Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.     

Novel synthesis and release of GABA in cerebellar granule cell cultures after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) is synthesized from glutamate in a single step by the enzyme glutamatic acid decarboxylase (GAD). We sought to determine whether viral vectors containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of CNS neurons. For this purpose, we generated double-cassette defective herpes simplex virus (HSV) vectors that expressed one of the two GAD isoforms (GAD65 or GAD67), and Escherichia coli LacZ. Infection of cerebellar granule cell (CGC) cultures with vectors containing GAD cDNA resulted in a significant increase in isoform-specific expression of GAD, synthesis of GABA, and stimulation-evoked GABA release. GAD65 and GAD67 vector-infected neurons exhibited a comparable profile of GABA levels, synthesis and release, as well as GAD protein distribution. In CGCs cultured for 6 days in vitro (DIV), GABA synthesized after vector-derived GAD expression was released by treatment with glutamate or veratridine, but only in a Ca2+-independent fashion. In more mature (10 DIV) cultures, both Ca2+-dependent, K+ depolarization-induced, as well as Ca2+-independent, veratridine-induced, GABA release was significantly enhanced by GAD vector infection. Treatment of CGCs with kainic acid, which destroys most of the GABAergic neurons (<1% remaining), did not prevent vector-derived expression of GAD nor synthesis of GABA. This suggests that defective HSV vector-derived GAD expression can be used to increase GABA synthesis and release in CNS tissue, even in the relative absence of GABAergic neurons. The use of such GAD vectors in the CNS has potential therapeutic value in neurologic disorders such as epilepsy, chronic pain, Parkinson's and Huntington's disease.     

Origin, course, and laterality of spinocerebellar axons in the North American opossum, Didelphis virginiana

Inhibition of neurons containing gamma-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned mu- and delta-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II-VI of the parietal cortex and in layers II-IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of mu-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, delta-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly.     

Retinal ganglion cell depletion alters the phenotypic expression of GABA and GAD in the rat retina

We have looked at the phenotypic expression of gamma-aminobutyric acid (GABA) and the two isoforms of its synthetic enzyme [glutamic acid decarboxylase (GAD)-65 and -67] in adult rat retinas that had the superior colliculus, pretectum and optic tract lesioned unilaterally at birth. It has been shown previously that this type of manipulation induces retrograde degeneration of retinal ganglion cells presumably without affecting other intraretinal neurons. We present evidence that GABAergic amacrine cells are affected by such manipulation. The number of cells immunoreactive for GABA, GAD-65 and GAD-67 decreased in the inner nuclear layer. In the retinal ganglion cell layer, however, the number of GABA- and GAD-65-labelled cells increased, while the number of GAD-67-labelled cells did not change. Biochemical assay showed that overall GAD activity was not altered in retinas of lesioned animals. Our results support the notion that, while neonatal lesion reorganizes the expression of GABA and GAD in the retina, enzyme activity is maintained within normal levels.     

CNS GABA neurons express the mu-opioid receptor: immunocytochemical studies

It has been proposed that mu-opioid receptors excite neurons in hippocampus and nucleus raphe dorsalis (NRD) by decreasing GABAergic tone. In the present study, we examined whether immunocytochemical evidence of interaction between GABAergic neurons and the mu-opioid receptor could be found in the CNS. Portions of rat brain were sectioned and stained for GABA and for the cloned mu-opioid receptor (MOR1) using two-color immunofluorescence. Neurons double-labeled for GABA and MOR1 were present in hippocampus and NRD, as well as in olfactory bulb, dorsal lateral periaqueductal gray matter, nucleus raphe medianis, nucleus raphe obscurus, and the spinal trigeminal nucleus and tract. We conclude that expression of the mu-opioid receptor by GABAergic neurons is common in the rat CNS.     

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.     

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.     

Suppression of IL-12 production by phosphodiesterase inhibition in murine endotoxemia is IL-10 independent

Limbic system-associated membrane protein (LAMP), a 64-kDa membrane protein, is an axon guidance adhesion molecule expressed by neurons in limbic system-related areas of the CNS. During development, LAMP is expressed on growing axons, growth cones, and their target neurons, but in adults it is restricted to membranes of somata and dendrites. In the adult spinal cord, LAMP immunoreactivity is found only on neurons of lamina II, lamina X, and the intermediolateral cell column and its ultrastructural localization is entirely postsynaptic. We studied changes in the expression of LAMP in lamina II of adult rat spinal cord after L1-S2 dorsal rhizotomy, a procedure that partially deafferents lamina II neurons and induces axonal sprouting by spared systems in lamina II. At the light microscopic level, LAMP immunoreactivity in lamina II was decreased in density at 3, 10, and 60 days postoperatively. This decrease in immunoreactivity suggests that LAMP expression by lamina II neurons may normally be regulated by specific afferent activity. Ultrastructurally, in control lamina II and after deafferentation in both control and deafferented lamina II at 3 and 60 days postoperatively, LAMP expression was restricted to postsynaptic membranes. Ten days after deafferentation, however, when axons are actively sprouting, LAMP was expressed on both axonal and postsynaptic membranes. The reexpression of LAMP on axonal profiles after deafferentation may identify axons that undergo sprouting in response to deafferentation.     

