Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.     

Differential expression of annexins I-VI in the rat dorsal root ganglia and spinal cord

The annexins are a family of Ca²⁻-dependent phospholipid-binding proteins. In the present study, the spatial expression patterns of annexins I-VI were evaluated in the rat dorsal root ganglia (DRG) and spinal cord (SC) by using indirect immunofluorescence. Annexin I is expressed in small sensory neurons of the DRG, by most neurons of the SC, and by ependymal cells lining the central canal. Annexin II is expressed by most sensory neurons of the DRG but is primarily expressed in the SC by glial cells. Annexin III is expressed by most sensory neurons, regardless of size, by endothelial cells lining the blood vessels, and by the perineurium. In the SC, annexin III is primarily expressed by astrocytes. In the DRG and the SC, annexin IV is primarily expressed by glial cells and at lower levels by neurons. In the DRG, annexin V is expressed in relatively high concentrations in small sensory neurons in contrast to the SC, where it is expressed mainly by ependymal cells and by small-diameter axons located in the superficial laminae of the dorsal horn areas. Annexin VI is differentially expressed by sensory neurons of the DRG, being more concentrated in small neurons. In the SC, annexin VI has the most striking distribution. It is concentrated subjacent to the plasma membrane of motor neurons and their processes. The differential localization pattern of annexins in cells of the SC and DRG could reflect their individual biological roles in Ca²⁻-signal transduction within the central nervous system. © 1996 Wiley-Liss, Inc.     

GABAB receptor protein and mRNA distribution in rat spinal cord and dorsal root ganglia

The presence of metabotropic receptors for GABA, GABAB, on primary afferent terminals in mammalian spinal cord has been previously reported. In this study we provide further evidence to support this in the rat and show that the GABAB receptor subunits GABAB1 and GABAB2 mRNA and the corresponding subunit proteins are present in the spinal cord and dorsal root ganglion. We also show that the predominant GABAB1 receptor subunit mRNA present in the afferent fibre cell body appears to be the 1a form. In frozen sections of lumbar spinal cord and dorsal root ganglia (DRG) GABAB receptors were labelled with [3H]CGP 62349 or the sections postfixed with paraformaldehyde and subjected to in situ hybridization using oligonucleotides designed to selectively hybridize with the mRNA for GABAB(1a), GABAB(1b) or GABAB2. For immunocytochemistry (ICC), sections were obtained from rats anaesthetized and perfused-fixed with paraformaldehyde. The distribution of binding sites for [3H]CGP 62349 mirrored that previously observed with [3H]GABA at GABAB sites. The density of binding sites was high in the dorsal horn but much lower in the ventral regions. By contrast, the density of mRNA (pan) was more evenly distributed across the laminae of the spinal cord. The density of mRNA detected with the pan probe was high in the DRG and distributed over the neuron cell bodies. This would accord with GABAB receptor protein being formed in the sensory neurons and transported to the primary afferent terminals. Of the GABAB1 mRNA in the DRG, approximately 90% was of the GABAB(1a) form and approximately 10% in the GABAB(1b) form. This would suggest that GABAB(1a) mRNA may be responsible for encoding presynaptic GABAB receptors on primary afferent terminals in a manner similar to that we have previously observed in the cerebellar cortex. GABAB2 mRNA was also evenly distributed across the spinal cord laminae at densities equivalent to those of GABAB1 in the dorsal horn. GABAB2 mRNA was also detected to the same degree within the DRG. Immunocytochemical analysis revealed that GABAB(1a), GABAB(1b) and GABAB2 were all present in the spinal cord. GABAB(1a) labelling appeared to be more dense than GABAB(1b) and within the superficial dorsal horn GABAB(1a) was present in the neuropil whereas GABAB(1b) was associated with cell bodies in this region. Both 1a and 1b immunoreactivity was expressed in motor neurons in lamina IX. GABAB2 immunoreactivity was expressed throughout the spinal cord and was evident within the neuropil of the superficial laminae.     

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

Substance P-like immunoreactivity in neurons in dissociated cell cultures of mammalian spinal cord and dorsal root ganglia

Dissociated cell cultures prepared from fetal mouse spinal cords and dorsal root ganglia were stained for endogenous substance P using the peroxidase-antiperoxidase technique. Substance P-like immunoreactivity was localized within a small percentage of rounded or multipolar neuronal somata and in varicose processes. The substance P-positive multipolar neurons were derived from spinal cord, while the small rounded neurons were possibly of spinal cord and/or sensory ganglion origin. Large dorsal root ganglion neurons were unreactive. These results are consistent with in vivo findings and indicate the feasibility of electrophysiologic studies in culture to analyze the synaptic connections between substance P neurons and their target cells.     

