Immunoreactivity for glutamic acid decarboxylase and several neuropeptides in the spinal cord of the raccoon.

The peroxidase-antiperoxidase method was used to examine major immunohistochemical features of the spinal cord of adult raccoons. The lateral portions of the ventral horn contained many large multipolar neurons that showed cholecystokinin-like immunoreactivity, suggesting the coexistence of cholecystokinin with acetylcholine in a subset of motoneurons. The dorsal horn revealed unique but overlapping patterns of immunoreactivity for glutamic acid decarboxylase, somatostatin, substance P, vasoactive intestinal polypeptide and cholecystokinin. The data imply that some of the peptides may coexist within the same dorsal root ganglion cells and their spinal cord processes.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

Development of the gabaergic phenotype in murine spinal cord-dorsal root ganglion cultures

Murine spinal cord and dorsal root ganglion GABAergic neurons, derived from 12-day-old fetuses, were examined autoradiographically, biochemically and immunocytochemically in vitro to determine the timecourse of appearance and maturation of this phenotype and the extent and mode of its innervation of target neurons. Specific ³H-GABA uptake into spinal cord neurons was the first property to develop and was present at the earliest time studied, one day in vitro. Immunocytochemical localization of glutamic acid decarboxylase (GAD) revealed positively stained neurons beginning at four days. At five days in vitro, electron microscopic immunocytochemistry revealed GAD-immunoreactive (GAD-IMR) boutons investing neuronal perikarya as well as neuronal processes. By one week in vitro, GAD-IMR neurons constituted 27% of the total population and GAD-IMR boutons could be seen contacting every cell with a neuronal morphology. The mode of investment of target neurons by GAD-IMR boutons was not circumscribed to either soma or dendrites but usually involved the entire neuronal perimeter and did not change with time in culture. Three morphologically distinct types of GAD-IMR neurons were evident: a small, bipolar type; a medium-sized multipolar neuron which was the most common and a large, multipolar type, resembling a motoneuron. A small population (8%) of dorsal root ganglion neurons was found to contain GAD both biochemicaly and immunocytochemically but was never invested by GAD-IMR boutons. GAD activity in vitro paralleled in vivo levels with maximal activity being reached at four weeks in vitro and 10 days postnatally in the intact mouse spinal cord. Murine spinal cord GABAergic neurons are a morphologically diverse and abundant neuronal population with extensive, precocious innervation of all other neuronal phenotypes in vitro suggesting that GABA has a widespread influence over other developing neuronal systems in the murine spinal cord.     

Nogo-A在成年大鼠脊髓和背根节的分布

目的研究Nogo—A在成年正常大鼠脊髓和背根节的分布。方法免疫组织化学方法(ABC法)和免疫荧光双标记法。结果正常成年大鼠的脊髓灰质分布有大量的Nogo—A免疫阳性的寡突胶质细胞、运动神经元和中间神经元，免疫阳性反应产物主要分布于细胞的胞体和部分突起中。Nogo—A广泛分布于穿行于脊髓白质的纤维包裹的髓鞘和轴突上。在脊髓前根、后根和坐骨神经的运动和感觉的有髓和无髓纤维也可观察到Nogo—A的表达。而背根神经节的神经元也大量表达Nogo—A，其强度由弱至强不等，广泛分布于大、中、小各类感觉神经元的胞质及突起中。结论Nogo—A在成年大鼠脊髓，背根神经节和外周神经纤维的广泛存在提示其在正常状态下的神经功能中可能起重要作用。     

Co-localization of basic fibroblast growth factor-like immunoreactivity and its receptor mRNA in the rat spinal cord and the dorsal root ganglion.

In the present study, we examined the localizations of basic fibroblast growth factor-like immunoreactivity (bFGF-LI) and its receptor mRNA in the spinal cord and the dorsal root ganglion of the rat. Anti-bFGF peptide antibody and cRNA probe were employed to visualize the localizations of bFGF-LI and FGF receptor (FGF-R) mRNA, respectively. In the spinal cord, we observed that a number of neurons including the motor neurons and interneurons were positive for both substances. In the dorsal root ganglion (DRG), the large neurons preferentially showed co-localization of bFGF-LI and FGF-R mRNA, while the small neurons were not always positive for both. Given the fact that FGF-R is a membrane-spanning protein, these findings suggest the following two possibilities: (1) bFGF acts on the neurons of the spinal cord and the DRG in an autocrine and/or paracrine manner; (2) FGF-R mRNA-positive neurons take up bFGF from innervating neurons and/or surrounding glias in a receptor-mediated fashion.     

