Technical note: a blind test of mandibular ramus flexure as a morphologic indicator of sexual dimorphism in the human skeleton

Following axotomy most medial septal neurons in the adult rat brain have dramatically reduced numbers of choline acetyltransferase (ChAT) positive neurons. Since leukemia inhibitory factor (LIF) promotes cholinergic expression in several neuronal populations, the aim of this study was to determine if LIF would continue to support cholinergic expression in axotomized medial septal neurons. Mini-osmotic pumps were used to infuse saline or LIF into the lateral cerebral ventricle. Counts of ChAT and low-affinity nerve growth factor (p75NGFR) immunostained neurons indicated that LIF-treated animals retained ChAT expression in > 90% of axotomized neurons whereas in saline-infused animals this was < 30%. Also, LIF was equally effective in maintaining p75NGFR expression levels in axotomized medial septal neurons.     

ADP-ribosylation of serum proteins: evaluation as a potential tumor marker

The low-affinity p75 receptor for nerve growth factor (p75NGFR) has been implicated in mediating neuronal cell death in vitro. A recent in vitro study from our laboratory showed that the death of sensory neurons can be prevented by reducing the levels of p75NGFR with antisense oligonucleotides. To determine if p75NGFR also functions as a death signal in vivo, we have attempted to reduce its expression in peripheral sensory neurons by applying antisense oligonucleotides to the proximal end of the transected sciatic or median and ulnar nerves. We report here that antisense oligonucleotides, when applied to the proximal stump of a transected peripheral nerve, are retrogradely transported and effectively reduce p75NGFR protein levels in sensory neurons located in the dorsal root ganglia. Furthermore, treatment of the proximal nerve stump with antisense p75NGFR oligonucleotides significantly reduced the loss of these axotomized sensory neurons. These findings further support the view that p75NGFR is a death signaling molecule and that it signals death in axotomized neurons in the neonatal sensory nervous system.     

Accelerated Nerve Regeneration in Mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after trauma

In this study we aimed to examine a role for interleukin 6 (IL-6) and its receptor (IL-6R) in peripheral nerve regeneration in vivo. We first observed that cultured mouse embryonic dorsal root ganglia exhibited dramatic neurite extension by simultaneous addition of IL-6 and soluble IL-6R (sIL-6R), a complex that is known to interact with and activate a signal transducing receptor component, gp130. After injury in the hypoglossal nerve in adult mice by ligation, immunoreactivity to IL-6 was upregulated in Schwann cells at the lesional site as well as in the cell bodies of hypoglossal neurons in the brain stem. In the latter, upregulation of the immunoreactivity to IL-6R was also observed. Regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6R antibody. Surprisingly, accelerated regeneration of the axotomized nerve was achieved in transgenic mice constitutively expressing both IL-6 and IL-6R, as compared with nontransgenic controls. These results suggest that the IL-6 signal may play an important role in nerve regeneration after trauma in vivo.     

Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.     

Calcitonin gene-related peptide and alpha-CGRP mRNA expression in cranial motoneurons after hypoglossal nerve injury during postnatal development

Changes in calcitonin gene-related peptide (CGRP) immunoreactivity and alpha-CGRP mRNA expression were determined in the hypoglossal nucleus after the nerve was crushed or transected in rats at 10, 14 and 21 days postnatal. alpha-CGRP mRNA expression was determined in normal, noninjured, hypoglossal nuclei at the three ages and after both injuries in 10 and 21 days postnatal rats. Reinnervation and neuronal survival were assayed. Although the three age groups expressed comparable levels of alpha-CGRP mRNA and its peptide in intact, hypoglossal nuclei, axonal injury produced age-dependent alterations in alpha-CGRP mRNA and CGRP. In the 21 days postnatal rats, changes in alpha-CGRP mRNA and peptide mimicked those reported in adult motoneurons after the same injuries. CGRP was elevated until reinnervation after nerve crush, whereas biphasic elevations occurred after nerve transection. In 21 days postnatal rats, increases in alpha-CGRP mRNA preceded elevations of the peptide but a greater increase resulted initially after nerve transection. An upregulation of alpha-CGRP mRNA also developed initially after both injuries in 10 days postnatal rats but subsequent elevations of alpha-CGRP mRNA did not materialize. In contrast, CGRP immunoreactivity did not increase after either injury in 10 days postnatal rats and, in fact decreased. Levels of CGRP immunoreactivity did not differ from normal amounts after either nerve injury in 14 days postnatal rats. Substantial neuronal cell loss occurred after each injury in 10 and 14 days postnatal rats but was not found in 21 days postnatal rats. Tongue reinnervation by surviving motoneurons was established after all injury paradigms except 10 days postnatal transection. The current findings demonstrate an age-dependent correlation between injury-induced expression of CGRP and hypoglossal motoneuron survival.     

