P2X3 is expressed by DRG neurons that terminate in inner lamina II

The P2X3 receptor subunit, a member of the P2X family of ATP-gated ion channels, is almost exclusively localized in sensory neurons. In the present study, we sought to gain insight into the role of P2X3 and P2X3-containing neurons in sensory transmission, using immunohistochemical approaches. In rat dorsal root ganglia (DRG), P2X3-immunoreactivity (-ir) was observed in small- and medium-sized neurons. Approximately 40% of DRG neuronal profiles in normal rats contained P2X3-ir. In rats that had received neonatal capsaicin treatment, the number of P2X3-positive neurons was decreased by approximately 70%. Analysis of the colocalization of P2X3-ir with cytochemical markers of DRG neurons indicated that approximately 94% of the P2X3-positive neuronal profiles were labelled by isolectin B4 from Bandeiraea simplicifolia, while only 3% contained substance P-ir, and 7% contained somatostatin-ir. In dorsal horn of rat spinal cord, P2X3-ir was observed in the inner portion of lamina II and was reduced subsequent to dorsal rhizotomy, as well as subsequent to neonatal capsaicin treatment. Finally, P2X3-ir accumulated proximal to the site of sciatic nerve ligation, and was seen in nerve fibres in skin and corneal epithelium. In summary, our results suggest that P2X3 is expressed by a functionally heterogeneous population of BSI-B4-binding sensory neurons, and is transported into both central and peripheral processes of these neurons.     

The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor

We have studied the distribution and regulation of the P2X3 receptor (a ligand-gated ion channel activated by ATP) in adult dorsal root ganglion (DRG) neurons using a polyclonal antibody. P2X3 receptor immunoreactivity was normally present in about 35% of L4/5 DRG neurons, virtually all small in diameter. In the dorsal horn, P2X3 receptor expression was restricted to the terminals of sensory neurons terminating in lamina IIinner. P2X3 receptors were expressed in approximately equal numbers of sensory neurons projecting to skin and viscera but in very few of those innervating skeletal muscle. P2X3 receptors were found mostly in sensory neurons that bind the lectin IB4. After sciatic nerve axotomy, P2X3 receptor expression dropped by more than 50% in L4/5 DRG. Glial cell line-derived neurotrophic factor (GDNF), delivered intrathecally, completely reversed axotomy-induced down-regulation of the P2X3 receptor. We conclude that P2X3 receptors are normally expressed in nociceptive primary sensory neurons, predominantly the nonpeptidergic nociceptors. P2X3 receptors are down-regulated following peripheral nerve injury and their expression can be regulated by GDNF.     

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.     

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.     

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.     

Ultrastructural localization of P2X3 receptors in rat sensory neurons

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.     

Expression and regulation of the neuropeptide Y Y2 receptor in sensory and autonomic ganglia

The Y2 subtype of neuropeptide tyrosine (NPY) receptors (Y2R) and some neuropeptides have been studied with in situ hybridization in sensory and autonomic neurons of rat and monkey. Between 10% and 20% of the lumbar dorsal root ganglion (DRG) neuron profiles (NPs) contain Y2R mRNA in the rat and monkey. In rat DRGs Y2R mRNA is expressed in calcitonin gene-related peptide (CGRP)-positive, medium-sized, and large neurons, that is in a complementary fashion to the Y1R that is located in small CGRP neurons. In monkey DRGs Y2R mRNA is expressed mainly in small neurons. Peripheral axotomy up-regulates the Y2R in small and large DRG neurons in both species. Y2R and NPY mRNAs are colocalized in many large neurons in axotomized rat DRGs. Y2R mRNA is expressed in 50% of the NPs in the nodose ganglion with a modest increase after axotomy. Y2R mRNA is detected in a few NPs in normal rat superior cervical ganglia, with a marked increase after transection of the carotid nerves. No Y2R mRNA-positive, but many (approximately 30%) weakly Y1R mRNA-positive NPs were found in the sphenopalatine ganglion. Finally, Y2R mRNA levels increase in rat spinal motoneurons after axotomy. Thus, under normal circumstances NPY may act on Y1 and Y2Rs expressed, respectively, in small and large CGRP-positive DRG neurons in the rat. Y2R may be an important receptor in the viscero-sensory neurons. Y2Rs may be particularly important after axotomy serving as presynaptic and/or autoreceptors on rat DRG, superior cervical ganglion, and nodose ganglion neurons and as presynaptic receptors in monkey DRG neurons.     

