Down-regulation of mu-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy

To understand the role of opioids and their receptors in chronic pain following peripheral nerve injury, we have studied the mu-opioid receptor in rat and monkey lumbar 4 and 5 dorsal root ganglion neurons and the superficial dorsal horn of the spinal cord under normal circumstances and after peripheral axotomy. Our results show that many small neurons in rat and monkey dorsal root ganglia, and some medium-sized and large neurons in rat dorsal root ganglia, express mu-opioid receptor-like immunoreactivity. Most of these neurons contain calcitonin gene-related peptide. The mu-opioid receptor was closely associated with the somatic plasmalemma of the dorsal root ganglion neurons. Both mu-opioid receptor-immunoreactive nerve fibers and cell bodies were observed in lamina II of the dorsal horn. The highest intensity of mu-opioid receptor-like immunoreactivity was observed in the deep part of lamina II. Most mu-opioid receptor-like immunoreactivity in the dorsal horn originated from spinal neurons. A few mu-opioid receptor-positive peripheral afferent terminals in the rat and monkey dorsal horn were calcitonin gene-related peptide-immunoreactive. In addition to pre- and post-junctional receptors in rat and monkey dorsal horn neurons, mu-opioid receptors were localized on the presynaptic membrane of some synapses of primary afferent terminals in the monkey dorsal horn. Peripheral axotomy caused a reduction in the number and intensity of mu-opioid receptor-positive neurons in the rat and monkey dorsal root ganglia, and of mu-opioid receptor-like immunoreactivity in the dorsal horn of the spinal cord. The decrease in mu-opioid receptor-like immunoreactivity was more pronounced in the monkey than in the rat dorsal root ganglia and spinal cord. It is probable that there was a parallel trans-synaptic down-regulation of mu-opioid-like immunoreactivity in local dorsal horn neurons of the monkey. These data suggest that one factor underlying the well known insensitivity of neuropathic pain to opioid analgesics could be due to a marked reduction in the number of mu-opioid receptors in the axotomized sensory neurons and in interneurons in the dorsal horn of the spinal cord.     

Partial and complete sciatic nerve injuries induce similar increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in primary sensory neurons and their central projections

Partial nerve injury is more likely to cause neuropathic pain than complete nerve injury. We have compared the changes in neuropeptide expression in primary sensory neurons which follow complete and partial injuries to determine if these might be involved. Since more neurons are damaged by complete injury, we expected that complete sciatic nerve injury would simply cause greater increases in neuropeptide Y and vasoactive intestinal peptide than partial injury. We examined neuropeptide Y and vasoactive intestinal peptide immunoreactivities in L4 and L5 dorsal root ganglia, the dorsal horn of L4-L5 spinal cord, and the gracile nuclei of rats killed 14 days after unilateral complete sciatic nerve transection, partial sciatic nerve transection and chronic constriction injury of the sciatic nerves. In all three groups of rats, neuropeptide Y- and vasoactive intestinal peptide-immunoreactive neurons were increased in the ipsilateral L4 and L5 dorsal root ganglion when compared with the contralateral side. Most neuropeptide Y-immunoreactive neurons were of medium and large size, but a few were small. Neuropeptide Y-immunoreactive axonal fibers were increased from laminae I to IV, and vasoactive intestinal peptide-immunoreactive axonal fibers were increased in laminae I and II, of the ipsilateral dorsal horn of L4-L5 spinal cord. The increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in the dorsal horn were similar among the three groups. However, only after constriction injury were some vasoactive intestinal peptide-immunoreactive neurons seen in the deeper laminae of the ipsilateral dorsal horn. Robust neuropeptide Y-immunoreactive axonal fibers and some neuropeptide Y-immunoreactive cells were seen in the ipsilateral gracile nuclei of all three groups of animals, but neuropeptide Y-immunoreactive cells were more prominent after constriction injury. Contrary to our expectations, partial and complete sciatic nerve injuries induced similar increases in neuropeptide Y and vasoactive intestinal peptide in lumbar dorsal root ganglion neurons and their central projections in the dorsal horn and the gracile nuclei two weeks after injury. Some neurons whose axons were spared by partial injury may also increase neuropeptide Y or vasoactive intestinal peptide expression. Altered neuropeptide release from these functional sensory neurons may play a role in neuropathic pain.     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury