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of the cat visual cortex

The postnatal development of GAD67 and GAD65 protein expression and of GAD67 positive neurons and GAD65 containing axon terminals in cat visual cortex was studied. Western blot analysis showed that the expression of both GAD67 and GAD65 increased to approximately two-thirds of the adult level during the first 5 postnatal weeks and gradually increased thereafter. In adult cats, immunohistochemistry showed that GABA and GAD67 containing neurons were found in all cortical layers. Faint cell body staining was seen with the antibody to GAD65, but it densely labeled puncta. In neonates, GABA and GAD67 immunoreactivity was most intense in two distinct bands, one superficial (Layer 1/Marginal zone), another deep (Layer VI/Subplate). Unlike in adults, GAD65 positive cell bodies were clearly evident in neonates and distributed similarly to, but less frequently than, GABA and GAD67. These GAD65 positive cells frequently had morphologies suggestive of embryonic cells and largely disappeared in older animals. During postnatal development, the neurochemical differentiation of GAD67 positive neurons and GAD65 positive axon terminals across visual cortical laminae followed an inside-outside developmental pattern, which reached adult levels after 10 weeks of age. These results suggest that postnatal development of the visual cortical GABA system involves three distinct processes: (A) a dying off of embryonic GABA cells which could play a role in formation of the cortical plate; (B) a period of relative quiescence of the VC GABA system in the first 5 postnatal weeks which could maximize excitatory NMDA effects during the rising phase of the critical period; (C) the prolonged postnatal maturation of the adult GABA system which could be involved in the crystallization of adult physiological properties and the disappearance of neural plasticity.     

Oxytocinergic and serotonergic innervation of identified lumbosacral nuclei controlling penile erection in the male rat

Penile erection is due to activation of proerectile neurons located in the sacral parasympathetic nucleus of the L6-S1 spinal cord in the rat. Contraction of the ischiocavernosus and bulbospongiosus striated muscles, controlled by motoneurons located in the ventral horn of the L5-L6 spinal cord, reinforces penile erection. Physiological and pharmacological arguments have been provided for a role of oxytocin and serotonin in the spinal regulation of penile erection. Immunohistochemistry of oxytocinergic and serotonergic fibres was performed at the lumbosacral level of the male rat spinal cord, and combined with retrograde tracing from the pelvic nerve or from the ischiocavernosus and bulbospongiosus muscles using wheat germ agglutinin-horseradish peroxidase. Sacral preganglionic neurons retrogradely labelled from the pelvic nerve formed a homogeneous population, predominant at the L6 level. Motoneurons retrogradely labelled from the ischiocavernosus and bulbospongiosus muscles were observed in the medial part of the dorsolateral and in the dorsomedial nuclei. Fibres immunoreactive for oxytocin were mainly distributed in the superficial layers of the dorsal horn, the dorsal gray commissure and the sacral parasympathetic nucleus. Some of these fibres were apposed to retrogradely-labelled sacral preganglionic neurons and at the ultrastructural level, some synapses were evidenced. Fibres immunoreactive for serotonin were largely and densely distributed in the dorsal horn, the dorsal gray commissure, the sacral parasympathetic nucleus and the ventral horn. Some serotonergic fibres occurred in close apposition with retrogradely-labelled sacral preganglionic neurons and motoneurons, and synapses were demonstrated at the ultrastructural level. This study provides morphological support for a role of oxytocin and serotonin on sacral preganglionic neurons innervating pelvic organs and motoneurons innervating the ischiocavernosus and bulbospongiosus muscles.     

TIMP1 and adverse prognosis in non-small cell lung cancer

Antisera were developed that specifically recognize orphanin FQ/nociceptin, the 17 amino acid peptide reported to be the endogenous ligand for the orphan opioid receptor. Immunocytochemical localizations in rat spinal cord demonstrated that orphanin FQ /nociceptin-immunoreactivity (-ir) was abundant in superficial dorsal horn, lateral spinal nucleus and the region dorsal to the central canal, areas that also exhibit prominent enkephalin-and dynorphin-ir. Orphanin FQ/nociceptin-ir was not affected by dorsal rhizotomy, indicating that in spinal cord the peptide is produced by central rather than primary afferent neurons. thus, the distribution of orphanin FQ/nociceptin-ir appeared in neuronal circuits that parallel those containing enkephalin- and dynorphin-ir, with only modest co-existence of these peptides.     

