Schwann cells modulate sodium channel expression in spinal sensory neurons in vitro

In order to study the factors that govern the expression of sodium channel α-, β1- and β2-subunits, the influence that Schwann cells (SC) exert in the expression of sodium channels in DRG neurons was examined with in situ hybridization, immunocytochemistry, and patch clamp recording. The expression of sodium channel α-, β1-, and β2-subunit mRNAs in DRG neurons isolated from E15 rats cultured in defined medium in the absence (control) or presence of SC, or in SC-conditioned medium, was examined with isoform-specific riboprobes for sodium channel α-subunits I, II, III, NaG, Na6, hNE/PN1, SNS, and β1- and β2-subunits. DRG neurons cultured in the presence of SC displayed a significant (P < 0.05) increase in the hybridization signal for NaG, Na6, SNS, and Naβ2 mRNAs in comparison to control DRG neurons. In contrast, in SC-conditioned medium, only the hybridization signal for SNS mRNA was significantly increased. The upregulation of sodium channel mRNAs in DRG neurons co-cultured with SC was paralleled by an increase in sodium channel immunoreactivity of these cells. An increase in the mean sodium current density in DRG neurons in the presence of SC was also observed. These results demonstrate that a SC-derived factor selectively upregulates sodium channel α- and β-subunit mRNAs in DRG neurons isolated from E15 rats that is reflected in an increase in functional sodium channels in these cells. This culture system may allow elucidation of the SC factor(s) that modulate the expression of sodium channels in DRG neurons. GLIA 21:339–349, 1997. © 1997 Wiley-Liss, Inc.     

Endogenous nerve growth factor is required for regulation of the low affinity neurotrophin receptor (p75) in sympathetic but not sensory ganglia

During development, many sympathetic and sensory neurons are dependent on nerve growth factor (NGF) for survival. The low affinity neurotrophin receptor (p75), expressed in these neurons, is regulated by exogenous NGF in vitro and in vivo. However, whether p75 expression in vivo is under the control of endogenous NGF has not been determined. The role of NGF in regulating the expression of p75 in sympathetic and sensory nerves was investigated in Sprague-Dawley rats treated with an antiserum specific for NGF. P75 was differentially regulated. P75 immunoreactivity (-ir) within sympathetic neurons in the superior cervical ganglia (SCG) was reduced after 2 days, and disappeared after 5 days, of treatment with the NGF antiserum. In contrast, a significant increase in p75-ir was detected in nerve bundles within and close to the SCG from 3 to 14 days after treatment. A similar pattern of p75 expression was observed in the stellate and coeliac ganglia. In contrast, p75 expression in nerve terminals of the mesenteric arteries and irides was reduced. However, in the same animals the expression of p75 was not significantly affected by the treatment in dorsal root, trigeminal or nodose ganglia, salivary gland or small intestine. In contrast to p75, the NGF high affinity receptor trkA was little affected in sympathetic neurons by depletion of endogenous NGF for 2 weeks. These results indicate that endogenous NGF is required in sympathetic ganglia for the expression of p75 but not trkA in neurons, but for the down-regulation of p75 in glia. In contrast, endogenous NGF is not essential for the regulation of p75 in neurons or glia within sensory ganglia. © Wiley-Liss, Inc.     