Pituitary adenylate cyclase activating peptide (PACAP) is expressed in a population of capsaicin-sensitive primary sensory neurons of small to medium size in the rat. In the present report we have examined the effect of sciatic nerve injury (unilateral transection) on PACAP expression (immunocytochemistry, radioimmunoassay, in situ hybridization and northern blot analysis) in dorsal root ganglia at the lumbar level and on immunoreactive PACAP in the spinal cord and in the sciatic nerve stump. For comparison, calcitonin gene-related peptide was examined. In dorsal root ganglia of the intact side immunoreactive PACAP and PACAP messenger RNA were localised to a population of nerve cell bodies of small to medium size. In dorsal root ganglia on the injured side, PACAP-immunoreactive nerve cell bodies were more numerous and PACAP messenger RNA was considerably more abundant as studied 14 days after sciatic nerve transection. By contrast, calcitonin gene-related peptide-containing nerve cell bodies were numerous and rich in calcitonin gene-related peptide messenger RNA in dorsal root ganglia on the intact side, while after transection both the number of immunoreactive nerve cell bodies and their content of messenger RNA were markedly reduced. There were indications of axotomy-induced expression of PACAP messenger RNA in larger neurons. In the dorsal horn of the spinal cord on the intact side PACAP and calcitonin gene-related peptide-immunoreactive fibres were densely accumulated in the superficial layers. On the transected side the densities of both PACAP and calcitonin gene-related peptide-immunoreactive nerve fibres were reduced in the medial part. The data obtained indicate a marked up-regulation of PACAP in sensory neurons following peripheral nerve injury. Since PACAP depresses a C-fibre evoked flexion reflex, this may have implications for sensory transmission. Further, in view of the known promoting effects of PACAP on neuronal survival and differentiation and non-neuronal cell growth as well as its proinflammatory effects a role of PACAP in the neuronal and periaxonal tissue restoration after injury is not inconceivable.     

Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: possible implications for sensory sprouting

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I-II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury.     

Differential expression of galanin immunoreactivities in the primary sensory neurons following partial and complete sciatic nerve injuries

Neuropeptide expression in primary sensory neurons is highly plastic in response to peripheral nerve axotomy. While neuropeptide changes following complete sciatic nerve injury have been extensively studied, much less is known about the effects of partial sciatic nerve injuries on neuropeptide plasticity. Galanin. a possible endogenous analgesic peptide, was up-regulated in primary sensory neurons following complete sciatic nerve injury. We investigated the effects of partial sciatic nerve injuries on galanin expression in primary sensory neurons, and compared this effect with that after complete sciatic nerve injury. Complete transection, partial transection and chronic constriction injury were made, respectively, on the sciatic nerves of three groups of rats at high thigh level. Animals were allowed to survive for four and 14 days before being killed. L4 and L5 dorsal root ganglia, L4 5 spinal cord and lower brainstem were processed for galanin immunocytochemical staining. After all three types of sciatic nerve injuries, galanin-immunoreactive neurons were significantly increased in the ipsilateral dorsal root ganglia, and galanin-immunoreactive axonal fibres were dramatically increased in the superficial laminae of the dorsal horn and the gracile nuclei, compared to the contralateral side. However, in partial injury models, the percentages of galanin-immunoreactive dorsal root ganglion neurons were significantly higher than in complete nerve transection. Size frequency distribution analysis detected that more medium- and large-size galanin-immunoreactive dorsal root ganglion neurons were present after partial nerve transection and constriction injury than after complete nerve transection. Using a combined approach of retrograde tracing of flurorescent dyes and galanin immunostaining, we found that a partial transection increased the proportions of galanin-immunoreactive neurons among both axotomized and non-axotomized neurons. Galanin-immunoreactive axonal fibres were not only detected in the superficial laminae, but also in the deeper laminae of the dorsal horn of partial injury animals. Furthermore, more galanin-immunoreactive axonal fibres were observed in the ipsilateral gracile nuclei of partially injured rats than in completely injured rats. We conclude that partial sciatic nerve injuries induced greater galanin up-regulation in medium- and large-size dorsal root ganglion neurons than complete sciatic nerve injury. Galanin expression in primary sensory neurons seems to be differentially regulated following partial and complete sciatic nerve injuries.     

