Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla.

The adrenal medulla is innervated by both cholinergic and substance P (SP)-containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known. In the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 +/- 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 +/- 19 in T4 to 372 +/- 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 +/- 26, distributed normally about a peak at T8 (33.8 +/- 6.3 cells) and T9 (33.3 +/- 6.8 cells) with the least at T3 (1.5 +/- 0.8) and T13 (5.2 +/- 2.0). No Fast blue-labelled cells were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelled cells were distributed from T7 to T9. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats.     

Expression of estrogen receptors in the dorsal root ganglia of the chick embryo

Estrogen receptors (ER) are widely distributed in the central nervous system (CNS). Recent studies, to date in rat only, have shown that ER are also expressed in neurons of the dorsal root ganglia (DRG) where they appear to have functional roles. However, no data yet exists about estrogen receptors in the embryonic DRG. In the present study, immunocytochemical staining for ER in the DRG of chick embryos from day 6.5 to 18.5 (Hamburger and Hamilton St. 30–45) of incubation was performed. ER+ cells were first consistently observed at day 8.5 (St. 34), more concentrated in the ventral-lateral portion of the DRG. From day 8.5 to 12.5 (St. 38), the density of ER+ cells and the staining intensity increased, with no obvious changes from day (E) 12.5 to 18.5. Although ER is detected mainly in the cytoplasm of embryonic DRG neurones, ER+ cells with nuclear staining are sometimes observed and gradually increase in number during development. ER-immunoreactivity in the DRG at cervical, thoracic and lumbo-sacral levels is similar and no obvious differences in staining were observed between male and female embryos. ER+ neurons are also present in the sympathetic ganglia from E8.5 and some primary spinal motoneurons are ER+ beginning at E14.5. The results suggest that estrogen may play a role in the embryonic development of the DRG.     

Expression of PDE5 splice variants during ontogenesis of chick dorsal root ganglia

Cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5) activity was found in chick dorsal root ganglia (DRG). PDE5 expression was studied at different stages of development: in embryonic day 10 (E10) and E18 embryos and in 5-day post-hatching chick (P5). The presence of PDE5 was suggested by the ion exchange chromatography elution profile in E18 DRG extracts, where cGMP-specific hydrolytic calmodulin-independent activity was found; in other stages, this activity coeluted with the PDE1 calmodulin-stimulated isoform characterized previously. Inhibition studies supported the hypothesis that the newly identified PDE activity belongs to the PDE5 isoform. Western blot analysis using a PDE5-specific antibody was also carried out and revealed the presence of three specific immunoreactive bands with apparent molecular weights of 98, 93, and 86 kDa, corresponding to the three described splice variants (PDE5A1, PDE5A2, and PDE5A3). The expression in DRG of the three PDE5 isoforms was also confirmed by RT-PCR. Developmental regulation of PDE5 was revealed by the immunoblot analysis at different stages; expression was very low at E10 but an overall substantial increase occurred between E10-18 (about 12-fold, considering the three PDE5 isoforms together). Differences were revealed, however, when a single PDE5 isoform was considered. PDE5A1 and PDE5A3 showed an increase at all stages although more pronounced between E10-18, whereas PDE5A2 underwent a marked increase (about 38-fold) in the first period and remained nearly constant between E18 and P5. This is the first evidence of PDE5 in sensory neurons, and the distinct temporal expression patterns of enzyme isoforms may indicate different physiologic roles in developing and mature chick DRG.     

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.     

