Sections Of Dorsal Root Ganglia Research Articles

Stereological analysis of Ca2+/calmodulin‐dependent protein kinase IIα‐containing dorsal root ganglion neurons in the rat: Colocalization with isolectin Griffonia simplicifolia, calcitonin gene‐related peptide, or vanilloid receptor 1

The enzyme Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is widely distributed in the nervous system. A previous report describes immunostaining for CaMKII alpha in dorsal root ganglion (DRG) neurons. In this study, CaMKII alpha is colocalized in the rat with three putative markers of nociceptive DRG neurons, isolectin Griffonia simplicifolia (I-B4), identifying small-diameter, "peptide-poor" neurons; calcitonin gene-related peptide (CGRP), identifying " peptide-rich" neurons; or the vanilloid receptor 1 (VR1), identifying neurons activated by heat, acid, and capsaicin. Lumbar 4 and 5 DRG sections were labeled using immunofluorescence or lectin binding histochemistry, and percentages of single and double-labeled CaMKIIalpha neurons were determined. Stereological estimates of total neuron number in the L4 DRG were 13,815 +/- 2,798 and in the L5 DRG were 14,111 +/- 4,043. Percentages of single-labeled L4 DRG neurons were 41% +/- 2% CaMKII alpha, 38% +/- 3% I-B4, 44% +/- 3% CGRP, and 32% +/- 6% VR1. Percentages of single-labeled L5 DRG neurons were 44% +/- 5% CaMKII alpha, 48% +/- 2% I-B4, 41% +/- 7% CGRP, and 39% +/- 14% VR1. For L4 and L5, respectively, estimates of double-labeled CaMKII alpha neurons showed 34% +/- 2% and 38% +/- 17% labeled for I-B4, 25% +/- 14% and 19% +/- 10% labeled for CGRP, and 37% +/- 7% and 38% +/- 5% labeled for VR1. Conversely, for L4 and L5, respectively, 39% +/- 14% and 38% +/- 7% I-B4 binding neurons, 24% +/- 12% and 23% +/- 10% CGRP neurons, and 42% +/- 7% and 35% +/- 7% VR1 neurons labeled for CaMKIIalpha. The mean diameter of CaMKII alpha - labeled neurons was approximately 27 microm, confirming that this enzyme was preferentially localized in small DRG neurons. The results indicate that subpopulations of DRG neurons containing CaMKII alpha are likely to be involved in the processing of nociceptive information. Thus, this enzyme may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory neurons.

Autometallographic mercury correlates with degenerative changes in dorsal root ganglia of rats intoxicated with organic mercury.

Organic mercury intoxication in rats produces degenerative changes in the dorsal root ganglia and dorsal nerve roots. In a previous study of rats treated with organic mercury (2 mg/kg ) for 19 days, significant losses of ganglion cells (especially A-cells) and myelinated axons were observed in dorsal nerve roots and there was qualitative evidence of glial cell proliferation and the formation of Nageotte bodies (1). In the present study, the autometallographic silver-enhancement technique, for tracing inorganic mercury bound to sulphide or selenide (AMG-Hg), was applied to tissue sections of dorsal root ganglia and dorsal nerve roots of the same rats used in the earlier study. Satellite cells and macrophages that surrounded ganglion cells and formed Nageotte bodies were heavily labelled by coarse deposits of AMG-Hg, while the labelling of ganglion cells was less pronounced. A-cells were consistently labelled, while B-cells were only occasionally labelled. In the dorsal nerve roots, only a few AMG-Hg deposits could be seen in macrophages. At the ultrastructural level, AMG-Hg was observed within lysosomes of target cells. It is concluded that AMG-Hg is primarily located in glial cells and that the pattern of deposition of AMG-Hg is the same as that for the morphological changes observed in rats intoxicated with organic mercury.

Binding patterns of lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord.

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.