Inflammation alters somatostatin mRNA expression in sensory neurons in the rat

Proinflammatory neuropeptides, such as substance P and calcitonin gene-related peptide, are up-regulated in primary afferent neurons in acute and chronic inflammation. While these neuropeptides have been intensively studied, potentially anti-inflammatory and/or anti-nociceptive neuropeptides such as somatostatin (SS) have been less widely investigated. Endogenous somatostatin is thought to exert a tonic antinociceptive effect. Exogenous SS is anti-inflammatory and antinociceptive and is thought to exert these actions through inhibition of proinflammatory neuropeptide release. In this study we have compared the expression of somatostatin in two inflammatory models: arthritis, a condition associated with increased nociception, and periodontitis, in which there is little evidence of altered nociceptive thresholds. In acute arthritis (< 24 h) SS mRNA was down-regulated in ipsilateral dorsal root ganglia (DRG; 52 +/- 7% of control, P < 0.05), and up-regulated in contralateral DRG (134 +/- 10% of control; P < 0.05). In chronic arthritis (14 days) this pattern of mRNA regulation was reversed, with SS being up-regulated ipsilaterally and down-regulated contralaterally. In chronic mandibular periodontitis (7-10 days), SS mRNA was up-regulated in only the mandibular division of the ipsilateral trigeminal ganglion (TG) (day 7, 219 +/- 9% and day 10, 217 +/- 12% of control; P < 0.02) but showed no change in other divisions of the trigeminal ganglion or in the mesencephalic nucleus. These data show that antinociceptive and anti-inflammatory neuropeptides are also regulated in inflammation. It is possible that the degree of inflammation and nociception seen may depend on the balance of pro- and anti-inflammatory and nociceptive peptide expression in a particular condition.     

Nerve growth factor and neurotrophin-3 modulate the rabies infection of adult sensory neurons in primary cultures

With the aim of determining if the proportion of rabies virus (RV)-infected adult neurons from dorsal root ganglion are affected by in vitro treatment with different neurotrophins, experiments using Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) or Neurotrophin-3 (NT-3) as supplements for cells in culture were performed. Cultures treated with three different concentrations of each of the neurotrophins mentioned were infected with Challenge Virus Standard RV strain. An indirect immunoperoxidase technique was performed for the detection and counting of infected cells. NGF (2 ngml(-1) and 10 ngml(-1)) and NT-3 (1 ngml(-1) and 5 ngml(-1)) induced a significant reduction of infected neurons. None of the cultures treated with BDNF showed changes in the percentage of infected neurons. Likewise, the proportion of infected non-neuronal cells (Schwann cells and fibroblasts) was not altered by the treatment with neurotrophins. In addition, morphometric analysis of total and virus-immunoreactive neurons in culture were carried out, the neurotrophin treatment induced variations in the profile of neurons preferentially infected, since cell diameters in the infected cell population are different in the presence of NGF and NT-3. Data presented here could indicate a putative participation of neurotrophin receptor or biochemical modifications induced by neurotrophin treatment that affect the infection. The primary culture of dorsal root ganglion cells from adult mice is a very useful model for studying the basic phenomena of the RV-neuron interaction.     

Effect of injury on nerve growth factor uptake by sensory ganglia

The level of the nerve growth factor protein, NGF, in vivo has a profound influence on axonal sprouting by sensory neurons of vertebrate dorsal root ganglia. There is evidence also that NGF may play similar roles in cholinergic central structures in brain. In both instances, retrograde transport of NGF has been demonstrated. Here we examined uptake of NGF by DRG neurons in response to contusion injury of the spinal cord. Under these conditions there was uptake and transport of NGF into large DRG neurons via central processes but no uptake by non-DRG central neurons. Thus, any effects of NGF on spinal neurons or their processes would be secondary to the direct effects of NGF on DRG neurons.     

Effects of systemically administered NT-3 on sensory neuron loss and nestin expression following axotomy

Previous work has shown that administration of the neurotrophin NT-3 intrathecally or to the proximal stump can prevent axotomy-induced sensory neuron loss and that NT-3 can stimulate sensory neuron differentiation in vitro. We have examined the effect of axotomy and systemic NT-3 administration on neuronal loss, apoptosis (defined by morphology and activated caspase-3 immunoreactivity), and nestin expression (a protein expressed by neuronal precursor cells) in dorsal root ganglia (DRG) following axotomy of the adult rat sciatic nerve. Systemic administration of 1.25 or 5 mg of NT-3 over 1 month had no effect on the incidence of apoptotic neurons but prevented the overall loss of neurons seen at 4 weeks in vehicle-treated animals. Nestin-immunoreactive neurons began to appear 2 weeks after sciatic transection in untreated animals and steadily increased in incidence over the next 6 weeks. NT-3 administration increased the number of nestin-immunoreactive neurons at 1 month by two- to threefold. Nestin-IR neurons had a mean diameter of 20.78 +/- 2.5 microm and expressed the neuronal markers neurofilament 200, betaIII-tubulin, protein gene product 9.5, growth associated protein 43, trkA, and calcitonin gene-related peptide. Our results suggest that the presence of nestin in DRG neurons after nerve injury is due to recent differentiation and that exogenous NT-3 may prevent neuron loss by stimulating this process, rather than preventing neuron death.     