NGF mRNA is expressed in the dorsal root ganglia after spinal cord injury in the rat

Nerve growth factor (NGF) mRNA is expressed in a variety of cell types in the injured spinal cord and its protein implicated in both positive and negative neurological outcomes of cord injury. Here we demonstrate that NGF mRNA is also upregulated in dorsal root ganglion (DRG) neurons after spinal cord injury and that the percentage of sensory neurons expressing NGF mRNA correlates with proximity to the lesion epicenter. Our data suggest that, in DRG, NGF gene expression may be upregulated by damage to the central processes of sensory neurons.     

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.     

Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation

Our recent study showed that peripheral inflammation induced an increased expression of brain-derived neurotrophic factor (BDNF) mRNA which was mediated by nerve growth factor (NGF) in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF immunoreactivity in the DRG and spinal cord following peripheral inflammation by means of immunohistochemistry. Significant increases in the percentage of BDNF-immunoreactive (IR) neuron profiles in the L5 DRG and marked elevation in the expression of BDNF-IR terminals in the spinal dorsal horn were observed following peripheral tissue inflammation produced by an intraplantar injection of Freund's adjuvant into the rat paws. These findings suggest that peripheral tissue inflammation induces an increased BDNF synthesis in the DRG and an elevated anterograde transport of BDNF to the spinal dorsal horn. The functional role of this increased BDNF was discussed briefly.     

Ontological study of calbindin-D28k-like and parvalbumin-like immunoreactivities in rat spinal cord and dorsal root ganglia

The calcium ion plays an important role in some critical developmental events in the nervous system, such as neurulation and neurite elongation. Therefore, as the intracellular calcium-binding proteins calbindin-D28k (CaB) and parvalbumin (PV) may be expressed in these developmental events. Accordingly, the ontological expression of CaB and PV was examined immunocytochemically in the spinal cord and dorsal root ganglia (DRG) of the rat, in order to evaluate the relationship between CaB and PV expression, and other important developmental events. During the ontogenesis of the spinal cord, the CaB-like immunoreactivity was mainly observed in the cell somata. The immunoreactive cells in the ventral horn of the cervical and thoracic, lumbar, and sacral segments first appeared at embryonic day (E)-12, E-13, and E-14, respectively. However, these cells were not detected in the intermediate gray matter of the same segments at E-14, E-15, and E-16, respectively, and in the dorsal horn at E-14-E-15, E-16, and E-17, respectively. The peak of immunoreactive cells, both as to number and intensity, occurred in the perinatal period. However, from postnatal day (P)-14 on, the number and intensity of the positive cells decreased, the adult levels being reached at P-35. The PV-like immunoreactivity was mainly detected in the fibers and punctata during the ontogenesis of the spinal cord. The immunoreactive fibers first appeared on the surface of the dorsal horn in the cervical and thoracic segments at E-14, then entered the dorsal horn at E-15, and reached the intermediate gray matter and ventral horn at E-16. The first appearance of these fibers in the same areas of the lumbar and sacral segments occurred 1 day later than in the cervical and thoracic segments. During the perinatal period, the maximum content of PV-like immunoreactive fibers, together with many punctata, was seen in the gray matter. However, between P-14 and P-17, most of them lost immunoreactivity rapidly, with the exception of the medial region of the intermediate gray matter, where the PV-immunoreactive punctata remained up to the adult stage. In DRG neurons, both CaB and PV was expressed, but in different neurons. Neurons labeled with anti-CaB and anti-PV sera were first detected at E-16 and E-14, respectively. These neurons were large or medium-sized in the prenatal period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of oxytocin on rat dorsal root ganglia in vitro

The influence of bath application of oxytocin (OT) was investigated on the isolated dorsal root ganglion (DRG) cells by means of intracellular recording. The results showed that OT evoked depolarization in most cells and affected predominantly the DRG neurons with biophysical+ characteristics inherent in small neurons.     

