first morphological identification of spinal afferent nerve endings in the mammalian urinary bladder.     

AMPA glutamatergic receptor-immunoreactive subunits are expressed in lumbosacral neurons of the spinal cord and neurons of the dorsal root and pelvic ganglia controlling pelvic functions in the rat

Sacral preganglionic neurons innervate the pelvic organs via a relay in the major pelvic ganglion. Pudendal motoneurons innervate striated muscles and sphincters of the lower urinary, genital and digestive tracts. The activity of these spinal neurons is regulated by sensory afferents of visceral and somatic origins. Glutamate is released by sensory afferents in the spinal cord, and interacts with a variety of receptor subtypes. The aim of the present study was to investigated the presence of AMPA glutamate receptor subunits (GluR1-GluR4) in the neural network controlling the lower urogenital and digestive tracts of male rats. We performed double-immunohistochemistry directed against a neuronal tracer, the cholera toxin beta subunit (Ctbeta) and each of the four receptor subunits. GluR1, GluR2 and GluR3 subunits were present in many sacral preganglionic neurons retrogradely labelled with Ctbeta applied to the pelvic nerve, and in some dorsolateral and dorsomedian motoneurons retrogradely labelled with Ctbeta injected in ischiocavernosus and bulbospongiosus muscles. The four subunits were detected in postganglionic neurons of the major pelvic ganglion retrogradely labelled with Ctbeta injected in the corpus cavernosum, and in some somata of sensory afferents of the L6 dorsal root ganglion labelled with Ctbeta applied to the dorsal penile nerve or injected in corpus cavernosum. The results provide a detailed knowledge of the neural targets expressing the various AMPA receptor subunits and suggest that part of the neural network that controls pelvic organs, including sensory afferents and postganglionic neurons, is sensitive to glutamate through the whole family of AMPA subunits.     

Visualization of spinal afferent innervation in the mouse colon by AAV8‐mediated GFP expression

Background Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno‐associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV‐driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. Methods Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. Key Results Analysis of colon from rAAV8‐GFP‐treated mice demonstrated GFP‐immunoreactivity (GFP‐ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP‐ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular‐like clusters surrounding nuclei within myenteric ganglia. In addition, GFP‐positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP‐ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. Conclusions & Inferences These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.     

Distribution in the central nervous system of Aplysia of afferent fibers arising from cell bodies located in the periphery

The present study used autoradiography to determine the location of the projections of presumptive peripheral afferent neurons into the central nervous system of Aplysia. Selected peripheral tissues (with an empliasis on structures involved in feeding behavior) were exposed to radioactive amino acids, and the distribution of macromolecules transported into the nervous system via afferent fibers was determined by autoradiography. Different regions of the body exhibited different patterns of projections, and within the neuropil of the cerebral ganglion, there was a loose topographical prganization of projections from the head. For some regions of the body, the projection was largely limited to the ganglion from which the nerve enters; for other regions, the projection was very widespread. In some cases (e.g., rhinophore to eye), there was evidence of projections from one peripheral structure to another. Experiments with all peripheral tissues that were studied resulted in extensive labeling of central ganglia, indicating that afferents with peripheral cell bodies may provide a major source of sensory input to the central nervous system and suggesting that many or all of the numerous ultrafine axons visualized via electron microscopy in the nerves of Aplysia may originate from first- or second-order sensory afferents whose cell bodies are located in the periphery. © 1995 Wiley-Liss, Inc.     

Vagal and spinal afferent innervation of the rat esophagus: A combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera. J. Comp. Neurol. 398:289–307, 1998. © 1998 Wiley-Liss, Inc.     

Adult Rat Dorsal Root Ganglion Neurons Extend Neurites on Predegenerated But Not on Normal Peripheral Nerves In Vitro

The abilities of embryonic and adult rat sensory neurons to regenerate were compared when cultured on cryostat sections of normal and lesioned sciatic nerve tissues. Differences in neurite growth, visualized by GAP-43 immunolabelling, were most pronounced on substrata consisting of longitudinal sections of normal versus predegenerated sciatic nerve. Adult dorsal root ganglion (DRG) neurons grew only on the lesioned nerves. Neurites extended along these sections in a characteristically longitudinal orientation, and this growth was not dependent on nerve growth factor. Embryonic DRG neurons extended neurites on sections from both types of nerves. These results highlight important differences in the regenerative abilities of embryonic and adult DRG neurons when grown on physiologically appropriate substrata.