Human and rat primary C-fibre afferents store and release secretoneurin, a novel neuropeptide

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and, lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (< 25 microns) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexistence of calcium-binding proteins in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of the calcium-binding proteins (CaBPs), calbindin D-28k, parvalbumin and S100 protein, were immunohistochemically examined in the glossopharyngeal and vagal sensory ganglia, the carotid body and taste buds. The CaBPs were found in each ganglion with the nodose ganglion containing the largest number of CaBP-immunoreactive (ir) cells (calbindin D-28k > or = S100 > parvalbumin). The coexistence of CaBPs was found in neurons of the nodose, petrosal, and jugular ganglia. Calbindin D-28k-ir neurons in the nodose and petrosal ganglia frequently colocalized S100-ir whereas calbindin D-28k-ir neurons in the jugular ganglion less frequently contained S100-ir. Only small percentages of calbindin D-28k-ir neurons in each ganglion colocalized parvalbumin. Similarly, S100-ir neurons in the nodose and petrosal ganglia frequently colocalized calbindin D-28k-ir whereas S100-ir neurons in the jugular ganglion less frequently contained calbindin D-28k-ir. Moderate to small percentages of S100-ir neurons in each ganglion colocalized parvalbumin. Parvalbumin-ir neurons nearly always colocalized S100-ir in the nodose, petrosal and jugular ganglia. Moderate to small percentages of parvalbumin-ir neurons in each ganglion colocalized calbindin D-28k. Whereas calbindin D-28k- and S100-ir were colocalized in nerve fibers and cells within taste buds of circumvallate papilla of the tongue, the coexistence of these CaBPs could not be determined in the carotid body. These findings suggest a co-operative role for CaBPs in the functions of subpopulations of nodose and petrosal ganglia neurons.     

The regulation mechanism of HLA class II gene expression at the level of mRNA stability

Sensory ganglia (trigeminal, jugular, nodose, cervical and lumbar dorsal root ganglia) of the guinea-pig were investigated for the presence of a constitutive carbon monoxide-generating enzyme, heme oxygenase-2 (HO-2). A 36-kDa HO-2 immunoreactive protein was identified by Western blotting in protein extracts from dorsal root ganglia and localized by immunohistochemistry to all neuronal perikarya, including both substance P-positive and substance P-negative neurons, in all ganglia investigated. This ubiquitous distribution points to a general requirement for HO-2 in primary afferent neurons rather than to an association with a specific functionally defined subpopulation. Neither the axons of the sensory neurons nor their peripheral terminals in the skin and around visceral arteries were HO-2 immunoreactive. Explants of dorsal root ganglia with crushes placed on the dorsal roots showed accumulation of the neuropeptide, substance P, at the ganglionic side of the crush, but these axons were non-reactive to HO-2, indicating that there is no substantial transport of HO-2 towards the central ending of these sensory neurons. Collectively, the findings suggest that HO-2 exerts it major effects within the sensory ganglia themselves.     

Comparison of desflurane with isoflurane or propofol in spontaneously breathing ambulatory patients

The coexistence of S100beta with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100beta immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100beta-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100beta-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100beta-ir. Many neurons in the nodose ganglion colocalized S100beta-ir and NADPH-d activity, whereas S100beta-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100beta- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100beta and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

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.     

