Neurogenesis in adult rat dorsal root ganglia

Nerve cells in mammalian species, including primary sensory neurons in the dorsal root ganglia (DRGs), are thought to be generated pre- or perinatally. The only known exceptions are olfactory receptor cells and some cortical microneurons. We now report results of experiments in which the number of neurons in the L4 and L5 DRGs of normal adult rats was counted from serial 10-micrometers paraffin sections stained with cresyl violet. Contrary to expectations, we found that there is a gradual increase in the number of DRG neurons as the animals age. The neuronal population nearly doubles over the adult life of the animal.     

Neurons and satellite glial cells in adult rat lumbar dorsal root ganglia express connexin 36

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-β-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.     

Proton-induced sodium current in frog isolated dorsal root ganglion cells

1. The proton-induced current was examined in isolated frog dorsal root ganglion (DRG) cells by the use of the "concentration-clamp" technique, which allows intracellular perfusion and rapid change of external solution with various pH (pHo) within 2 ms under single-electrode voltage-clamp condition. 2. Over one-half of the examined neurons showed no response for a "step" reduction of pHo even in a Ca2(+)-free external solution. In smaller neurons having a diameter less than 20 microns, the persistent and reliable proton-induced responses were obtained, though the current amplitude and the activation and inactivation varied considerably for each cell. 3. The decrease of external Na+ concentration ([Na+]o) reduced the proton response. The proton response reversed the direction and the Na+ equilibrium potential (ENa). 4. With decreasing pHo from 7.4, proton response increased in a sigmoidal fashion. The threshold was around pH 7.0 and the maximum response appeared at pH 5.2, whereas pKa and Hill coefficient were 6.0 and 1.97, respectively. 5. The activation and inactivation phases of the proton-induced current behaved as a single exponential function. The time constants of activation (tau a) and inactivation (tau i) were not affected by changing either the holding membrane potential (VH) or the low external Ca2+ concentration [( Ca2+]o) between 10(-6) and 5 X 10(-3) M. But the decrease of pHo up to 5.2 decreased both tau a and tau i in a saturable manner. 6. In the inactivation curve of proton-induced current obtained by decreasing pHo from various conditioning pHo to 5.5, half inactivation occurred at pHo 7.45.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological properties of subpopulations of rat dorsal root ganglion neurons in vitro

Intracellular recordings were made from rat dorsal root ganglion neurons in vitro. Action potentials with an inflection on the falling phase occurred in all cells conducting up to 5.2 m/s and in a proportion of faster conducting cells which decreased with increasing conduction velocity, until no cells conducting faster than 31 m/s had an inflection. Overall, all C-cells (less than 1.3 m/s), 61% of A delta-cells (1.3-12 m/s) and 23% of A alpha/beta-cells (greater than 12 m/s) had inflections. A-cells with inflections were found to be electrophysiologically distinct from those without as they differed in the mean and distribution of every action potential and afterhyperpolarization parameter measured. C-cells differed from all A-cell groups, but the means and distributions of most parameters were closer to those of A-cells with inflections than of A-cells without. In addition, all A- and C-cell action potentials with inflections were tetrodotoxin resistant, while all those without were sensitive. The only parameters whose means differed between A alpha/beta- and A delta-neurons were ones which correlated with conduction velocity (action potential duration, overshoot and maximum rate of rise and fall). The response pattern to prolonged current injection did not correlate with conduction velocity, but slightly more A-cells with inflections were single firing. A-cells with long afterhyperpolarizations always fired singly, while those with shorter durations fired singly or multiply. Somatic following frequency was most strongly limited by long afterhyperpolarization duration; it was also slightly lower in A delta- than in A alpha/beta-cells, and lower in A-cells with inflections than in those without. Fibre following frequencies were highest in the fastest conduction neurons.     