Lectin binding identifies a subpopulation of neurons in chick dorsal root ganglia

We screened a variety of lectins with different sugar specificates to determine whether subpopulations of dorsal root ganglion (DRG) neurons in the chick can be distinguished by the carbohydrates they express. Of the 15 lectins tested only those that recognize N-acetylgalactosamine (galNac) residues labeled a subset of DRG neurons. For example, Dolichos biflorus (DBA) labeled a population of small-diameter neurons in the dorsomedial DRG and their terminals in the dorsal horn in hatchling chicks. Staining of live neurons in vitro demonstrated that DBA was binding to the cell surface. Labeling first appeared in sensory neurons at about St.38 (E12) and in dorsal horn laminae 1 and 2 at about St.42 (E16). Fainter labeling appeared somewhat later in lamina 3, after hatching. Labeling of the tissue sections was eliminated by chloroform: methanol extraction and reduced by alpha-N-acetylgalactosaminidase digestion, but survived trypsinization. Together these results suggest that a subset of DRG neurons in the chick can be identified by the presence of a cell surface glycoconjugate, perhaps a glycolipid, containing terminal alpha-linked galNac residues.     

Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal subpopulation but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia. © 1994 Wiley-Liss, Inc.     

Acetylcholinesterase in the development of chick dorsal root ganglia

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development. The specific activity of acetylcholinesterase in chick dorsal root ganglia increases, during in ovo development, from day E5 to day E13; after day E13 there is a decrease. Conversely, when acetylcholinesterase activity was expressed on a per ganglion basis, a continuous increase in the level of the enzyme until day E20 was observed. Acetylcholinesterase is a polymorphic enzyme and its molecular forms have different cellular localizations. Two globular forms, a tetramer (G4) and a dimer (G2), are present in the ganglia, as in chick brain. G4 is the major form at day E5, where it represents about 85% of the activity. This form shows a progressive decrease since day E8, and at day E20 exhibits activity levels similar to those of G2. It is known that acetylcholinesterase-producing cells are also able to release the enzyme in the extracellular space. We determined the release of acetylcholinesterase by cultured dorsal root ganglia neurons at various developmental stages: acetylcholinesterase release is significantly increased at day E20, as compared to younger stages, and 90% of the enzyme released is G4.     

Adult dorsal root ganglia sensory neurons express the early neuronal fate marker doublecortin

It has been widely accepted that doublecortin (DCX) may represent a neuronal fate marker transiently expressed by immature neurons during development of the central and peripheral nervous tissue and in neurogenic areas of the adult brain. Previous work described the presence of DCX in the developing dorsal root ganglia (DRG), structures of the peripheral nervous system originating from the neural crest, but no information is available on its expression in adulthood. To this purpose, we have performed an immunohistochemical and biochemical analysis for DCX expression in DRG from adult male mice and rats. To our surprise, we demonstrated that the majority of DRG neurons do express DCX, both in somata and in fibers. DCX(+) cells have been characterized morphologically and phenotypically with well-established markers of DRG neuronal subpopulations. A large number of DCX(+) cells belong to the small and medium-sized nociceptive neurons. Additionally, DCX immunoreactivity is present in the spinal cord dorsal horns, the projection area of DRG neurons. The novel and unexpected localization for DCX protein opens up new, interesting vistas on the functional role of this protein in mature neurons and in particular in sensory neurons.     

Calbindin-immunoreactive sensory neurons of dorsal root ganglion project to skeletal muscle in the chick

In the chicken dorsal root ganglia, two neuronal subpopulations referred to as A1 and B1 share in common an immunoreactivity to antisera raised to calbindin D-28k but are distinguished by their cytological and ultrastructural characteristics. To determine the peripheral targets innervated by calbindin-immunoreactive neurons in lumbosacral dorsal root ganglia, cryostat sections of various hindlimb tissues were treated with anticalbindin antisera. Calbindin-immunostained axons were clearly detected in skeletal muscle. Large myelinated nerve fibres and afferent axon terminals in neuromuscular spindles were calbindin-immunoreactive; thin unmyelinated nerve fibres were also immunostained in nerve bundles of the perimysium. Since motoneurons and neurons of the autonomic nervous system were devoid of calbindin immunostaining, it was suggested that the immunoreactive axons found in skeletal muscle originate from sensory neurons expressing a calbindin immunoreaction in the dorsal root ganglia. This hypothesis was corroborated after introduction of wheat germ agglutinin coupled with horseradish peroxidase or colloidal gold particles into the sartorius muscle. The retrogradely transported tracer was collected only in ganglion cell bodies which displayed the ultrastructural characteristics of A1 and B1 sensory neurons. On the basis of calbindin immunoreaction and of tracer retrograde transport, it is concluded that ganglion cells of subclasses A1 and B1 contribute to the sensory innervation of skeletal muscle in the chicken.     