GAP-43 and p75NGFR immunoreactivity in presynaptic cells following neuromuscular blockade by botulinum toxin in rat

Peripheral nerve lesion results in changes in protein expression by neurons and denervated Schwann cells. In the present study we have addressed the question whether similar changes take place following functional denervation. Using immunohistochemistry and immunoelectron microscopy we examined changes in growth-associated protein (GAP-43) and low-affinity nerve growth factor receptor (p75NGFR) in rat gastrocnemius muscle following botulinum toxin-induced paralysis. GAP-43 and p75NGFR were selected because they are not expressed by mature intact motor neurons or Schwann cells, but are expressed following nerve lesion in both motor neurons and denervated Schwann cells. In control muscle, GAP-43 and p75NGFR immunoreactivity was seen only in nerve fibres near blood vessels. Two weeks after toxin injection, GAP-43 immunoreactivity could be seen at the motor endplates and in axons. Intensity of staining increased with longer survival and reached a peak between 4 and 8 weeks post-injection. Ultrastructurally, GAP-43 immunoreactivity was confined to nerve terminals and axons, whereas Schwann cells remained negative. Immunostaining for p75NGFR also increased following toxin injection and was detected in some terminal Schwann cells and in perineurial cells of small nerve fascicles near the paralyzed target cells, but not in axons. These results show that changes in expression of GAP-43 in motor neurons following functional denervation closely resemble the changes following anatomical interruption of nerve-muscle contact. GAP-43 was not expressed in Schwann cells, indicating that its upregulation in these cells is induced by loss of axonal contact or nerve degeneration products. There is no support for a role of p75NGFR in incorporation of neurotrophins in axons. The restriction of p75NGFR expression to terminal Schwann cells and perineurial cells in close proximity to the paralyzed target suggests a role for a target-derived signal or, alternatively, macrophages in eliciting this expression.     

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.     

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.     

Hydroxyapatite paste (BoneSource) used as an onlay implant for supraorbital and malar augmentation

Calbindin D28K, an intracellular calcium-binding protein, acts as Ca2+ buffering system in the cytoplasm. By means of this property, calbindin may protect neurons against large fluctuations in free intracellular Ca2+ and, hence, may prevent cell death. Although axotomy causes a massive influx of calcium into the lesioned neurons, resection of the hypoglossal nerve does not induce extensive neuronal cell death in rats. Even several weeks after axotomy, about 70% of the motoneurons survive despite permanent target deprivation. The mechanisms responsible for this remarkable survival rate are unknown. In this study, we have looked at the modification of calbindin immunoreactivity in axotomized hypoglossal motoneurons. In non-axotomized motoneurons, no calbindin is detectable by immunocytochemistry. Axotomy induced an increase of calbindin immunoreactivity in lesioned motoneurons. This increase, visualised by the number of calbindin-immunoreactive neurons extended from 1 day to 28 days. At this time most, but not all, motoneurons located on the side of the lesion were calbindin-positive as shown by retrograde labeling and immunoquenching. From 14 days post operation, calbindin immunoreactivity decreased and reached its basal value after 35 days post operation. At that time, only fibres were still calbindin immunoreactive. Interestingly, calbindin-immunoreactivity was also increased in almost all cell nuclei, compatible with a nuclear regulation. These data are consistent with the hypothesis that, as a reaction to axotomy, motoneurons trigger an increase in calbindin expression which acts as a compensatory Ca(2+)-buffering system, enabling neurons to maintain Ca2+ homeostasis and the survival of many motoneurons after axotomy.     