The time-course of abeta-evoked c-fos expression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury

We have examined the mechanisms underlying Abeta-evoked c-fos expression in the dorsal horn and gracile nucleus following either sciatic nerve section or crush injury. The results indicate that in the spinal cord Abeta-evoked c-fos does not depend on primary afferent sprouting but is associated with the disconnection from the peripheral target since its expression in the dorsal horn reverts to normal after crush injury when regeneration occurs but persists after cut and ligation where regeneration is prevented. In contrast, however, Abeta-evoked c-fos expression in the gracile nucleus may be under some other control since its expression appears independent of peripheral nerve regeneration.     

Changes in brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia, spinal cord, and gracile nuclei following cut or crush injuries

In the present study, we evaluated changes in brain-derived neurotrophic factor (BDNF) immunoreactivity in the rat lumbar (L) 5 dorsal root ganglion (DRG) and areas where afferents from the DRG terminate, the L5 spinal cord and gracile nuclei, following unilateral sciatic nerve transection or crush. From 3 days to 4 weeks following cut or crush injury, the percentage of medium and large BDNF-immunoreactive neurons in the ipsilateral DRG increased significantly compared with those on the contralateral side. Following cut injury, there was no significant change in the percentage of small BDNF-immunoreactive neurons in the ipsilateral DRG; however, the intensity of immunoreactivity of these cells decreased. Following crush injury, however, both the percentage and intensity of small BDNF-immunoreactive neurons in the ipsilateral DRG significantly increased. Following cut injury, the expression of BDNF-immunoreactive axonal fibers decreased markedly in the ipsilateral superficial laminae of the L5 spinal cord and increased significantly in the ipsilateral deeper laminae of the spinal cord and gracile nuclei. Crush injury induced a marked increase in the expression of BDNF-immunoreactive axonal fibers in the superficial laminae of the spinal cord and gracile nuclei. These differences in BDNF response in the DRG and spinal cord after cut or crush injuries may reflect differences in trophic support to the injured DRG neurons and altered neuronal activity in the spinal cord and gracile nuclei following different types of peripheral nerve injury.     

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.     

Immunocytochemical co-localization of substance P and calcitonin gene-related peptide in afferent renal nerve soma of the rat

Substance P, calcitonin gene-related peptide and somatostatin immunoreactivities have been demonstrated in putative afferent renal nerve fibers in the rat. Utilizing retrograde-tracing and immunohistochemistry, we labeled afferent renal nerve soma throughout dorsal root ganglia T9 to L1. Most (85%) of afferent renal nerve perikarya were immunoreactive for calcitonin gene-related peptide, 21% had substance P immunoreactivity and none had somatostatin immunoreactivity. All renal afferents immunoreactive for substance P also contained calcitonin gene-related peptide. These results provide evidence that calcitonin gene-related peptide and substance P are present and co-localized in afferent renal nerves, and therefore, mediate transmission of afferent renal input to the spinal cord in the rat.     