S100 protein-immunoreactive primary sensory neurons in the trigeminal and dorsal root ganglia of the rat

The cell body size (cross-sectional area) of S100-immunoreactive (-ir) primary neurons was measured in the trigeminal (TG) and lumbar dorsal root ganglia (DRG). About a half of neurons exhibited S100-immunoreactivity (-ir) in the DRG (44.0%) and TG (59.0%). DRG neurons with cell bodies >1200 μm² mostly exhibited S100-ir (96.5%), whereas S100-ir DRG neurons <600 μm² were rare (8.0%). 36.6% of DRG neurons in the cell size range 600–1200 μm² showed the ir. TG neurons >800 μm² mostly exhibited S100-ir (93.1%), whereas those <400 μm² were devoid of it (positive cells 10.5%). 58.3% of TG cells in the range 400–800 μm² contained S100-ir. Double-immunofluorescence method revealed the co-expression of S100 and other calcium-binding proteins. Parvalbumin-ir neurons mostly exhibited S100-ir in the DRG (97.4%) and TG (97.0%). The co-expression of S100 and calbindin D-28k was very rare in the DRG, because the DRG contained few calbindin D-28k-ir neurons. Unlike in the DRG, numerous neurons co-expressed S100- and calbindin D-28k-ir in the TG. Most calbindin D-28k-ir TG neurons were also immunoreactive for S100 (90.7%). Sub-populations of calretinin (CR)-ir neurons co-expressed S100-ir in both the DRG (68%) and TG (50.0%). Virtually all CR-ir neurons >1400 μm² co-expressed S100-ir in the DRG (100%) and TG (95.9%). CR-ir neurons <800 μm² were rarely exhibited S100-ir (DRG 18.0%, TG 21.9%). 71.3 and 60.5% of CR-ir neurons in the range 800–1400 μm² co-expressed S100-ir in the DRG and TG, respectively. The present study indicates that S100 is closely correlated to the primary neuronal cell size in the DRG and TG.     

Quox 1 homeobox protein is expressed in postmitotic sensory neurons of dorsal root ganglia

The expression of vertebrate homeoproteins has been extensively studied in a variety of normal and cancerous tissues, but little is known on the role of vertebrate homeoproteins in the proliferation and differentiation of cells from these tissues. In the present study, we investigate the relationship between Quox 1 protein (a quail homeodomain containing protein) expression and the proliferation and differentiation of quail dorsal root ganglia (DRG) and neural crest cells. In vivo [³H]TdR labeling experiments demonstrate that the postmitotic sensory neuroblasts appear before the formation of the ganglion, and that more than half of sensory neuroblasts from DRG have already terminated their proliferation in embryos of 2 days of incubation (E2). All DRG neurons have completely ceased to proliferate from E6.5 onwards. By means of immunocytochemistry, we observe that Quox 1 protein is accumulated exclusively in all bipolar neurons in culture of DRG from E9–E11, and in all postmitotic sensory-like neuroblasts during in vitro cell differentiation of the neural crest. The Quox 1 immunoreactive neurons express simultaneously neurofilaments or substance P, and they are never labeled by anti-bromodeoxyuridine. These observations together with the morphology of Quox 1 positive cells, demonstrate that Quox1 protein is expressed in the postmitotic sensory neurons of DRG. Our previous experiments have shown that between E4 and E6, the accumulation of Quox 1 protein increases in DRG in vivo, but decreases in the central nervous system in which cell proliferation decreases (Xue et al., (1993) Mech. Dev. 43, 149–158). Taken together, our results show that the accumulation of Quox 1 protein in DRG is tightly linked to the increase in the number of postmitotic neurons, whereas in the central nervous system the level of expression of Quox 1 seems concomitant with the extent of cell proliferation.     