Sympathetic axons surround neuropeptide-negative axotomized sensory neurons

Nerve injury can lead to sympathetically dependent neuropathic pain. A possible site of sympathetic-sensory interaction is the dorsal root ganglion (DRG), where sympathetic axons form pericellular 'baskets' around a subpopulation of DRG neurons. Since these structures possibly represent functional units of sympathetic pain, we attempted to characterize the neuropeptidergic phenotype of basketed DRG neurons. We performed double-labeling immunohistochemistry for tyrosine hydroxylase and neuropeptides on DRG sections, 2 weeks following L5 spinal nerve ligation (a well-characterized animal model of sympathetic pain). We found that basketed DRG neurons typically do not contain substance P, calcitonin gene-related peptide, galanin, neuropeptide tyrosine, or vasoactive intestinal polypeptide, and we conclude that if sympathetic baskets contribute to neuropathic pain, the involvement of these neuropeptides is unimportant.

Small sensory neurons in the rat dorsal root ganglia express functional NK-1 tachykinin receptor.

The tachykinins substance P (SP) and neurokinin A, released by the C-type primary afferent fibre terminals of the small dorsal root ganglion (DRG) neurons, play important roles in spinal nociception. By means of non-radioactive in situ hybridization and whole-cell recording, we showed that the small rat DRG neurons also express the NK-1 tachykinin receptor. In situ hybridization demonstrated that the positive neurons in rat DRG sections were mainly small cells (85.9%) with diameters less than 25 microm. The remaining positive neurons (14.1%) were cells with medium diameters between 26 and 40 microm. No positive large neurons (diameters > 40 microm) were observed. Expression in small DRG neurons (diameter < 21 microm) was confirmed by in situ hybridization of isolated cells, which were demonstrated to express NK-1 receptor mRNA at a very high frequency (> 90% of small DRG neurons) and therefore were subjected to whole-cell recording. In 57 of 61 cells recorded, SP or the selective NK-1 receptor agonist [Sar9, Met(O2)11]SP (Sar-SP, 1 or 2 microM) produced a delayed vibrating inward current (50-300 nA) with a long duration of 0.5-2 h. These currents were blocked by co-application of the NK-1 receptor antagonist L-668, 169 (1 microM), but were not affected by the NK-2 antagonist L-659, 877 (2 microM). Both current-clamp recording and cell-attached single-channel recording demonstrated that the long-lasting response was due to the opening of a channel with an inward current. Employment of non-Ca2+ and Ca2+ + choline solutions revealed that this channel might be a Ca2+-permeable, non-selective cation channel. The prolonged NK-1 tachykinin response exhibited extreme desensitization. This work suggests that presynaptic NK-1 autoreceptors may be present on the primary afferent terminals in the spinal cord, where they could contribute to the chronic pain and hyperalgesia.

Preprovasopressin mRNA is not present in dorsal root ganglia of the rat

Immunohistochemical studies on colchic ine-treated rats have suggested that more than half of the neurons in dorsal root ganglia (DRG) contain vasopressin. Thus, vasopressin would be the most commonly found peptide in DRG neurons. In the present study we have reexamined the presence of vasopressin in DRG neurons, using a sensitive in situ hybridization method employing long riboprobes that will detect very small amounts of mRNA. The C3, C6, T2, T12, L2 and L5 DRG were studied. None of these ganglia contained any preprovasopressin mRNA. Yet, dense labeling for preprovasopressin mRNA was seen on simultaneously processed hypothalamic sections and a heavy preprotachykinin mRNA expression was seen in adjacent DRG sections. These findings demonstrate that vasopressin is not produced in DRG in normal rats.

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: relationship with neuronal development.

Previous studied from the laboratory demonstrated the presence of a UDP-galactose:Gb3Cer alpha 1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Gal alpha 1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the alpha-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Gal alpha 1-3Gb3Cer in DRGN, we examined the expression of Gal alpha 1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Gal alpha 1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cells bodies and neurites. The expression of Gal alpha 1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Gal alpha 1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development.

A simple method for a specific retrograde labelling of dorsal root and sympathetic ganglion cells innervating the knee joint of the cat

To examine the biochemical characteristics of primary afferents and sympathetic efferents innervating the normal and inflamed knee joint of the cat, the corresponding perikarya have to be labelled retrogradely. In the present study we used an injection of the fluorescent dye Fast Blue through the intact skin and the patellar ligament into the centre of the knee joint cavity and examined the efficiency of this kind of labelling. The segmental distributions of labelled cell profiles in cryostat sections of dorsal root ganglia (DRG) and paravertebral ganglia of the sympathetic trunk agreed with data obtained previously after applying the dye at the transected end of the two main articular nerves. The soma size distribution of labelled DRG cells was identical to previous results indicating that the perikarya labelled in the present study may be a representative proportion of the knee joint innervation. Axotomy of the medial and posterior articular nerves reduced the number of labelled cell profiles of about 85%. Taken together, these data indicate that an injection of Fast Blue into the knee joint cavity of the cat revealed a highly specific and efficient retrograde labelling of the neuronal cell bodies innervating the knee joint.