Brain-derived neurotrophic factor and neurotrophin-4 increase neurotrophin-3 expression in the rat hippocampus

Hippocampal levels of mRNA encoding nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are rapidly induced by enhanced neuronal activity following seizures and glutamate or muscarinic receptor activation. However, the levels of neurotrophin-3 (NT-3) mRNA acutely decrease after limbic seizures suggesting that a different mode of regulation may exist for these neurotrophins. Here we show that BDNF and neurotrophin-4 (NT-4), but not NT-3 itself, up-regulate NT-3 mRNA in cultured hippocampal neurons. In the rat hippocampus, the muscarinic receptor agonist, pilocarpine increased BDNF mRNA levels rapidly and those of NT-3 with a delay of several hours. Injection of BDNF into neonatal rats elevated NT-3 mRNA in the hippocampus which demonstrates that BDNF is able to enhance NT-3 expression in vivo. The regulation of NT-3 by BDNF and NT-4 enlargens the neurotrophic spectrum of these neurotrophins to include neuron populations responsive primarily to NT-3.     

Skeletal muscle extract and nerve growth factor have developmentally regulated survival promoting effects on distinct populations of mammalian sensory neurons

Neurotrophic factors appear to be relevant to the therapy of degenerative diseases as well as neural regeneration. In this respect, we have investigated the neurotrophic effects of skeletal muscle extract on DRG neuron survival by examining the survival and neurite outgrowth promoting activity of factor(s) present in skeletal muscle extracts (SME) on dissociated cultures of embryonic or early postnatal mouse dorsal root ganglion (DRG) sensory neurons. The numbers of surviving neurons resulting from SME addition increased continuously from embryonic day 13 (15%) to birth (55%), then decreased up to 7 days after hatching (0%). Preliminary characterization of the factor(s) present in SME suggests that the active molecule is a protein different from the known neurotrophic factors NGF, BDNF, NT3, CNTF, and bFGF, and that its neurotrophic effect is not mediated by direct interaction with the substratum. © 1993 John Wiley & Sons, Inc.     

Embryonic expression of pituitary adenylate cyclase-activating polypeptide in sensory and autonomic ganglia and in spinal cord of the rat

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that is related structurally to vasoactive intestinal polypeptide (VIP), has been shown to stimulate neuronal growth and differentiation, indicating a possible function in the development of the nervous system. Studies have indicated that the PACAP receptor is expressed during development, but data on PACAP expression are limited mainly to postnatal development. In the present study, we used immunohistochemistry and in situ hybridization histochemistry to examine the expression of PACAP in autonomic and sensory ganglia and spinal cord of rat fetuses at embryonic days 12–21 (E12–E21). PACAP immunoreactivity was visualized by using a specific monoclonal anti-PACAP antibody to detect both PACAP-38 and PACAP-27, and PACAP mRNA was visualized by using a [³³P]-labeled cRNA-probe. PACAP⁺ nerve fibers were observed in the spinal cord as early as E13. At E14, PACAP-immunoreactive nerve fibers projected to the sympathetic trunk, where few PACAP⁺ nerve cell bodies were seen from E15. On the same embryonic day, PACAP-immunoreactive nerve cell bodies appeared in the intermediolateral column of the spinal cord. From E15 to E16, PACAP-immunoreactive nerve cell bodies were visible within sensory and autonomic ganglia, such as the dorsal root, the trigeminal, the sphenopalatine, the otic, the submandibular, and the nodose ganglia. At E16, PACAP⁺ nerve fibers were innervating the adrenal medulla, and immunoreactive fibers could also be observed in the superior cervical ganglion, in which PACAP-immunoreactive cell bodies were detected occasionally from E18. The synthesis of PACAP in neuronal cell bodies was confirmed by the demonstration of PACAP mRNA with in situ hybridization histochemistry. Thus, in all of the structures examined, PACAP appeared at roughly the same embryonic stage and, thereafter, increased to the adult level before birth. Because PACAP occurred with the same distribution pattern as that described in the adult rat, there is no evidence for transient expression. The early expression of PACAP suggests a possible role for the peptide in the developing nervous system. J. Comp. Neurol. 394:403–415, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of central sensory axons in transgenic mice overexpressing nerve growth factor and lacking functional p75 neurotrophin receptor expression