Sympathetic axons surround neuropeptide-negative axotomized sensory neurons

Nerve injury can lead to sympathetically dependent neuropathic pain. A possible site of sympathetic-sensory interaction is the dorsal root ganglion (DRG), where sympathetic axons form pericellular 'baskets' around a subpopulation of DRG neurons. Since these structures possibly represent functional units of sympathetic pain, we attempted to characterize the neuropeptidergic phenotype of basketed DRG neurons. We performed double-labeling immunohistochemistry for tyrosine hydroxylase and neuropeptides on DRG sections, 2 weeks following L5 spinal nerve ligation (a well-characterized animal model of sympathetic pain). We found that basketed DRG neurons typically do not contain substance P, calcitonin gene-related peptide, galanin, neuropeptide tyrosine, or vasoactive intestinal polypeptide, and we conclude that if sympathetic baskets contribute to neuropathic pain, the involvement of these neuropeptides is unimportant.     

Corticotropin releasing factor-like immunoreactivity in afferent projections to the sacral spinal cord of the cat

Distribution and origin of corticotropin releasing factor (CRF) in the thoraco-lumbar and sacral spinal cord of the cat has been studied using immunohistochemical method. CRF immunoreactive (CRF-IR) nerve fibers and terminals were most prominent in dorsal part of sacral spinal cord. In the sacral segments of the spinal cord, immunoreactivity for CRF was detected in a prominent bundle of axons and varicosities extending from Lissauer's tract (LT) along the lateral edge of the superficial dorsal horn (laminae I and II) to laminae V at the base of the dorsal horn. Individual CRF-IR fibers passed from the bundle in ventral medial and ventrolateral directions to the dorsal commissure and the sacral preganglionic nucleus (SPN), respectively. The bundle of CRF-IR axons closely resembled vasoactive intestinal polypeptide (VIP) containing fibers in LT and on the lateral edge of the dorsal horn. Sacral dorsal root transection eliminated both the CRF and VIP fiber staining in the dorsal horn. Spinal transection at the T12-T13 segmental level did not influence the CRF- or VIP-IR. Less intense CRF-IR was also present in fibers in: (1) the dorsal lateral funiculus adjacent to LT, (2) the superficial layers of the dorsal horn and intermediolateral nucleus at thoracolumbar spinal levels, (3) the ventral horn, including Onuf's nucleus, (4) the intermediate gray matter including the dorsal gray commissure, and (5) the SPN. The similarity in the distribution of CRF-IR and pelvic nerve afferent projections in the sacral spinal cord raises the possibility that CRF may be a transmitter in afferent neurons innervating the pelvic viscera.     

An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats

The acid sensing ion channel (ASIC) identified in rat brain and spinal cord is potentially involved in the transmission of acid-induced nociception. We have developed polyclonal antisera against ASIC, and used them to screen rat brain and spinal cord using immunocytochemistry. ASIC-immunoreactivity (-ir) is present in but not limited to the superficial dorsal horn, the dorsal root ganglia (DRG) and the spinal trigeminal nucleus, as well as peripheral nerve fibers. These observations, combined with the disappearance of ASIC-ir following dorsal rhizotomy, suggest localization of ASIC to primary afferents. DRG ASIC-ir co-localizes with substance P (SP) and calcitonin gene-related peptide (CGRP)-ir in small capsaicin-sensitive cell bodies, suggesting that ASIC is poised to play a role in the transduction of noxious stimuli.     

Central projection of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive trigeminal primary neurons in the rat

Substance P (SP) is implicated in transmission of primary afferent nociceptive signals. In primary neurons, SP is colocalized with calcitonin gene-related peptide (CGRP), which is another neuropeptide marker for small to medium primary neurons. CGRP coreleased with SP augments the postsynaptic effect of SP and thereby modulates the nociceptive transmission. This study demonstrates the distribution of CGRP-like immunoreactivity (-ir) and SP-ir in the lower brainstem of normal rats and after trigeminal rhizotomy or tractotomy at the level of subnucleus interpolaris (Vi). By comparing the results obtained from normal and deafferented rats, we analyzed the central projection of trigeminal primary nociceptors. The CGRP-immunoreactive (-ir) trigeminal primaries projected to the entire rostrocaudal extent of the spinal trigeminal nucleus, the principal nucleus (PrV), the paratrigeminal nucleus (paraV), and the lateral subnucleus of solitary tract nucleus (STN) on the ipsilateral side. The trigeminal primaries projecting to the spinal trigeminal nucleus, paraV and STN also contained SP-ir. The ipsilateral trigeminal primaries were the exclusive source of CGRP-ir terminals in the PrV, the Vi and the dorsomedial nucleus within the subnucleus oralis (Vo). The medullary dorsal horn (MDH) and the lateral edge of Vo received convergent CGRP-ir projection from the ipsilateral trigeminal primaries and other neurons. The glossopharyngeal and vagal primaries are candidates for the source of CGRP-ir projection to the Vo and the MDH, while the dorsal root axons supply the MDH with CGRP-ir terminals. In addition, contralateral primary neurons crossing the midline appear to contain CGRP and to terminate in the MDH.     