Neurons of the peripheral nervous system express thymidine phosphorylase

Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor which recently has been shown to be identical to thymidine phosphorylase. We describe here, high levels of expression of PD-ECGF/thymidine phosphorylase in neurons of the peripheral nervous system (PNS) but very little in the central nervous system (CNS). Monoclonal and polyclonal antibodies were used for the staining of sections of dorsal root ganglia, sympathetic cervical ganglia and the enteric plexus as well as the brain and spinal cord. In addition, in situ hybridisation confirmed the results of immunohistochemistry. The possible role of thymidine phosphorylase in the PNS is discussed.

Expression of Preproenkephalin A Gene and Presence of Met‐Enkephalin in Dorsal Root Ganglia of the Adult Rat

The expression of the preproenkephalin A gene was investigated in adult rat dorsal root ganglia (DRG). A radioimmunoassayable Met-enkephalin (ME)-like material was detected in 0.1 M HCl extracts of rat DRG, representing approximately 60 pg of ME equivalents/mg of protein. Chromatographic analyses indicated that the major component of the ME-like material coeluted with authentic ME. In northern blot experiments on total RNA extracted from DRG, a cDNA probe corresponding to the entire coding region of rat preproenkephalin A mRNA yielded a single band of the expected size for this mRNA, i.e., 1.5 kb. Polymerase chain reaction (PCR) experiments were carried out with DRG, striatum, and liver cDNAs using two primers flanking the 1,371-1,771 base region of the preproenkephalin A gene. Thirty PCR cycles performed on both striatum and DRG cDNAs generated a single band of 400 bp, as expected, whereas only trace amounts of this product were detectable using liver cDNAs. Nucleotide sequencing of the PCR product obtained with DRG cDNAs revealed a 100% homology with the 1,371-1,771 sequence of the preproenkephalin A gene. In situ hybridization with a cRNA probe showed that about 3.5% of DRG cells expressed the preproenkephalin A transcript. However, most of these cells probably did not process proenkephalin to enkephalins, as thorough immunohistochemical investigations with anti-ME antibodies allowed the detection of only one in approximately 6,000 cells (in 30 sections of DRG) that exhibited ME-like immunoreactivity. Cells expressing preproenkephalin A mRNA were intermediate-sized neurons, suggesting that primary afferent ME-containing fibers belong to the A category and may participate in a local (spinal) inhibitory control of nociception.

Autometallographic detection of gold in dorsal root ganglia of rats treated with sodium aurothiomalate

Ultraviolet light autometallography, a very sensitive method for gold detection, was applied to sections of dorsal root ganglia from adult male Wistar rats treated with intraperitoneal injections of sodium aurothiomalate. Silver-amplified traces of gold were detected within the cytoplasm of ganglion cells, satellite cells, Schwann cells, macrophages, endothelial cells, and fibroblasts throughout the ganglia. Gold was never detected in axons nor myelin sheaths. In the electron microscope, gold deposits were restricted to the lysosomes irrespective of cell type or dosage.

Simultaneous existence of galactose-containing glycoprotein and several neuropeptides in DRG of the rat.

By use of light microscopic immunohistochemical and lectin histochemical methods, the interrelation of galactose-containing glycoprotein (GCGP), calcitonin gene-related peptide (CGRP)-like, leu-enkephalin (L-ENK)-like, and substance P (SP)-like peptides has been evaluated on consecutive sections of dorsal root ganglia from colchicine-treated rats. The results showed that GCGP, CGRP, L-ENK and SP exist simultaneously in individual neurons of the dorsal root ganglia in rats. Almost all small neurons in dorsal root ganglion contained both GCGP and CGRP. The stronger peanut lectin affinity with small neurons, the weaker CGRP immunoreactivity, and vice versa. Some neurons of medium size were of strong CGRP-like immunoreactivity; however, they lacked in affinity with peanut lectin. The large spinal ganglionic cells rarely showed CGRP immunoreactivity and affinity with peanut lectin. The results suggested that there was a negative interrelation between GCGP and CGRP in small primary sensory neurons. From the above it may be suggested that the GCGP plays an important role in recognizing and transmitting information in primary sensory neurons.