Developmental shift of vanilloid receptor 1 (VR1) terminals into deeper regions of the superficial dorsal horn: correlation with a shift from TrkA to Ret expression by dorsal root ganglion neurons

The cloned vanilloid receptor VR1 can be activated by capsaicin and by thermal stimuli. The pattern of nerve terminals that contain VR1 in adult rat spinal cord does not correspond to axons that arise from a single subset of dorsal root ganglion neurons. Thus, we postulated that the basis underlying this complexity might be better understood from a developmental perspective. First, using capsaicin-induced hyperalgesia as a measure of VR1 function, we found that vanilloid receptors were functional as early as postnatal day 10 (P10), although hyperalgesia was of longer duration in adult. Interestingly, the appearance of VR1 protein in terminals of dorsal root ganglion neurons shifts over this postnatal period. From embryonic day 16 to P20, the majority of VR1 protein in the spinal cord was observed in lamina I. As animals matured, VR1 protein became more abundant in lamina II, particularly in the inner portion. Consistent with these observations, the number of dorsal root ganglion neurons coexpressing VR1 and isolectin B4 binding sites doubled while the number of neurons that had both VR1 and substance P remained relatively constant from P2 to P10. In peripheral processes, the number of VR1-positive nerve fibres and terminals in cutaneous structures in postnatal day 10 was half of that in adults. We also show that the association of VR1 with Ret is the reciprocal of the association of VR1 with Trk A. These results suggest that neurotrophins may regulate the extent to which populations of dorsal root ganglion neurons express VR1.     

Ontogeny of enkephalin- and VIP-containing neurons in dissociated cultures of embryonic mouse spinal cord and dorsal root ganglia

The ontogeny of vasoactive intestinal polypeptide (VIP), and Met-enkephalin in primary cultures of spinal cord/dorsal root ganglia from 12-day mouse embryos was examined by radioimmunoassay and immunohistochemistry. Met-enkephalin levels rose from less than 5 to 700 pg/culture over 26 days and were half maximal by day 16-18 in culture. VIP levels rose from less than 1 to 30 pg/culture over the same period, but were already half maximal by day 9. Met-enkephalin immunoreactivity was localized in multipolar medium sized neurons while VIP immunoreactivity was visualized both in neurons with extensively branched processes and in bipolar cells some of which appeared to be dorsal root ganglion cells. Tetrodotoxin (TTX)-sensitive spontaneous release of both peptides developed in parallel with the ability to stimulate peptide release with elevated potassium. Factors affecting the ontogeny of neuropeptide expression in, and release from, spinal cord neurons can now be examined in vitro in a strictly defined neurochemical environment.     

SSeCKS immunolabeling in rat primary sensory neurons

SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.     

The ontogeny of carbonic anhydrase in chick dorsal root ganglia and spinal cords

We examined the distribution of carbonic anhydrase in dorsal root ganglia and spinal cords of chick embryos from Stage 26 (5 days) through hatching using the Hansson technique (Hansson, H.P., Histochemistry, 11 (1967) 112-128; Lannerholm, G., Ann. N.Y. Acad. Sci., 429 (1984) 369-381). Although reactivity was apparent in this tissue at all stages (e.g. in endothelial cells, erythrocytes, and cell nuclei) carbonic anhydrase-positive dorsal root ganglion neurons were not detected until Stage 35 (8.5-9 days). At this stage fewer than 1% of neurons were reactive. The number and proportion of carbonic anhydrase-positive dorsal root ganglion neurons increased steadily from Stage 35 to hatching, when about 60% of the neurons were reactive. In hatchlings carbonic anhydrase-positive neurons spanned the entire size spectrum of ganglion cells and appeared to be randomly distributed throughout the dorsal root ganglion. No carbonic anhydrase-positive neurons were found in the spinal cord at any stage. However, in hatchlings the background staining was markedly lighter in laminae 1 and 2 and in Lissauer's tract than in the rest of the cord, suggesting that unmyelinated axons in the CNS are not reactive.     

Slow excitatory transmission in rat dorsal horn: possible mediation by peptides

Repetitive stimulation of a dorsal root elicited a slow depolarization in about half of the dorsal horn neurons examined in the rat spinal cord slice preparation. The response was markedly depressed or abolished in the presence of substance P, substance P antagonists and capsaicin. In some dorsal horn neurons a slow hyperpolarization was also observed.     