This study examined the roles of nerve growth factor (NGF) and the p75 neurotrophin receptor (p75NTR) in the growth of dorsal root ganglion (DRG) central processes in the dorsal horn. Two genetically modified mouse strains were used: transgenic mice that overexpress NGF in the CNS under the control of the glial fibrillary acidic protein promoter, and p75NTR exon III null mutant mice that express a hypomorphic form of this receptor. In both NGF transgenic and nontransgenic mice with hypomorphic expression of p75NTR, there is a significant loss of DRG neurons compared to mice with normal p75NTR expression. This reduction in neuron number has been shown to underlie a corresponding decrease in peripheral nociceptive sensory innervation. Within the CNS, however, nociceptive innervation of the dorsal horn appears to be unaffected by hypomorphic expression of p75NTR. Comparisons of calcitonin gene-related peptide immunoreactivity in the dorsal horn revealed that the area occupied by DRG central processes was not significantly different between p75NTR hypomorphic mice and wild-type siblings, or between NGF transgenic mice with either hypomorphic or normal expression of p75NTR. We propose that DRG central processes arborize extensively in both NGF-transgenic and nontransgenic p75NTR hypomorphic mice in order to compensate for the loss of DRG neurons and restore dorsal horn innervation to normal levels. We also present evidence suggesting that NGF plays only a minor role in the growth of DRG central processes.     

Expression of Neuronal Nicotinic Acetylcholine Receptor Subunit Genes in the Rat Autonomic Nervous System

In the autonomic nervous system efferent signals are relayed in sympathetic and parasympathetic ganglia. Fast synaptic transmission between pre- and postsynaptic neurons is achieved by neuronal nicotinic acetylcholine receptors (nAChRs). There is still little known about the subunit composition of these receptors. Establishing the subunit composition of native neuronal nAChRs is important for the understanding of their functional properties both in vivo and after expression in heterologous expression systems. We have combined in situ hybridization and autoradiography to detect the presence of mRNAs encoding subunits of neuronal nAChRs in sympathetic and parasympathetic ganglia. Inspection of the autoradiographs showed that the hybridization signal of five riboprobes (α3, 014–1, α7, β2 and β4) was significantly higher than the unspecific signal obtained with sense riboprobes. The distribution of α7 was tissue-dependent: a7 riboprobe binding was detected in the neurons of the superior cervical ganglion, adrenal medulla and ciliary ganglion. In contrast, the α7 hybridization signal was found only in a small fraction (1 -3%) of the neurons of the sphenopalatine and otic ganglia. Our results are consistent with the idea that α3 mRNA expression levels are somewhat higher than those of α7, α4 -1, β2 and β4.     

The immunomodulatory effect of nerve growth factor

A prolonged application of nerve growth factor (NGF) to neonatal prepuberal rats induced selective hypertrophy and hyperfunction of ganglionic sympathetic neurons. This was accompanied by higher density and fluorescence intensity of varicose adrenergic fibers and terminals in the immunocompetent organs, total increase of catecholamines in them, catecholamine diffusion into the intercellular space, and their accumulation by macrophages and mast cells. The plaque-forming and lymphocyte blast transformation responses were activated, signs of increased lymphocyte migration to peripheral lymphoid tissues appeared, and histamine concentration in the thymus and spleen increased. All of the foregoing indicate the immunomodulatory effect of NGF and the dependence of immune responsiveness of the organism on the functional state of the adrenergic moiety. The net effect of the increase is attributed to non-specific protein sensitization, changes in the sympathetic (and, probably, somatic) nervous system, and direct action on immunocompetent cells.     

