Morphological and biochemical characterisation of sensory neurons infected in vitro with rabies virus

This work was aimed at the morphological and biochemical characterisation of the most susceptible neuronal subpopulation to rabies virus (RABV) infection. Adult mouse DRG cultures were infected with RABV and double-processed for viral antigen detection and neuropeptides: calcitonine gene-related peptide (CGRP), galanin (GAL), substance P (SP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). It was found that 56% of the neurons in culture were small (diameter <20 μm) but, in spite of this, 69% of the infected neurons had intermediate and large diameters (≥20 μm). More than 50% of infected neurons expressed NPY, VIP or SP, whereas no association was found between infected neurons and the presence of CGRP or GAL. Despite SP having been shown to be a small neuron marker, it was found that RABV infects medium and large-sized SP positive cells. RABV preference for larger neurons could explain part of the neuropathogenesis since it can be suggested that, following a rabid accident, the virus uses large neurons (mainly innervating muscle and joints) in vivo to be transported later on to the central nervous system.     

Prostacyclin enhances the evoked-release of substance P and calcitonin gene-related peptide from rat sensory neurons

Prostacyclin (PGI₂) is a potent prostanoid producing various symptoms of inflammation, including an increased sensitivity to noxious stimulation. One component of these PGI₂-mediated actions may involve activation or sensitization of sensory neurons to enhance release of neuroactive peptides. We, therefore, examined whether PGI₂ and carra prostacyclin (CPGI₂), a stable analog of PGI₂, could alter the resting and evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) from embryonic rat sensory neurons grown in culture. Treating isolated sensory neurons with CPGI₂ (10–1000 nM) for 30 min caused a 3-fold increase in the resting release of both peptides. One nM CPGI₂, a concentration that did not alter the resting release, significantly enhanced neuropeptide release evoked by capsaicin, 100 nM bradykinin, or 40 mM KCl. Similarly, 10 nM PGI₂ did not alter resting release, but augmented capsaicin-stimulated release of SP and CGRP 2–3 fold. In contrast, prostaglandin F_2α was ineffective in altering either resting or capsaicin-evoked peptide release. Our results demonstrate that low concentrations of PGI₂ sensitize sensory neurons to other stimuli, whereas higher concentrations evoke release directly. This PGI₂-induced augmentation of neuropeptide release may be one mechanism contributing to neurogenic inflammation.     

Apoptosis of primary sensory neurons in GD1b-induced sensory ataxic neuropathy

Experimental autoimmune sensory ataxic neuropathy was induced in three of six rabbits sensitized with GD1b ganglioside (GD1b-SAN). TUNEL assay was performed on sections of dorsal root ganglia in the cauda equina. The results showed the presence of TUNEL-positive neurons in all three rabbits affected with GD1b-SAN. In contrast, no such neurons were observed in any of the sections from the unaffected rabbits that had been inoculated with GD1b, rabbits inoculated with adjuvant alone or those without inoculation. These data support that an apoptotic mechanism is involved in the pathogenesis of GD1b-SAN.     

Embryonic sensory development: Local expression of neurotrophin-3 and target expression of nerve growth factor

Development and maintenance of peripheral sensory and sympathetic neurons are regulated by target-derived neurotrophins, including nerve growth factor (NGF). To determine whether trophins are potentially critical prior to and during target innervation, for neuronal survival or axon guidance, in situ hybridization was performed in the rat embryo. We examined the expression of genes encoding NGF, neurotrophin-3 (NT-3), and their putative high-affinity receptors, trk A and trk C, respectively. Trks A and C were detected in dorsal root sensory ganglia (DRG) on embryonic day 12.5 (E12.5), implying early responsiveness to NGF and NT-3. NGF mRNA was expressed in the central spinal cord target and by the peripheral somite, at this early time, which thereby may function as a transient “guidepost” target for sensory fibers. Somitic expression was transient and was undetectable by E17.5. NT-3 was expressed in the DRG itself from E13.5 to 17.5, suggesting local transient actions on sensory neurons. NT-3 was also expressed in the ventral spinal cord at low levels on E13.5. We examined the trigeminal ganglion to determine whether cranial sensory neurons are similarly regulated. Trk A was detected in the trigeminal ganglion, while NGF was expressed in the central myelencephalon target, paralleling observations in the DRG and spinal cord. However, NT-3 and trk C were undetectable, in contrast to DRG, suggesting that the environment or different neural crest lineages govern expression of different trophins and trks. Apparently, multiple trophins regulate sensory neuron development through local as well as transient target mechanisms prior to innervation of definitive targets.     