Differential effect of thyroid hormone deficiency on the growth of calretinin-expressing neurons in rat spinal cord and dorsal root ganglia

The development of spinal cord or dorsal root ganglia neurons expressing calretinin (CR) was studied in thyroid hormone-deficient rats. Immunocytochemical and morphometric analyses showed that the hypothyroidism induced a significant decrease in the number and size of immunoreactive neurons in the spinal cord, as well as stunted growth and arborization of the axons and dendrites. These alterations were observed at different embryonic ages and persisted during the whole postnatal life. In adult hypothyroid rats, the mean number of CR-positive neurons per spinal cord section (31.2 +/- 2.3 in laminae I and II and 30.5 +/- 5.5 in laminae III-X) was significantly decreased (P < 0.001 and P = 0.024, respectively) compared with adult normal rats (68.7 +/- 8.9 and 50.0 +/- 11.0, respectively). In the peripheral nervous system, hypothyroidism altered the growth of sensory neurons expressing CR protein mainly during embryonic life. In comparison with normal rats, hypothyroid embryonic animals showed not only reduced cell size but also a significantly decreased percentage of CR-positive neurons (6.6 +/- 0. 9% in normal, 2.1 +/- 0.3% in hypothyroid rats, P < 0.001). In contrast, although the size of neurons was reduced in hypothyroid young and adult rats, there was no reduction in the percentage of CR-positive neurons. These results showed that thyroid hormone deficiency altered differentially the development of neurons expressing CR protein in the central and peripheral nervous systems. This suggests that central and peripheral neurons are heterogeneous in their sensitivity to thyroid hormone.     

Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.     

Inflammation of rat dorsal root ganglia below a mid-thoracic spinal transection

Macrophages and T-lymphocytes invade the spinal cord in and around a lesion and spinal microglia are converted into macrophages. After spinal transection at T8 in rats, T-lymphocyte and major histocompatibility complex II+ (MHC II+) macrophage numbers were increased within dorsal root ganglia (DRGs) below the lesion. Inflammation was greater in DRGs closer to the site of transection. After 8 weeks, MHC II+cell density had fallen by 30% but T-lymphocyte numbers were undiminished. In lumbosacral DRGs, inflammation preceded inflammation within the spinal cord. The responses in distant DRGs are hard to reconcile with the limited damage to sensory neurons produced by the lesion. Inflammation of DRGs after spinal injury may contribute to hyper-reflexia and pain.     

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.     

Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S₁–S₃ segments but not in adjacent segments (L₆–L₇ and Cx₁) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S₁–S₃), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L₇–S₂ dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat. © 1994 Wiley-Liss, Inc.     

Insulin-like growth factor-II/Mannose-6-phosphate receptor in the spinal cord and dorsal root ganglia of the adult rat

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a multifunctional transmembrane glycoprotein, which interacts with a number of molecules, including IGF-II and M6P-containing lysosomal enzymes. The receptor is widely distributed throughout the brain and is known to be involved in lysosomal enzyme trafficking, cell growth, internalization and degradation of IGF-II. In the present study, using autoradiographic, Western blotting and immunocytochemical methods, we provide the first report that IGF-II/M6P receptors are discretely distributed at all major segmental levels of the spinal cord and dorsal root ganglia of the adult rat. In the spinal cord, a high density of [(125)I]IGF-II binding sites was evident in the ventral horn (lamina IX) and in areas around the central canal (lamina X), whereas intermediate grey matter and dorsal horn were associated with moderate receptor levels. The dorsal root ganglia exhibited rather high density of [(125)I]IGF-II binding sites. Interestingly, meninges present around the spinal cord displayed highest density of [(125)I]IGF-II binding compared to any given region of the spinal grey matter or the dorsal root ganglia. Western blot results indicated the presence of the IGF-II/M6P receptor at all major levels of spinal cord and dorsal root ganglia, with little segmental variation. At the cellular level, spinal motorneurons demonstrated the most intense IGF-II/M6P receptor immunoreactivity, followed by interneurons in the intermediate region and deeper dorsal horn. Some scattered IGF-II/M6P immunoreactive fibers were found in the superficial laminae of the dorsal horn and dorsolateral funiculus. The meninges of the spinal cord also seemed to express IGF-II receptor immunoreactivity. In the dorsal root ganglia, receptor immunoreactivity was evident primarily in a subset of neurons of all diameters. These results, taken together, provide anatomical evidence of a role for the IGF-II/M6P receptor in general cellular functions such as transport of lysosomal enzymes and/or internalization followed by clearance of IGF-II in the spinal cord and dorsal root ganglia.