Isolation and characterization of neural crest progenitors from adult dorsal root ganglia

After peripheral nerve injury, the number of sensory neurons in the adult dorsal root ganglia (DRG) is initially reduced but recovers to a normal level several months later. The mechanisms underlying the neuronal recovery after injury are not clear. Here, we showed that in the DRG explant culture, a subpopulation of cells that emigrated out from adult rat DRG expressed nestin and p75 neurotrophin receptor and formed clusters and spheres. They differentiated into neurons, glia, and smooth muscle cells in the presence or absence of serum and formed secondary and tertiary neurospheres in cloning assays. Molecular expression analysis demonstrated the characteristics of neural crest progenitors and their potential for neuronal differentiation by expressing a set of well-defined genes related to adult stem cells niches and neuronal fate decision. Under the influence of neurotrophic factors, some of these progenitors gave rise to neuropeptide-expressing cells and protein zero-expressing Schwann cells. In a 5-bromo-2'-deoxyuridine chasing study, we showed that these progenitors likely originate from satellite glial cells. Our study suggests that a subpopulation of glia in adult DRG is likely to be progenitors for neurons and glia and may play a role in neurogenesis after nerve injury. Disclosure of potential conflicts of interest is found at the end of this article.     

Gap junctional communication between the satellite cells of rat dorsal root ganglia

Many studies have described the ultrastructure of the dorsal root ganglia in various embryonic and adult animals, but in spite of the efforts of many investigators the functional role of the satellite cells in this tissue is not clearly understood. In this study, we discuss the function of this cell type based on the concept of cell-to-cell interaction through gap junctions. Five male 60 day-old Wistar strain rats were used. All animals were anesthetized with pentobarbital and perfused with glutaraldehyde fixative, then the dorsal root ganglia in levels L4, L5 and L6 were taken from each rat. After postosmication, the specimens were prepared for observation by transmission electron microscopy. All nerve cells were completely surrounded by satellite cell cytoplasmic expansions. The boundaries between adjacent nerve cells and satellite cells were complicated due to the presence of perikaryal projections of nerve cells. Gap junctions which showed the typical trilamellar structure of plasma membranes were found mainly between satellite cell processes belonging to the same nerve cell. On the other hand, some gap junctions were found between the satellite cell projections belonging to different nerve cells. The size of the gap junctions ranged from 300 to 400 nm. No gap junctions were associated with the plasma membrane of any nerve cell. In conclusion, only satellite cells can share free transcellular exchange of cytoplasmic molecules such as ions, amino acids, sugars and several second messengers including cAMP and inositol 1,4,5-triphosphate by way of gap junctions in dorsal root ganglia.     

Epidermal growth factor receptor in adult human dorsal root ganglia

Transforming growth factor- (TGF) enhances neuronal survival and neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons. It binds a membrane protein, denominated epidermal growth factor receptor (EGFr). EGFr has been localized in developing and adult human DRG. However, it remains to be elucidated whether all DRG neurons express EGFr or whether differences exist among neuronal subtypes. This study was undertaken to investigate these topics in adult human DRG using immunoblotting, and combined immunohistochemistry and image analysis techniques. A mouse monoclonal antibody (clone F4) mapping within the intracytoplasmic domain of EGFr was used. Immunoblotting revealed two main proteins with estimated molecular masses of - 65 kDa and 170 kDa, and thus consistent with the full-length EGFr. Additional protein bands were also encountered. Light immunohistochemistry revealed specific immunoreactivity (IR) for EGFr-like proteins in most (86%) primary sensory neurons, the intensity of immunostaining being stronger in the small- and intermediate-sized ones. Furthermore, EGFr-like IR was also observed in the satellite glial cells of the ganglia as well as in the intraganglionic and dorsal root Schwann cells. Taken together, our findings demonstrate that EGFr, and other related proteins containing the epitope labeled with the antibody F4, are responsible for the EGFr IR reported in DRG. Furthermore, we demonstrated heterogeneity in the expression of EGFr-like IR in adult human primary sensory neurons, which suggests different responsiveness to their ligands.     