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Tα1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.     

Pathological involvement of primary sensory neurons in Werdnig-Hoffmann disease

Summary Seven patients with the typical clinical picture and muscle biopsy findings of classical Werdnig-Hoffmann disease showed Wallerian degeneration in their biopsied sural nerves. In dorsal root ganglia of one patient there were residual nodules and several chromatolytic neurons. By electron microscopy the changes of chromatolysis were confirmed and found to be consistent with an axonal reaction. Involvement of the primary sensory neuron is probably a regular pathologic feature of Werdnig-Hoffmann disease. The type of abnormality suggests an initial failure of the axon distal to the nerve roots.     

A subpopulation of chicken primary sensory neurons defined by complete co-localization of Peripherin- and ovalbumin-immunoreactivities

In a previous study, we have demonstrated that an ovalbumin-like antigen is present within approximately one-half of all neurons of chicken spinal ganglia. The current study demonstrates this antigen co-localizes absolutely with neural intermediate filament protein (Peripherin) in small to medium-sized neurons of spinal ganglia. While the function of ovalbumin in neurons is unknown, its precise co-localization with Peripherin suggests a functional role restricted to neurons of a defined phenotype.     

Localization of neurotrophin-3-like immunoreactivity in peripheral tissues of the rat

Neurotrophin-3 (NT-3) mRNA is widely distributed in both the peripheral and central nervous systems but neither the distribution of the native factor nor its physiological function is known. In the present study we produced and characterized an antibody to a synthetic peptide and showed that it specifically recognised endogenous rat and recombinant human NT-3 (rNT-3), but not mouse nerve growth factor and recombinant brain derived-neurotrophic factor. NT-3-like immunoreactivity (NT-3-ir) was detected within the distal tubular cells of the kidney, the zona glomerulosa and reticularis of the adrenal cortex, ganglion cells of the adrenal medulla, red pulp of the spleen, epithelial cells of the intestinal villi and parenchymal cells of the liver. Within peripheral ganglia, NT-3-ir was observed in a subpopulation of large sensory neurons of dorsal root, trigeminal and cochleovestibular ganglia but not in principle neurons of the sympathetic ganglia. These results provide the first evidence for the localization of NT-3-ir and indicate its presence in various peripheral organs and large sensory neurons. We conclude that NT-3 may function outside the nervous system in addition to a neurotrophic role within large sensory neurons.     

Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons

We have investigated the effects of nerve growth factor (NGF, 2.5 ng/ml for 1–2 weeks) on enriched adult rat dorsal root ganglion (DRG) neurons maintained in cell culture in defined media. Whole-cell recordings in cells cultured in the absence and presence of NGF revealed no significant difference in resting membrane potential and input resistance. However, the threshold for spike generation was significantly lower in untreated cells than in treated cells; −25 ± 1.1mV vs−19 ± 2.2mV, respectively. The sensitivity of the Na⁺ spike to tetrodotoxin (TTX, 1 μM) was different in cells cultured in the absence or presence of NGF. For example, spikes were abolished by TTX in 100% of untreated cells, while in NGF-treated cells the spike was abolished in only 41% of the neurons. Chemosensitivity of DRG neurons was also different in the absence and presence of NGF. For example, the percent of neurons in which a current activated by 8-methyl-N-vanillyl-6-nonenamide (capsaicin, 500 nM) was detected, increased from 18% in untreated cells to 55% in NGF-treated cells. NGF did not influence the number of cells surviving. The results indicate that NGF can regulate TTX and capsaicin sensitivity in these adult rat sensory neurons. Our experimental protocol indicates that this effect is not mediated by a factor in the serum or released from non-neuronal cells.     

Peripheral projections of calretinin-immunoreactive primary sensory neurons in chick hindlimbs

In chicken dorsal root ganglia, calretinin immunoreactivity is expressed by a subpopulation of large A-neurons, most of which co-express calbindin D-28k. The myelinated axons of these neurons selectively innervate all muscle spindles and most Herbst corpuscles associated to feathers in hindlimbs. It is suggested that the presence of calretinin in primary afferents may be correlated with the electrophysiological properties of rapidly adapting mechanoreceptors.     