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.     

Heat shock protein 27: developmental regulation and expression after peripheral nerve injury

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.     

Aberrant reinnervation following hypoglossal nerve damage

Hypoglossal nerve damage is a known complication of carotid endarterectomy, occurring in approximately 5% of endarterectomies. The vast majority of these patients recover without functional disability from this injury even if the tongue remains hemiplegic. We report 2 patients who suffered hypoglossal nerve section during neck surgery. Although they were initially mildly symptomatic, they developed increasingly severe dysarthria and dysphagia beginning 4 months after surgery. EMG revealed abnormal coactivation of the genioglossus and styloglossus muscles on the affected side, suggesting aberrant reinnervation. Aberrant reinnervation is a well-known complication of facial nerve injury, but has not been previously recognized in hypoglossal nerve injury. Like the face, the tongue is composed of many muscles that must perform complex movements. Normally, injury to one hypoglossal nerve causes little or no disability, but when aberrant reinnervation occurs, the tongue no longer moves in a coordinated manner, and significant dysarthria ensues.     

Motor denervation induces altered muscle fibre type densities and atrophy in a rat model of neuropathic pain

Loose ligation of a sciatic nerve in rats (chronic constriction injury; CCI) provokes sensory, autonomic, and motor disturbances like those observed in humans with partial peripheral nerve injury. So far, it is unknown whether these motor disturbances result from (mechanical) allodynia or from damage to the motor neuron. These considerations prompted us to assess, in CCI rats, the density of motor axons in both the ligated sciatic nerve and the ipsilateral femoral nerve. To this end, we determined the number of cholinesterase positive fibres. It has been demonstrated previously that muscle fibre type density may be used as a measure of motor denervation and/or hypokinesia. Therefore, the myofibrillar ATPase reaction was employed to assess fibre type density in biopsies obtained from the lateral gastrocnemius muscle (innervated by sciatic nerve) and rectus femoris muscle (innervated by femoral nerve). We observed axonal degeneration of motor fibres within the loosely ligated sciatic nerve, both at an intermediate (day 21) and at a late stage (day 90) after nerve injury. The reduction in the number of motor nerve fibres was more pronounced distal to the site of the ligatures than proximal. A (less pronounced) reduction of motor fibres was observed in the ipsilateral (non-ligated) femoral nerve. In line with these findings, we observed altered fibre type densities in muscle tissue innervated by the ligated sciatic nerve as well as the non-ligated femoral nerve indicative of motor denervation rather than hypokinesia. The findings of this study suggest that the motor disorder induced by partial nerve injury involves degeneration of motor nerve fibres not only within the primarily affected nerve but also within adjacent large peripheral nerves. This spread outside the territory of the primarily affected nerve suggests degeneration of motor neurons at the level of the central nervous system.     

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.     

Somatotopic redistribution of c-fos expressing neurons in the superficial dorsal horn after peripheral nerve injury