GABAA-receptor-activated current in dorsal root ganglion neurons freshly isolated from adult rats

1. gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter affecting dorsal root ganglion (DRG) neurons. This study compares properties of current activated by the GABAA receptor in two populations of DRG neurons. DRG neurons were isolated from adult rat with the use of enzymatic and mechanical means. Within hours of being isolated, neurons were recorded from with the use of the whole-cell variant of the patch-clamp technique. 2. One population of neurons exhibited an afterdepolarizing potential (ADP), a low threshold for action-potential generation (-45 to -50 mV), a short-duration action potential (less than 2 ms) that was abolished in the presence of 1-2 microM tetrodotoxin (TTX), and an insensitivity to 50 nM capsaicin. The second population of neurons exhibited a high threshold for action-potential generation (less than -40 mV), a shoulder on the falling phase of the action potential, insensitivity of action-potential generation to TTX (1-2 microM), and a depolarizing response to application of 50 nM capsaicin. 3. Sensitivity to GABA (over the range of 1-1,000 microM) was comparable for the two populations of neurons. 4. GABA-activated current was greater in ADP neurons than in non-ADP-type neurons of a comparable diameter (30-50 microns). The mean +/- SE amplitude of current activated by 10 microM GABA in ADP neurons was 0.310 +/- 0.050 nA (range = 0.110-0.460 nA, n = 8), and 0.037 +/- 0.016 nA (range = 0.010-0.130 pA, n = 7) in non-ADP neurons. Ten microM GABA elicited cell firing in ADP neurons but not in non-ADP neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diverse immunocytochemical expression of opioid receptors in electrophysiologically defined cells of rat dorsal root ganglia

The development of opiate analgesics that do not produce adverse side effects is hampered by the difficulty in developing drugs that are tissue/sensory cell-specific. Previously, our laboratory has demonstrated that small- and medium-diameter dorsal root ganglia (DRG) cells can be subclassified into at least nine distinct cell types based upon their patterns of voltage activated currents [Petruska, J.C., Napaporn, J., Johnson, R.D., Gu, J.G., Cooper, B.Y., 2000. Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents. J. Neurophysiol. 84 (5), 2365–2379; Petruska, J.C., Napaporn, J., Johnson, R.D., Cooper, B.Y., 2002. Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion. Neuroscience 115 (1), 15–30.] Based on their responses to algesic compounds and histochemical phenotype, eight of the nine subtypes are likely nociceptors. In the present study, we examined the immunoreactivity (IR) of delta-, kappa- and mu-opioid receptors (DOR, KOR and MOR, respectively), in 164 electrophysiologically subclassified DRG neurons.

The expression of opioid receptors in the DRG cell types was diverse. Type 1 (25–30 μm cell diameter) and type 9 (35–45 μm) expressed MOR-IR, but were negative for DOR-IR and KOR-IR. Type 2 (25–30 μm) co-expressed DOR-IR and MOR-IR, but did not express KOR-IR. Type 3 (15–20 μm), the non-nociceptive cell type, was not immunoreactive. Type 4 (35–45 μm), type 6 (35–45 μm), and type 7 (15–20 μm) expressed all three opioid receptors. Type 5 (35–45 μm) and type 8 (35–45 μm), co-expressed KOR-IR and MOR-IR, but did not express DOR-IR. The co-expression of opioid receptors in some of the cell types suggests that these sensory afferents might contain heteromeric opioid receptors. Additionally, the diverse expression patterns of opioid receptors between cell types and the consistency of these patterns maintained within each cell type provides further evidence of distinct functional properties of DRG nociceptors.     