Expression and regulation of tyrosine hydroxylase in adult sensory neurons in culture: Effects of elevated potassium and nerve growth factor

To determine whether similar molecular mechanisms regulate the same proteins in diverse neuronal populations, the present study compared regulation of tyrosine hydroxylase (TOH) in placodal sensory and neural crest-derived sympathetic neurons in tissue culture. Long-term explant cultures of adult nodose and petrosal sensory ganglia (NPG) contained abundant TOH-immunoreactive neurons and exhibited TOH catalytic activity, as in vivo. After an initial decline during the first week of culture, enzyme activity was maintained at a stable plateau of 60% of zero time values for at least 3 weeks. However, exposure of 2-week-old cultures to depolarizing concentrations of potassium (K+; 40 mM) increased TOH activity approximately two-fold; total protein was unchanged, suggesting that the rise was due to increased TOH specific activity. Therefore, membrane depolarization in vitro appears to regulate this specific catecholaminergic (CA) trait in sensory, as in sympathetic CA cells. In sympathetic neurons, NGF regulates TOH activity throughout life. In marked contrast, TOH activity in adult NPG cultures was unchanged in the presence of 0, 10 or 100 units NGF/ml or in the presence of high concentrations of antiserum against the beta-subunit of NGF. Adult sympathetic neurons, however, grown under identical conditions, exhibited a 5- to 10-fold rise in TOH activity in the presence of NGF. Thus, unlike sympathetics, CA metabolism in adult NPG neurons is not regulated by NGF in vitro; NGF is therefore unlikely to mediate target effects on CA metabolism in placodal sensory neurons in vivo. Our findings indicate that certain mechanisms of CA regulation are shared by placodal sensory and neural crest-derived sympathetic neurons, whereas others are not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental co-expression and functional redundancy of tyrosine phosphatases with neurotrophin receptors in developing sensory neurons

Receptor-type protein tyrosine phosphatases (RPTPs) have been implicated as direct or indirect regulators of neurotrophin receptors (TRKs). It remains less clear if and how such RPTPs might regulate TRK proteins in vivo during development. Here we present a comparative expression profile of RPTP genes and Trk genes during early stages of murine, dorsal root ganglion maturation. We find little if any specific, temporal mRNA co-regulation between individual RPTP and Ntrk genes between E12.5 and E14.5. Moreover, a double fluorescent in-situ hybridization and immunofluorescence study of seven Rptp genes with Ntrks revealed widespread co-expression of RPTPs in individual neurons, but no tight correlation with Trk expression profiles. No Rptp is expressed in 100% of Ntrk1-expressing neurons, whereas at least 6 RPTPs are expressed in 100% of Ntrk2- and Ntrk3-expressing neurons. An exception is Ptpro, which showed very selective expression. Short hairpin RNA suppression of Ptprf, Ptprs or Ptpro in primary, E13.5 DRG neurons did not alter TRK signalling. We therefore propose that TRK signalling may not be simply dependent on rate-limiting regulation by individual RPTP subtypes during sensory neuron development. Instead, TRK signalling has the potential to be buffered by concurrent inputs from several RPTPs in individual neurons.     

The effect of endoneurial nerve growth factor on calcitonin gene-related peptide expression in primary sensory neurons

Recent findings indicate that calcitonin gene-related peptide (CGRP) is involved in neuropathic pain, this peptide being up-regulated in a small population of large- and medium-sized primary sensory neurons after peripheral nerve injury. In adult animals, the expression of CGRP is regulated by nerve growth factor (NGF). After nerve injury, NGF is up-regulated at the injury site for several weeks, and this up-regulation contributes to the onset of neuropathic pain. Using immunohistochemistry, we investigated the time course of the effect of an endoneurial injection of NGF on the expression of CGRP in primary sensory neurons. NGF increased the percentage of medium- to large-sized DRG neuron profiles expressing CGRP, did not modify the percentage of small-sized neurons expressing CGRP, and increased CGRP expression in the laminae III and IV of the dorsal horn. The effects of NGF were evident as soon as 1 day after endoneurial injection, and lasted for 5 days. Ten days after the injection of NGF, the patterns of CGRP expression in the DRG were normal, whereas a slight decrease in CGRP content was observed in the dorsal horn. The injection of vehicle did not produce any change on CGRP expression in primary sensory neurons. These results suggest that endoneurial NGF is responsible for the increase in CGRP expression in some large-sized neurons and their central processes observed after nerve injury in animal models of neuropathic pain. Our findings contribute to the understanding of the role of NGF in neuropathic pain.     