Tibial intramedullary nailing without open drilling

There are presently two competitive theories that attempt to explain the etiology of multiple sclerosis (MS). Briefly summarized, they are: 1. An infection, probably of viral type, may attack the oligodendroglia of the central nervous system; or, 2. An autoimmune process may begin with an infection of the peripheral lymphatic immune system, producing antibodies that cross the blood-brain barrier, leading to myelinoclasia. Since 1935, research has been directed toward myelin of the central nervous system and the myelin sheaths of peripheral nerve; however, dorsal root and cranial sensory ganglia (DRG) have apparently not been studied. The present hypothesis states that an infectious agent (probably viral) finds privileged sanctuary in the dorsal root and cranial sensory ganglia (DRG): thereafter periodically invading the spinal cord, brain, or peripheral nerve. Previously reported erratic spinal fluid viral titers and cultures can be explained by differences in the anatomy of the DRG in which there is a variable and limited contact of spinal fluid with sensory ganglia. Clues to this hypothesis were noted by the author during routine neurological examinations of patients with MS, in which sensory signs and symptoms were frequently encountered. This clinical observation has also been reported by others who found such symptoms in 75% of MS patients, ranking second only to incoordination.     

Peripheral and central target requirements for survival of embryonic rat dorsal root ganglion neurons in slice cultures

Developmental cell death in the nervous system usually is controlled by the availability of target-derived trophic factors. It is well established that dorsal root ganglia (DRG) neurons require the presence of their peripheral target for survival, but because of their central projections, it is possible that the spinal cord also may be required. Before examining this possibility in rat embryos, we first used terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to determine that thoracic DRG cell death occurred from embryonic day 15 (E15) to E18. To determine the target requirements of DRG neurons, we used organotypic slice cultures of E15 thoracic trunk segments. After peripheral target removal, essentially all DRG neurons disappeared within 5 d. In contrast, after removal of the spinal cord, approximately half of the DRG neurons survived for at least 8 d. Hence, some E15 DRG neurons could survive without the spinal cord. However, those DRG neurons that died after spinal cord ablation apparently required trophic factors from both central and peripheral targets, because the presence of only one of these tissues was not adequate by itself to support this cell group. Addition of neurotrophin-3 (NT-3) to the culture medium rescued some DRG neurons after CNS removal, suggesting a possible role for NT-3 in vivo. In other experiments, cultures were established from older (E16) embryos, and essentially all neurons survived after spinal cord ablation, even without added factors. These and other experiments indicated that approximately 65% of DRG neurons are transiently dependent on the CNS early in development.     

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.     

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.     

Sensory axons are guided by local cues in the developing dorsal spinal cord

During development, different classes of sensory neurons establish distinctive central projections within the spinal cord. Muscle spindle afferents (Ia fibers) grow ventrally through the dorsal horn to the ventral cord, whereas cutaneous sensory collaterals remain confined to the dorsal horn. We have studied the nature of the cues used by Ia fibers in establishing their characteristic projections within the dorsal horn. An organotypic culture preparation of embryonic chicken spinal cord and sensory ganglia was used to test the influence of ventral spinal cord and local cues within the dorsal spinal cord on the growing Ia afferents. When the ventral half of the spinal cord was replaced with an inverted duplicate dorsal half, Ia fibers entering through the dorsal columns still grew ventrally within the host dorsal horn. After the fibers entered the duplicate dorsal half, they continued growing in the same direction. With respect to the duplicate dorsal tissue, this was in an opposite, ventral-to-dorsal, direction. In both cases, however, Ia collaterals remained confined to the medial dorsal laminae. Restriction to these laminae was maintained even when the fibers had to change their direction of growth to stay within them. These results show that cues from the ventral cord are not required for the development of correct Ia projections within the dorsal horn. Local, rather than long-range directional, cues appear to determine the pattern of these projections. When the ventral half of the spinal cord was left intact but sensory axons were forced to enter the dorsal gray matter growing rostrally or caudally, their collateral axons grew in random directions, further showing the absence of directional cues even when the ventral cord was present. Taken together, these observations suggest that Ia fibers are guided by local positional cues that keep them confined to the medial gray matter within the dorsal horn, but their direction of growth is determined primarily by their orientation and position as they enter the dorsal gray matter.     

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.     

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.