Somatostatin is increased in the dorsal root ganglia of adjuvant-inflamed rat

To determine whether biosynthesis of somatostatin is enhanced in the primary sensory neurons by inflammatory pain, we examined the effects of adjuvant inoculation on the content of immunoreactive somatostatin, mainly composed of somatostatin-14 and somatostatin-28, in the dorsal root ganglia and the spinal cord of the rat. The adjuvant inoculation, which produced long-lasting inflammation and hyperalgesia, increased the content of immunoreactive somatostatin, especially somatostatin-14, in the dorsal root ganglia at L4-L6 levels with no change in the dorsal and ventral horns of lumbar enlargement. Such an increase was enhanced by an intrathecal injection of colchicine (0.2 mg) that inhibits axonal flow of somatostatin. Chronic administration of the anti-inflammatory analgesic, sodium diclofenac (3 mg.kg-1.d-1), abolished an adjuvant-induced increase in the content of immunoreactive somatostatin in the dorsal root ganglia. These results suggest that the turnover (biosynthesis and axonal flow) of somatostatin in the primary sensory neurons is enhanced in the presence of persisting inflammatory pain, and support the idea that somatostatin-containing primary afferents are involved in the transmission of pain in the spinal dorsal horn.     

Action of GABA on neurones and satellite glial cells of cultured rat dorsal root ganglia

The action of GABA was tested on the membrane potential of neurones and satellite glial cells of rat dorsal root ganglia (DRG) in tissue culture. GABA added to the bathing fluid at concentrations of 10^?4 and 10^?5 M caused a depolarization of neurones which was blocked by bicuculline (10^?5 and 10^?6 M). The depolarizing action of GABA clearly showed signs of receptor desensitization. Similarly, GABA caused a depolarization of satellite glial (SG) cells which also revealed desenitization was blocked by bicuculline.     

Responses of macrophages in rat dorsal root ganglia following peripheral nerve injury

Summary Immunohistochemical studies with monoclonal antibodies to macrophage antigens were performed on sections of rat lumbar dorsal root ganglia. In confirmation of previous observations, cells with macrophage antigenicity were detected in normal ganglia. Many of these presumptive macrophages were perineuronal in contact with the neuron/satellite cell complex, a few were perivascular, and others were in interstitial position not in apparent contact with either blood vessels or neurons. The number of macrophages in lumbar dorsal root ganglia started to increase 2–4 days after sciatic nerve transection and remained elevated for four weeks. Perineuronal macrophages resembled satellite glial cells in light microscope appearance but were distinguished from glial cells by their lack of S-100 immunoreactivity. Following this sciatic nerve injury, macrophage counts were modestly increased in contralateral lumbar dorsal root ganglia but not in cervical dorsal root ganglia. Thus peripheral nerve injury induces a recruitment and/or proliferation of macrophages in the corresponding dorsal root ganglion. Although the functions of these macrophages are unclear, those in perineuronal position could contribute to the survival or regeneration of axotomized neurons.     

神经生长颗粒含药血清对大鼠背根神经节生长的影响

目的: 研究神经生长颗粒(NGG)含药血清对体外培养新生大鼠背根神经节生长及高分子量神经丝蛋白(NF-H)和生长相关蛋白43(GAP43)表达的影响.方法: 采用体外大鼠背根神经节(DRG)植块培养,通过免疫荧光细胞化学法,观察不同剂量的含药血清对DRG神经突起生长的影响;采用DRG单细胞分离培养,通过实时荧光定量PCR和免疫印迹法分别观察不同剂量的含药血清对DRG细胞NF-H和GAP43基因及蛋白表达的影响.结果: 免疫荧光细胞化学法提示NGG含药血清能促进DRG神经突起的生长;实时荧光定量PCR和免疫印迹法结果提示NGG含药血清能增加体外培养的DRG细胞NF-H、 GAP43 mRNA和蛋白的表达.结论: NGG含药血清能促进体外培养DRG神经突起的生长并促进NF-H和GAP43的表达,表明NGG对发育期感觉神经元具有一定的神经营养作用.