Localization of angiotensin IV binding sites to motor and sensory neurons in the sheep spinal cord and hindbrain

In the sheep spinal cord, a high density of [¹²⁵I]angiotensin IV binding sites was localized to the perikaryon and processes of all somatic motor neurons, the autonomic motor neurons in the lateral horns of thoracic and lumbar segments and all dorsal root ganglia, but was low in lamina II of all dorsal horns. At supraspinal levels, [¹²⁵I]angiotensin IV binding was abundant in numerous motor associated regions, with weaker binding observed in the sensory regions. This wide distribution pattern suggests an important role for the binding site in the central nervous system.     

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.     

Change in excitability and phenotype of substance P and its receptor in cat Aβ sensory neurons following peripheral inflammation

The effect of peripheral inflammation on spontaneous firing and level of substance P (SP) and its receptor in electrophysiologically identified cat Aβ neurons of dorsal root ganglion (DRG) was studied in vivo using a combination of intracellular recording, dye injection and immunohistochemical techniques. Following injection of carrageenan (Carg) into cat hindpaw, the number of Aβ neurons with spontaneous firing was enhanced significantly (42.9%, n=182) in comparison with control (16.8%, n=149, P<0.01). DRG Aβ neurons became less depolarized 2–4 h following Carg injection. After identifying the cell properties, Lucifer Yellow was injected and SP-like immunoreactivity (SP-LI) was then detected. A total of 17% of Aβ sensory neurons exhibited SP-LI in inflammatory cat. We also found in rat DRGs that the number of SP-LI positive large cells (>35 μm) was also significantly increased in Carg-treated DRG (11.8±1.2, n=8) compared with untreated DRG (1.8±0.8, n=8, P<0.01). In control cat, the topical use of SP in DRG did not induce any response of Aβ neurons. However, in Carg-treated cat, SP depolarized the membrane potential in most Aβ neurons (68.2%, n=22). L668,169, an antagonist of SP receptor, completely blocked the SP-induced responses. Furthermore, repeated application of SP did not induce obvious desensitization of Aβ neurons. These data suggest that peripheral inflammation increased the excitability, SP level and sensitivity of SP receptor of Aβ neurons. Therefore, we concluded that Aβ sensory neurons appear to contribute to inflammatory allodynia.     

Inhibitor of cyclic AMP-dependent protein kinase blocks opioid-induced prolongation of the action potential of mouse sensory ganglion neurons in dissociated cell cultures

The duration of the calcium component of the action potential (APD) of dorsal root ganglion (DRG) neurons in mouse spinal cord-ganglion explants has been shown to be dually modulated via excitatory and inhibitory opioid receptors. In order to determine if opioid-induced APD prolongation is modulated by receptors that are positively coupled to the adenylate cyclase (AC)/cyclic AMP second messenger system, whole-cell recordings were made from mouse DRG neurons grown in dissociated cell cultures. Tests for opioid responsivity were carried out after intracellular dialysis of an inhibitor of cAMP-dependent protein kinase (PKI). In control recordings, both DADLE-induced APD prolongation as well as shortening were prevented by co-perfusion with the opioid antagonist, diprenorphine (10 nM). Intracellular dialysis of PKI in these neurons completely blocked opioid-induced APD prolongation but did not attenuate APD shortening generally elicited by higher opioid concentrations. Bath perfusion of 10 nM DADLE elicited APD prolongation in 59% of the DRG neurons (n = 34) tested with control solution in the recording pipette, whereas none showed APD prolongation when the pipette contained PKI (n = 18). In control tests with 1 microM DADLE, the APD was prolonged in 37% of the cells and shortened in 26% (n = 19); in contrast, a matched group of PKI-treated cells showed no APD prolongation, whereas 42% showed APD shortening (n = 26). The results support the hypothesis that opioid-induced APD prolongation in DRG neurons is mediated by opioid receptor subtypes that are positively coupled via Gs to AC/cAMP-dependent voltage-sensitive ionic conductances.