The functional somatotopic reorganization of the lumbar spinal cord dorsal horn after nerve injury was studied in the rat by mapping the stimulus-evoked distribution of neurons expressing proto-oncogene c-fos. In three different nerve injury paradigms, the saphenous nerve was electrically stimulated at C-fibre strength at survival times ranging from 40 h to more than six months: 1) Saphenous nerve stimulation from three weeks onwards after ipsilateral sciatic nerve transection resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous territory in laminae I-II, and an expansion of the saphenous territory into the denervated sciatic territory until 14 weeks postinjury. 2) Saphenous nerve stimulation from five days onwards after ipsilateral sciatic nerve section combined with saphenous nerve crush resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous nerve territory, and an expansion of the saphenous nerve territory into the denervated sciatic nerve territory. 3) Stimulation of the crushed nerve (without previous adjacent nerve section) at five days, but not at eight months resulted in a temporary increase in the number of Fos-immunoreactive neurons within the territory of the injured nerve, and no change in area at either survival time. The results indicate that nerve injury results in an increased capacity of afferents in an adjacent uninjured, or regenerating nerve, to excite neurons both in its own and in the territory of the permanently injured nerve in the dorsal horn. The onset and duration of the increased postsynaptic excitability and expansion depends on the types of nerve injuries involved. These findings indicate the complexity of the central changes that follows in nerve injuries that contain a mixture of uninjured, regenerating and permanently destroyed afferents.     

Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury

Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury.     

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.     

Introduction of the glutamate receptor subunit 1 into motor neurons in vitro and in vivo using a recombinant herpes simplex virus

We developed and characterized a recombinant herpes simplex virus vector and used it to introduce the complementary DNA encoding glutamate receptor subunit 1 flip into postmitotic motor neurons. Infection of purified motor neurons in vitro with this vector resulted in selective, high-level expression of glutamate receptor subunit 1 immunoreactivity in nearly 100% of the neurons. Patch-clamp experiments demonstrated that the protein product of the glutamate receptor subunit 1 flip transgene assembles into functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels. Herpes simplex virus-glutamate receptor subunit 1 flip was introduced into spinal cord cells by direct injection into the ventral horn and selectively into motor neurons by sciatic nerve injection. High levels of expression were sustained for at least one week and were accompanied by changes in the ionic permeability of AMPA receptors in transgene-expressing neurons. Throughout the first week of infection, there was little evidence for toxicity. Herpes simplex virus provides a versatile tool for manipulating the glutamate receptor phenotype of postmitotic neurons and will permit study of the role of individual glutamate receptor subunits in neuronal physiology and pathophysiology.     

Expression of glutamate receptor subunit 1 and nitric oxide synthase in the hypoglossal nucleus and dorsal vagal nucleus in the rat after neurectomy

Using nitric oxide synthase (NOS) and glutamate receptor subunit 1 (GluR1) immunohistochemistry, the present study demonstrated changes in the expression of NOS and GluR1 in the hypoglossal (HN) and dorsal vagal nucleus (DVN) after neurectomy. Two and 7 days after sectioning the left hypoglossal nerve, NOS expression was seen in a few neurons but GluR1 immunoreactivity was drastically reduced in the ipsilateral HN. The upregulation of NOS immunoreactivity in the HN appeared to peak at 14 days postoperation (dpo). At this period, however, the GluR1 immunoreactivity almost completely disappeared. Twenty-one, 35 and 56 days after neurectomy, NOS immunoreactivity was still expressed in the ipsilateral HN; at the same time, GluR1 immunoreactivity reappeared in a few neurons of the nucleus. Ninety days after operation, NOS immunoreactivity completely disappeared on the operated side of the nucleus, but GluR1 immunoreactivity was re-expressed in many hypoglossal neurons. The number of such neurons was obviously less than that on the unoperated side. After sectioning the left vagus nerve in the same animals, the expression of NOS immunoreactivity in the ipsilateral DVN resembled that in the HN. On the unoperated side, NOS immunoreactivity was demonstrated in some neurons in the DVN, like that in the normal. In both normal and operated rats, only a few neurons expressed GluR1 immunoreactive products on both the operated and unoperated sides of the DVN. Combining with previous results on protein synthesis observed at 14 dpo, the present investigation suggested that in the early stages after neurectomy, the expression of NOS immunoreactivity and loss of GluR1 expression in the HN may indicate the organism's double protective mechanism. Lastly, the reappearance of GluR1 in the same nucleus from 21 to 90 days after operation may reflect functional recovery of the hypoglossal neurons.