Ultrasound appearance of gas bubbles in hepatic veins after orthotopic liver transplantation: a phenomenon with consequences?

Neuropathic pain is not well understood. Although central dorsal horn remodelling is likely important in maintaining chronic neuropathic pain, afferent activity from injured nerves or ganglia may initiate these changes. It is suggested, in this review that the peripheral nerve trunk is capable of sustaining a "flare" response as observed in injured skin and other tissues. The injury response may be associated with local vasodilatation, plasma extravasation and the generation of painful local afferent activity sustained by locally originating peptidergic fibers (nervi nervorum). These fibers contain substance P, calcitonin gene-related peptide and other peptides that have been linked to nociceptive transmission. Manipulation of the local injury response of the nerve trunk by pharmacologic means may provide one strategy in the treatment of neuropathic pain.     

Localization of bradykinin-like immunoreactivity in the rat spinal cord: effects of capsaicin, melittin, dorsal rhizotomy and peripheral axotomy

A putative role for bradykinin has been proposed in the processing of sensory information at the level of the spinal cord. Autoradiographic studies have demonstrated the presence of B2 kinin receptor binding sites in superficial laminae of the dorsal horn and a down-regulation of those receptors in rat models of pain injury. In this study, classical immunocytochemistry and confocal microscopy immunofluorescence were used first to localize bradykinin-like immunoreactivity in all major spinal cord segments of naive rats; second, to assess bradykinin-like immunoreactivity changes that occur in animals subjected to various chemical treatments and surgical lesions. High densities of bradykinin-like immunoreactivity were observed in motoneuron of the ventral horn, deeper laminae and nucleus dorsalis of the dorsal horn. Higher magnification of ventral horn showed strong immunostaining of motoneuron perikaryas and their proximal processes. Two types of bradykinin-like immunoreactivity immunostained cellular bodies were observed in deeper laminae of the dorsal horn. These interneurons, morphologically corresponding to islets and antenna-type cells project dendrites to adjacent laminae. Furthermore, numerous strongly marked dendrites, transversally cut, suggest the presence of projection neurons to higher cervical centres. Following unilateral lumbar dorsal rhizotomy (L1-L6) or peripheral lesion of the sciatic nerve, important increases of bradykinin-like immunoreactivity were found in laminae III and IV of the ipsilateral dorsal horn. In contrast, significant decreases of immunodeposits were observed in both cell bodies and numerous dendrites of motoneuron surrounding neuropil. Specific destructions of sensory afferent fibres with capsaicin or selective activation of kallikreins with melittin caused increases of bradykinin-like immunoreactivity in both the dorsal and ventral horns of the spinal cord. These results which demonstrate the cellular localization of bradykinin-like immunoreactivity in both dorsal and ventral horns of the rat spinal cord, further reveal the plasticity of this non-sensory peptidergic system following various chemical and surgical treatments. Hence, these anatomical findings along with earlier functional and receptor autoradiographic studies reinforce the putative role of bradykinin in sensory function.     