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.     

Nitric oxide in the stress axis

Calcitonin gene-related peptide in sensory primary afferent neurons has an excitatory effect on postsynaptic neurons and potentiates the effect of substance P in the rat spinal dorsal horn. It has been established that calcitonin gene-related peptide expression in dorsal root ganglion neurons is depressed, and the effect of calcitonin gene-related peptide on dorsal horn neurons is attenuated, following peripheral nerve injury. We report here that a subpopulation of injured dorsal root ganglion neurons show increased expression of calcitonin gene-related peptide. Using in situ hybridization and the retrograde tracer, FluoroGold, we detected an increased number of medium- to large-sized rat dorsal root ganglion neurons projecting to the gracile nucleus that expressed alpha-calcitonin gene-related peptide messenger RNA following spinal nerve transection. Immunohistochemistry revealed a significant increase in calcitonin gene-related peptide immunoreactivity in the gracile nucleus and in laminae III-IV of the spinal dorsal horn. These results indicate that a subpopulation of dorsal root ganglion neurons express alpha-calcitonin gene-related peptide messenger RNA in response to peripheral nerve injury, and transport this peptide to the gracile nucleus and to laminae III-IV of the spinal dorsal horn. The increase of the excitatory neuropeptide, calcitonin gene-related peptide, in sites of primary afferent termination may affect the excitability of postsynaptic neurons, and have a role in neuronal plasticity following peripheral nerve injury.     

Peripheral nerve stimulation increases Fos immunoreactivity without affecting type II Ca2+/calmodulin-dependent protein kinase, glutamic acid decarboxylase, or GABAA receptor gene expression in cat spinal cord

Expression patterns of the immediate early gene c-fos and of other genes including those for the alpha-subunit of type II Ca2+/calmodulin-dependent protein kinase (CaMKII alpha), 67-kDa glutamic acid decarboxylase (GAD), and the alpha 1-, beta 2-, and gamma 2-subunits of the GABAA receptor were described in the spinal cord of normal cats and following peripheral nerve stimulation. As revealed by in situ hybridization histochemistry, CaMKII alpha messenger RNA (mRNA) is normally distributed only in cells of Rexed's laminae I-IV, whereas GAD mRNA is expressed by subpopulations of cells in all laminae, with the heaviest hybridization signal found in laminae I-III and medial parts of laminae V and VI. The three GABAA receptor subunits have varying expression patterns in the laminae. All of them are expressed by many cells located in the base of the dorsal horn and the intermediate zone, but only the gamma 2-subunit is intensely expressed by motoneurons. Single-pulse, electrical stimulation of the sciatic or median and ulnar nerve of anesthetized cats at a pulse rate of 1/s for 6-8 h failed to induce observable changes in gene expression for CaMKII alpha, GAD, or for the three subunits of the GABAA receptor; although immunoreactivity for the protein products of c-fos (or c-fos-related genes) was markedly upregulated in some neurons of the dorsal horn and the intermediate zone. Therefore, under the present experimental conditions, upregulation of the immediate early gene c-fos (or c-fos-related genes) is not associated with changes in expression of late-effector genes potentially involved in central nervous system plasticity.     

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.     

Castration decreases single cell levels of mRNA encoding glutamic acid decarboxylase in the diagonal band of broca and the sexually dimorphic nucleus of the preoptic area

Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.     

Dynorphin mRNA expression in dorsal horn neurons after traumatic spinal cord injury: temporal and spatial analysis using in situ hybridization

Dynorphin, an endogenous opioid, may contribute to secondary nervous tissue damage following spinal cord injury. The temporal and spatial distribution of preprodynorphin (PPD) mRNA expression in the injured rat spinal cord was examined by in situ hybridization. Rats were subjected to traumatic spinal cord injury at the T13 spinal segment using the weight-drop method. Motor function of these rats was evaluated by their ability to maintain their position on an inclined plane. Two double-labeling experiments revealed that increased PPD mRNA and dynorphin peptide expression were found exclusively in dorsal horn neurons. Neurons exhibiting an increase in the level of PPD mRNA were concentrated in the superficial laminae and the neck of dorsal horn within several spinal segments from the epicenter of the injury at 24 and 48 h after injury. A number of neurons showing increased PPD mRNA were found in gray matter adjacent to the injury areas. Segments caudal to the injury site exhibited a long-lasting elevation of PPD mRNA in neurons, compared to the rostral segments. The number of neurons expressing PPD mRNA in each rat was significantly positively correlated with its motor dysfunction. These findings suggest that increased expression of dynorphin mRNA and peptide in dorsal horn neurons occurs after traumatic spinal cord injury. This also supports the hypothesis that the dynorphin has a pathological role in secondary tissue damage and neurological dysfunction after spinal cord injury.     

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.     

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.