Galectin-1 is involved in the potentiation of neuropathic pain in the dorsal horn

Galectin-1 is one of the endogenous-galactoside-binding lectins, suggested to be involved in a variety of functions, such as neurite outgrowth, synaptic connectivity, cell proliferation and apoptosis. This protein is expressed in the dorsal root ganglion (DRG) and the spinal cord in the developing and adult rats, especially intensely in small DRG neurons. In the present study, we examined whether galectin-1 is colocalized with TrkA or c-Ret mRNA in small DRG neurons and the effect of axotomy on the expression of galectin-1 in the spinal cord. About 20% of the DRG neurons showed intense galectin-1-immunoreactivity (IR). Of the intensely galectin-1-IR DRG neurons, 93.9% displayed c-Ret mRNA positive signals. On the other hand, only 6.8% displayed TrkA mRNA positive signals. Galectin-1-IR was increased in the dorsal horn at 1 to 2 weeks after axotomy. Intrathecal administration of anti-recombinant human galectin-1 antibody (anti-rhGAL-1 Ab) partially but significantly attenuated the upregulation of substance P receptor (SPR) in the spinal dorsal horn and the mechanical hypersensitivity induced by the peripheral nerve injury. These data suggest that endogenous galectin-1 may potentiate neuropathic pain after the peripheral nerve injury at least partly by increasing SPR in the dorsal horn.     

Apoptosis of spinal interneurons induced by sciatic nerve axotomy in the neonatal rat is counteracted by nerve growth factor and ciliary neurotrophic factor

We have previously shown that not only motoneurons and dorsal root ganglion cells but also small neurons, presumably interneurons in the spinal cord, may undergo apoptotic cell death as a result of neonatal peripheral nerve transection in the rat. With the aid of electron microscopy, we have here demonstrated that apoptosis in the spinal cord is confined to neurons and does not involve glial cells at the survival time studied (24 hours). To define the relative importance of the loss of a potential target (motoneuron) and a potential afferent input (dorsal root ganglion cell) for the induction of apoptosis in interneurons in this situation, we have compared the distributions and time courses for TUNEL labeling, which detects apoptotic cell nuclei, in the L5 segment of the spinal cord and the L5 dorsal root ganglion after sciatic nerve transection in the neonatal (P2) rat. In additional experiments, we studied the effects on TUNEL labeling of interneurons after treatment of the cut sciatic nerve with either ciliary neurotrophic factor (CNTF) to rescue motoneurons or nerve growth factor (NGF) to rescue dorsal root ganglion cells. The time courses of the TUNEL labeling in motoneurons and interneurons induced by the lesion show great similarities (peak at 8-48 hours postoperatively), whereas the labeling in dorsal root ganglion cells occurs later (24-72 hours). Both CNTF and NGF decrease the number of TUNEL-labeled interneurons, but there is a regional difference, in that CNTF preferentially saves interneurons in deep dorsal and ventral parts of the spinal cord, whereas the rescuing effects of NGF are seen mainly in the superficial dorsal horn. The results are interpreted as signs of a trophic dependence on both the target and the afferent input for the survival of interneurons neonatally.     

Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons

Following sciatic nerve axotomy, vasoactive intestinal polypeptide (VIP) immunoreactivity increases dramatically in the central terminal areas of the nerve whereas other primary afferent neuropeptides are depleted. The contribution of the peripheral nerve to VIP increases in the spinal cord was investigated by performing sciatic nerve section alone, dorsal rhizotomy of the lumbar roots, axotomy and rhizotomy in combination or section of other peripheral nerves terminating in the same segments as the sciatic nerve. VIP, and for comparison, substance P (SP), cholecystokinin (CCK), somatostatin (SOM), were localized in the lumbar spinal cord and corresponding sensory ganglia using unlabeled antibody immunohistochemistry. After sciatic nerve section, SP, CCK and SOM were depleted in the lumbar dorsal horn whereas VIP increased. After rhizotomy alone all neuropeptide staining including VIP was depleted; axotomy followed by rhizotomy prodiced the same result. Axotomy of other peripheral nerves terminating in the lumbar cord increased the area of neuropeptide depletion but correspondingly increased the area of VIP staining. A large proportion of small and medium diameter dorsal root ganglion cells were stained for VIP after nerve section or axotomy but not after rhizotomy alone. A radical change in neuropeptide metabolism of dorsal root ganglion cells occurs after peripheral axotomy, in the form of a maked increase in VIP synthesis. An intact dorsal root is necessary for increases in VIP in the spinal cord indicating the primary afferent origin of the response.     