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats. J. Neurophysiol. 80: 3356-3360, 1998. Glutamate is a major fast transmitter between primary afferent fibers and dorsal horn neurons in the spinal cord. Recent evidence indicates that ATP acts as another fast transmitter at the rat cervical spinal cord and is proposed to serve as a transmitter for nociception and pain. Sensory synaptic transmission between dorsal root afferent fibers and neurons in the superficial dorsal horn of the lumbar spinal cord were examined by whole cell patch-clamp recording techniques. Experiments were designed to test if ATP could serve as a transmitter at the lumbar spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were completely abolished after the blockade of both glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and N-methyl--aspartate receptors. No residual current was detected, indicating that glutamate but not ATP is a fast transmitter at the dorsal horn of the lumbar spinal cord. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a selective P2x receptor antagonist, produced an inhibitory modulatory effect on fast EPSCs and altered responses to paired-pulse stimulation, suggesting the involvement of a presynaptic mechanism. Intrathecal administration of PPADS did not produce any antinociceptive effect in two different types of behavioral nociceptive tests. The present results suggest that ATP P2x2 receptors modulate excitatory synaptic transmission in the superficial dorsal horn of the lumbar spinal cord by a presynaptic mechanism, and such a mechanism does not play an important role in behavioral responses to noxious heating. The involvement of other P2x subtype receptors, which is are less sensitive to PPADS, in acute nociceptive modulation and persistent pain remains to be investigated.     

NK1, NMDA, 5HT1a, and 5HT2 receptor binding sites in the rat lumbar spinal cord: modulation following sciatic nerve crush

Quantitative receptor binding autoradiography was used to study the NK1, NMDA, 5HT1a, and 5HT2 receptor binding densities in the adult rat lumbar spinal cord from 3 days to 20 weeks following a unilateral crush lesion of the sciatic nerve. NK1 binding density increased unilaterally in the superficial dorsal horn on the side of the sciatic crush to reach levels 60% above controls by 4 weeks following the lesion and returned to control values by 12 weeks. NMDA binding density increased bilaterally and equally in both the dorsal and ventral horns to reach 300% of control values at 2 weeks following the crush and returned to near control values by 20 weeks following the lesion. Serotonergic receptor binding did not change. The changes in NK1 receptor binding density on postsynaptic dorsal horn cells are consistent with a response to the decrease and recovery in the synthesis and transport of tachykinins by the dorsal root ganglion cells following peripheral nerve injury. the bilateral changes in NMDA receptor binding are more likely mediated by polysynaptic pathways in the spinal cord that respond to the changes in metabolic events of the dorsal root ganglion cells evoked by axotomy and regeneration.     

A confocal microscopic survey of serotoninergic axons in the lumbar spinal cord of the rat: co-localization with glutamate decarboxylase and neuropeptides

Patterns of co-localization of serotonin with glutamate decarboxylase (the synthetic enzyme for GABA) or each one of eight neuropeptides (calcitonin gene-related peptide, dynorphin, enkephalin, galanin, neuropeptide Y, neurotensin, substance P and somatostatin) were investigated with dual-colour confocal laser scanning microscopy in the lumbar spinal cords of three adult rats. Four regions of the gray matter were studied (laminae I-II, V, IX and X). The extent of co-localization was estimated by direct assessment of merged pairs of optical sections and by automated image analysis. Co-localization of serotonin and glutamate decarboxylase was found only in a few axons of laminae I-II but was not detected in other laminae. Peptides were not co-localized with serotonin in the superficial dorsal horn but considerable co-localization was found in motor nuclei and sparse co-localization was found in laminae V and X. Galanin and substance P frequently co-existed with serotonin in lamina IX but some co-localization with dynorphin, somatostatin, [Met]enkephalin and neuropeptide Y was also detected. Galanin, substance P and dynorphin were also co-localized with serotonin in a few axons of the deep dorsal horn and in the gray matter around the central canal. Neurotensin and calcitonin gene-related compound did not co-exist with serotonin in any of the laminae investigated. This evidence suggests that different populations of serotoninergic axons project to different regions of the spinal gray matter. Those containing glutamate decarboxylase terminate in the superficial dorsal horn and are likely to be involved in antinociception, whereas those containing peptides terminate principally in motor nuclei and are likely to modulate motor activity.