Aspartate-like immunoreactivity in primary afferent neurons

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia. Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy: the great majority of cells immunopositive for aspartate were small (15–30 μm in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors. This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.

Cytochrome oxidase immunohistochemistry in rat brain and dorsal root ganglia: Visualization of enzyme in neuronal perikarya and in parvalbumin-positive neurons

Histochemical detection of cytochrome oxidase activity has been widely used to deduce patterns of neuronal electrical activity in the CNS. Here we investigated the utility of cytochrome oxidase localization by immunohistochemistry and compared immunostaining with histochemical staining patterns in dorsal root ganglia of the rat. In addition, a limited survey of cytochrome oxidase immunostaining density within what are thought to be highly active parvalbumin-immunoreactive neurons was conducted. The immunohistochemical approach produced granular cytoplasmic immunolabelling in neuronal cell bodies and allowed identification of individual labelled cells in all brain regions including those within dense immunoreactive networks of neuropil. Neuronal somata exhibited a wide range of staining densities which were particularly evident in the hippocampus and dorsal root ganglia. The distribution of neurons intensely immunoreactive for cytochrome oxidase within various structures was consistent with previous histochemical descriptions of enzyme activity. Densitometric measurements of immunohistochemical reaction product in individual neurons of hippocampus, substantia nigra, cerebellum and dorsal root ganglia showed that the rate of product deposition was linear with time under conditions chosen for comparisons of staining density. Quantitative analysis of cytochrome oxidase immunohistochemical and histochemical staining densities within the same cells in adjacent sections of dorsal root ganglion gave a correlation coefficient of r = 0.75 (P < 0.001). In sections processed immunohistochemically for both cytochrome oxidase and parvalbumin, most but not all parvalbumin-containing cells displayed dense cytochrome oxidase immunolabelling. Conversely, many examples were found of neurons that were densely stained for cytochrome oxidase, but lacked parvalbumin. Immunohistochemistry for cytochrome oxidase reveals the enzyme in neuronal cell bodies with a clarity not usually seen with the histochemical method. Combination of this immunohistochemical approach with simultaneous immunolabelling of other neuronal markers, as shown here in the case of parvalbumin, is expected to assist the elucidation of patterns of activity in neurochemically identified cell types and anatomically defined neural systems.

Substance P and calbindin D‐28k‐immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co‐localization

During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.

Nicotinic acetylcholine receptor mRNA in dorsal root ganglion neurons

AbstractNicotinic acetylcholine receptor (AChR) genes are expressed in subpopulations of chick dorsal root ganglion (DRG) neurons. In 18‐day embryonic ganglia, 19% of the neurons have material homologous to neuronal AChR α3 gene mRNA, and 8% have material homologous to α4 mRNA as seen with in situ hybridization. RNAase protection experiments confirm that DRG RNA contains α3 and α4 transcripts, and Northern blot analysis establishes the size of the transcripts as being 3.5 and 3.3 kb, respectively. The proportion of DRG neurons containing α3 mRNA does not decline up through 1 year post‐hatch, indicating that α3 gene expression is not a developmentally transient event in the ganglion. An antiAChR monoclonal antibody detects cross‐reacting material in 16% of the DRG neurons from 18‐day embryos, indicating that AChR mRNA is translated into protein. Electrophysiological measurements confirm the presence of functional AChRs on DRG neurons freshly isolated from 18‐day embryos: 24% of the neurons have substantial ACh sensitivities, whereas another 23% have small but detectable responses. Staining dorsal root ganglion sections with an anticholine acetyltransferase antiserum reveals cross‐reactive material localized in axons in the ganglion; no evidence suggests the presence of cholinergic synaptic structures or AChR clusters on neuronal somata in the ganglion. It is possible that AChRs on DRG neurons participate in a diffuse form of transmission between the cholinergic fibers and a subpopulation of neuronal somata in the ganglion. Alternatively, AChRs on the somata may represent an ectopic distribution of receptors whose primary function is at the terminals of central or peripheral DRG processes.