Dependence on nerve growth factor of early human fetal dorsal root ganlion neurons in organotypic cultures

Dorsal root ganglion (DRG) neurons explanted from human embryos, at stages less than about 8 weeks in utero, appeared to be strongly dependent on nerve growth factor (NGF) for their long-term survival. In cultures containing a high concentration of NGF (1000 units/ml, added only at explantation), most of the DRG neurons survived and developed for many weeks in vitro. In contrast, extensive degeneration of DRG neurons was evident within the 1st week after explantation of these immature ganglia in our normal culture medium without added NGF. On the other hand, although introduction of NGF in cultures of 10- to 12-week-old human fetal DRG neurons enhanced the early outgrowth of neurites, these ganglia showed relatively good growth and maintenance in long-term culture even when NGF was omitted from the medium. DRGs from human fetuses estimated to be between 9 and 10 weeks in utero showed intermediate degrees of survival when NGF was omitted from the culture medium (about 10 to 25% of the DRG neurons survived compared with those in paired cultures treated with NGF). The data demonstrate the existence of a critical period during which human DRG neurons may require high NGF concentrations to ensure long-term survival and maturation. Human fetal DRG cultures may provide a useful model system for studies related to familial dysautonomia where drastic deficits in sensory and sympathetic ganglia occur in utero.     

p75NTR expression in rat urinary bladder sensory neurons and spinal cord with cyclophosphamide-induced cystitis

A role for nerve growth factor (NGF) in contributing to increased voiding frequency and altered sensation from the urinary bladder has been suggested. Previous studies have examined the expression and regulation of tyrosine kinase receptors (Trks) in micturition reflexes with urinary bladder inflammation. The present studies examine the expression and regulation of another receptor known to bind NGF, p75(NTR), after various durations of bladder inflammation induced by cyclophosphamide (CYP). CYP-induced cystitis increased (P < or = 0.001) p75(NTR) expression in the superficial lateral and medial dorsal horn in L1-L2 and L6-S1 spinal segments. The number of p75(NTR)-immunoreactive (-IR) cells in the lumbosacral dorsal root ganglia (DRG) also increased (P < or = 0.05) with CYP-induced cystitis (acute, intermediate, and chronic). Quantitative, real-time polymerase chain reaction also demonstrated significant increases (P < or = 0.01) in p75(NTR) mRNA in DRG with intermediate and chronic CYP-induced cystitis. Retrograde dye-tracing techniques with Fastblue were used to identify presumptive bladder afferent cells in the lumbosacral DRG. In bladder afferent cells in DRG, p75(NTR)-IR was also increased (P < or = 0.01) with cystitis. In addition to increases in p75(NTR)-IR in DRG cell bodies, increases (P < or = 0.001) in pericellular (encircling DRG cells) p75(NTR)-IR in DRG also increased. Confocal analyses demonstrated that pericellular p75(NTR)-IR was not colocalized with the glial marker, glial fibrillary acidic protein (GFAP). These studies demonstrate that p75(NTR) expression in micturition reflexes is present constitutively and modified by bladder inflammation. The functional significance of p75(NTR) expression in micturition reflexes remains to be determined.     

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.     

Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat

Injury to a peripheral nerve induces in the dorsal root ganglia (DRG) sprouting of sympathetic and peptidergic terminals around large-diameter sensory neurons that project in the damaged nerve. This pathological change may be implicated in the chronic pain syndromes seen in some patients with peripheral nerve injury. The mechanisms underlying the sprouting are not known. Using in situ hybridization and immunohistochemical techniques, we have now found that nerve growth factor (NGF) and neurotrophin-3 (NT3) synthesis is upregulated in satellite cells surrounding neurons in lesioned DRG as early as 48 h after nerve injury. This response lasts for at least 2 months. Quantitative analysis showed that the levels of mRNAs for NT3 and NGF increased in ipsilateral but not contralateral DRG after nerve injury. Noradrenergic sprouting around the axotomized neurons was associated with p75-immunoreactive satellite cells. Further, antibodies specific to NGF or NT3, delivered by an osmotic mini-pump to the DRG via the lesioned L5 spinal nerve, significantly reduced noradrenergic sprouting. These results implicate satellite cell-derived neurotrophins in the induction of sympathetic sprouting following peripheral nerve injury.