Evidence for glycoconjugate in nociceptive primary sensory neurons and its origin from the Golgi complex

Glycoconjugates with terminal galactose residues were localized in rat spinal cord and spinal ganglia using lectin-HRP conjugates of Griffonia simplicifolia and Glycine max agglutinins. Alternate staining of serial sections with HRP-labelled lectins and an antibody for substance P (SP) showed staining in identical primary sensory neurons with both methods. Similarly, lectin-reactive as well as SP-positive fibers were found in Rexed laminae I and II, Lissauer's tract, the spinal nucleus and tract of the trigeminal nerve, the nucleus commissuralis and a small bundle of fibers just ventral to the central canal. Administration of capsaicin to neonatal rats produced a significant decrease in lectin-reactive fibers of the substantia gelatinosa, and in the number of lectin-reactive sensory neurons. The coexistence of SP with galactose-containing glycoconjugates in spinal ganglion neurons, as well as sensitivity of these cells to capsaicin, provided a basis for classifying the reactive neurons as nociceptive in type. Ligation of dorsal roots resulted in disappearance of lectin reactivity in the spinal cord and caused accumulation of lectin-positive material proximal to the ligature, indicating somatofugal transport of galactose-containing glycoconjugates. Colchicine injection caused an increase in SP reactivity in dorsal ganglion neurons but no change in lectin staining of galactoconjugate. At the ultrastructural level affinity for the lectin conjugates was confined to the Golgi cisternae and the plasmalemma of B-type sensory neurons in the dorsal ganglion. The axolemma of unmyelinated processes stained selectively in dorsal roots and the substantia gelatinosa of the spinal cord. These findings provide evidence for the presence in certain sensory cells of a characteristic galactosylconjugate which may prove to be of significance in nerve function.     

Carbonic anhydrase activity in the normal and injured peripheral nervous system of the rat

The carbonic anhydrase reactivity of primary neurons and axons of the L4 and L5 lumbar levels was studied in rats before and after various surgical procedures including transection of the spinal cord, removal of dorsal root ganglia, and transection of ventral or dorsal roots or spinal nerves. In normal animals, carbonic anhydrase reactivity was confined to large and medium size neurons of the dorsal root ganglia, and was also present in a sizeable percentage of cells scattered throughout the thoracolumbar sympathetic chain and in the celiac ganglion. At root level, enzymatic staining could be detected in 48.7% of the dorsal root myelinated axons of most sizes, whereas in ventral roots, it was restricted to small myelinated axons, in a proportion much higher at the L4 than in the L5 level. Spinal motoneurons remained unlabeled, despite procedures aimed at increasing the somal concentration of carbonic anhydrase, such as ventral root ligation and blocking of the fast or slow axoplasmic transport using colchicine or iminodiproprionitrile. However, it is likely that reactive ventral root axons originate from neurons situated segmentally in the spinal cord, and do not constitute aberrant sensory fibers, as carbonic anhydrase activity remained unchanged in the L4 and L5 ventral roots after removal of the corresponding spinal ganglia, whereas it disappeared after damage to the spinal cord at the lumbar level, or at a site distal to a ventral root section. Enzymatic staining of neurons of the dorsal root ganglia was not modified by a dorsal rhizotomy, but showed a marked decrease after transection of the spinal nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional distribution of substance P, neurokinin alpha and neurokinin beta in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection