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 μm. The remaining positive neurons (14.1%) were cells with medium diameters between 26 and 40 μm. No positive large neurons (diameters > 40 μm) were observed. Expression in small DRG neurons (diameter < 21 μm) 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 μm ) 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 μm ), but were not affected by the NK-2 antagonist L-659, 877 (2 μm ). 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.     

Serum antibodies to Purkinje cells and dorsal root ganglia neurons in sensory neuronopathy without malignancy

Anti-Purkinje cell antibodies (APCA), believed to be markers of paraneoplastic cerebellar degeneration in females, have been identified in the serum of 3 men with subacute sensory neuronopathies and no evidence of tumors 5 years after the onset of the neurological signs. By indirect immunohistochemistry on sections of rat cerebellum and dorsal root ganglia, the patients' IgG bound to the cytoplasms of both Purkinje cells and dorsal root ganglia neurons. By western blot analysis on whole human cerebellum and whole human dorsal root ganglia homogenates, the IgG from 2 patients bound to a 62-kd protein in both homogenates and the IgG from 1 patient bound to a 110-kd protein in the cerebellum homogenate only. Yo autoantibody test was negative in all patients. Our study provides evidence that non-anti-Yo APCA may be associated with subacute sensory neuronoopathies and are not necessarily markers of an underlying tumor. The previously described anti-Yo APCA has only occurred in females with cancer.     

Ontogeny of calretinin in chick dorsal root ganglion neurons.

Calretinin immunostaining was performed on chick lumbosacral dorsal root ganglia during embryonic development. Calretinin-immunopositive neurons were first observed at around the 9th day of incubation. Quantitative evaluation revealed a close correlation between the number of immunopositive cells and the duration of incubation. Morphometric measurements disclosed that calretinin-immunoreactive cells belong in the large or intermediate categories of dorsal root ganglion neurons. It was concluded that the appearance of calretinin immunopositivity in spinal ganglion cells during development may be associated with both the morphological and functional maturation of this particular population of primary sensory neurons.     

Prolonged sympathetic innervation of sensory neurons in rat thoracolumbar dorsal root ganglia during chronic colitis

Background Peripheral irritation‐induced sensory plasticity may involve catecholaminergic innervation of sensory neurons in the dorsal root ganglia (DRG). Methods Catecholaminergic fiber outgrowth in the thoracolumbar DRG (T13‐L2) was examined by tyrosine hydroxylase (TH) immunostaining, or by sucrose‐potassium phosphate‐glyoxylic acid histofluorescence method. TH level was examined by Western blot. Colonic afferent neurons were labeled by retrograde neuronal tracing. Colitis was induced by intracolonic instillation of tri‐nitrobenzene sulfonic acid (TNBS). Key Results The catecholaminergic fibers formed ‘basket‐like’ structures around the DRG cells. At 7 days following TNBS treatment, the number of DRG neurons surrounded by TH‐immunoreactive fibers and the protein levels of TH were significantly increased in T13, L1, and L2 DRGs (two‐ to threefold, P < 0.05). The DRG neurons that were surrounded by TH immunoreactivity were 200 kDa neurofilament‐positive, but not isolectin IB4‐positve or calcitonin gene‐related peptide‐positive. The TH‐immunoreactive fibers did not surround but adjoin the specifically labeled colonic afferent neurons, and was co‐localized with glial marker S‐100. Comparison of the level of TH and the severity of colonic inflammation showed that following TNBS treatment, the degree of colonic inflammation was most severe at day 3, subsided at day 7, and significantly recovered by day 21. However, the levels of TH in T13‐L2 DRGs were increased at both 3 days and 7 days post TNBS treatment and persisted up to 21 days (two‐ to fivefold increase, P < 0.05) as examined. Conclusions & Inferences Colonic inflammation induced prolonged catecholaminergic innervation of sensory neurons, which may have relevance to colitis‐induced chronic visceral hypersensitivity and/or referred pain.     

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.