Nicotinic acetylcholine receptor mRNA in dorsal root ganglion neurons

Nicotinic acetylcholine receptor (AChR) genes are expressed in subpopulations of chick dorsal root ganglion (DRG) neurons. In 18-day embryonic ganglia, 19% of the neurons have material homologous to neuronal AChR alpha 3 gene mRNA, and 8% have material homologous to alpha 4 mRNA as seen with in situ hybridization. RNAase protection experiments confirm that DRG RNA contains alpha 3 and alpha 4 transcripts, and Northern blot analysis establishes the size of the transcripts as being 3.5 and 3.3 kb, respectively. The proportion of DRG neurons containing alpha 3 mRNA does not decline up through 1 year post-hatch, indicating that alpha 3 gene expression is not a developmentally transient event in the ganglion. An antiAChR monoclonal antibody detects cross-reacting material in 16% of the DRG neurons from 18-day embryos, indicating that AChR mRNA is translated into protein. Electrophysiological measurements confirm the presence of functional AChRs on DRG neurons freshly isolated from 18-day embryos: 24% of the neurons have substantial ACh sensitivities, whereas another 23% have small but detectable responses. Staining dorsal root ganglion sections with an anticholine acetyltransferase antiserum reveals cross-reactive material localized in axons in the ganglion; no evidence suggests the presence of cholinergic synaptic structures or AChR clusters on neuronal somata in the ganglion. It is possible that AChRs on DRG neurons participate in a diffuse form of transmission between the cholinergic fibers and a subpopulation of neuronal somata in the ganglion. Alternatively, AChRs on the somata may represent an ectopic distribution of receptors whose primary function is at the terminals of central or peripheral DRG processes.

Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract.

Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in vivo, it is suggested that microenvironmental factors influence the expression of GS. More specifically, the repression of GS by primary sensory neurons grown in vitro may be strongly induced by soluble factors present in skeletal muscle, and to a lesser extent in brain, and potentiated by non-neuronal cells.

Ezrin immunoreactivity in neuron subpopulations: cellular distribution in relation to cytoskeletal proteins in sensory neurons.

Ezrin was first identified as a low-abundance phosphoprotein associated with the cytoskeletal core of microvilli, where it may function as a regulatory protein. We report immunocytochemical evidence for expression of ezrin, or an ezrin-like protein of molecular mass near 80 KD, confined to select populations of neurons, including sensory, motor, and autonomic, during chick embryonic development. We have compared the distribution of anti-ezrin staining with that of other major cytoskeletal proteins in sensory neurons in an effort to identify a possible association of the neural homologue of ezrin with the neuronal cytoskeleton. The diffuse distribution of anti-ezrin staining in the cell soma bore little resemblance to the filamentous staining observed with antibodies to the 68 KD neurofilament protein and alpha-tubulin. F-actin staining with fluorescein-conjugated phalloidin was indistinguishable from the anti-ezrin staining pattern in the soma of cultured neurons, including a peak in staining intensity around the periphery of the cell. Microfilaments in growth cones did not stain with the ezrin antibody. A close correspondence between the anti-ezrin and anti-spectrin staining patterns was found on cryostat sections of dorsal root ganglia, but the anti-spectrin staining was weak and could not be demonstrated in culture. Our findings, primarily from cultured neurons, are not inconsistent with ezrin associating with F-actin, although not with microfilaments found in motile structures such as growth cones.

Co-expression of α-CGRP and β-CGRP mRNAs in the rat dorsal root ganglion cells

We examined the expression of α-calcitonin gene-related peptide (α-CGRP) and β-CGRP mRNAs in the rat dorsal root ganglion (DRG) using in situ hybridization histochemistry with oligonucleotide probes. Analysis of autoradiograms showed that α-CGRP mRNA labeled 36.4% of all DRG neurons and β-CGRP mRNA labeled 30.8%. Small and medium-sized neurons expressed both types of mRNA, but large neurons showed a predominance of α-CGRP mRNA expression. The colocalization of α-CGRP and β-CGRP mRNAs was shown at a cellular level in consecutive DRG sections. Thus, we demonstrated that α-CGRP and β-CGRP were distributed differently in the DRG neurons, although genes coding for these neuropeptides coexist in the small or medium-sized neurons.