The regional distribution of 3 mammalian tachykinins (substance P, neurokinin alpha and neurokinin beta) in the rat spinal cord and related structures was investigated using a method of radioimmunoassay combined with high performance liquid chromatography. Substance P and neurokinin alpha were found to be distributed in a very similar manner with fairly constant molar ratios i.e. ratios of substance P to neurokinin alpha were 3.69 in the dorsal root ganglia, 3.49 in the dorsal root and 3.09 in the dorsal horn of the cervical spinal cord. On the other hand, the distribution of neurokinin beta was different from other tachykinins; although concentrated in the dorsal horn, neurokinin beta in the dorsal root ganglia or in the dorsal roots was negligibly small in amount. When the cervical dorsal roots were sectioned unilaterally, substance P and neurokinin alpha were decreased in a parallel fashion in the dorsal horn, whereas neurokinin beta was not. In addition neurokinin alpha was selectively and significantly decreased in the dorsal horn of the intact side when compared to that in the unoperated control rat. Since the magnitude of a decrease of neurokinin alpha in molar basis was approximately the same as a decrease of substance P, these findings suggest that the neurokinin alpha and substance P-containing primary afferent fibres could project partly to the contralateral dorsal horn as well. When the thoracic spinal cord was transected, substance P (and neurokinin alpha) was decreased in the ventral part of the lumbar spinal cord, suggesting the presence of tachykinin(s)-containing descending fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diazepam and its Anomalousp-Chloro-derivative Ro 5-4864: Comparative effects on mouse neurons in cell culture

The actions of diazepam and its p-chloro-derivative Ro 5-4864 were compared on mouse spinal cord and dorsal root ganglion neurons in cell culture. Diazepam enhanced but Ro 5-4864 reduced iontophoretic GABA responses in a concentration-dependent manner. Both diazepam and Ro 5-4864 limited sustained, high frequency repetitive firing of spinal cord neurons but diazepam was more potent. Ro 5-4864 was, however, more potent than diazepam in inhibiting spontaneous neuronal activity of spinal cord neurons and reducing the duration of calcium-dependent action potentials of dorsal root ganglion neurons. The differing actions of diazepam and Ro 5-4864 may account for the contrasting pharmacological spectra of the two benzodiazepines.     

Effect of Brn-3a deficiency on CGRP-immunoreactivity in the dorsal root ganglion

Immunohistochemistry for calcitonin gene-related peptide (CGRP) was performed on the dorsal root ganglion (DRG) and spinal cord in wildtype and knockout mice for Brn-3a. CGRP-immunoreactive (-IR) neurons were abundant in the DRG of wildtype, heterozygous and knockout mice. Cell size analysis revealed that CGRP-IR neurons were of various sizes in wildtype and heterozygous mice. In the knockout mice, however, most of CGRP-IR neurons were small. In the spinal cord of knockout mice, the number of CGRP-IR fibers increased in the dorsal column but decreased in the deep part of the dorsal horn. The loss of Brn-3a may have different effects on CGRP-IR expression in small and large DRG neurons.     

Localization of efferent sympathetic neurons and afferent neurons in dorsal root ganglia innervating the heart

The retrograde transport of horseradish peroxidase (HRP) has been used to study the localization and the number of neurons innervating the heart in the right stellate ganglion and accessory cervical ganglion, spinal cord and dorsal root ganglia of the cat. HRP was applied to the central cuts of anastomose of the stellate ganglion with the vagal nerve, of the vagosympathetic trunk caudal to anastomose and of the inferior cardiac nerve. HRP-labelled neurons were detected in the stellate ganglion in the regions which give off nerves, whereas in the accessory cervical ganglion labelled neurons were distributed throughout the whole ganglion. HRP-stained cells were found in the anastomose. In the spinal cord labelled neurons were detected in the lateral horn of T1-T5 segments. In the dorsal root ganglion the greatest number of neurons was observed in T2-T4 segments.     

Neurotrophic activity in the central and peripheral nervous systems of the cat. Effects of injury

Neurotrophic activity for ciliary ganglion neurons in culture was found in both central and peripheral nervous system of the cat. The activity found in extracts of spinal cord supported the survival of 100% of the test neurons during 24 h and was characterized by a slope of -56 +/- 13 in the linear portion of the dose-response curve. Sciatic nerve extract supported the survival of only 60% of the test neurons; it dose-response curve had a slope of -20 +/- 4. Extracts of meninges, spinal rootlets, dorsal root ganglia and muscle supported 100% of the test neurons; two slopes were observed in their dose-response curves, which coincided with those of spinal cord and sciatic nerve dose-response curves. The two different slopes may correspond to two different active molecules, tentatively denominated I and II, having distinct distributions in the assayed tissues. In the spinal cord, both direct injury and deafferentation led to increases in neurotropic activity. In the peripheral nervous system, transections leading to death of dorsal root ganglion neurons or to degeneration of their axons were accompanied by decreases in activity II. Activity I in dorsal roots and dorsal root ganglia was unaffected by injury and may be associated with non-neuronal cells or extracellular matrix components.