Prostaglandin E2 increases the proportion of neonatal rat dorsal root ganglion neurons that respond to bradykinin

Prostaglandins sensitize some nociceptors to noxious mechanical, thermal and chemical stimuli; however, not all nociceptors are sensitized by prostaglandins. We used cultures of dorsal root ganglion neurons from neonatal rats to determine whether prostaglandins differentially alter the responsiveness of populations of neurons to the chemical stimulus bradykinin. Groups of dorsal root ganglion neurons were defined by size of the cell soma and by the presence of immunoreactivity for substance P. An increase in the concentration of free intracellular Ca2+ was used as an indicator of responsiveness to bradykinin. Pretreatment (5 min) with prostaglandin E2 (100 nM) increased the proportion of intermediate-size neurons (somal areas of 240-320 microns2) that responded to 30 nM bradykinin by two-fold but did not alter the proportion of small-size neurons (somal areas of 160-239 microns2) that responded. Pretreatment with prostaglandin E2 had no effect on the maximum increase in free intracellular Ca2+ evoked by 30 nM bradykinin in either population of neurons, defined by size. Although pretreatment with PGE2 did not increase the proportion of intermediate-size neurons that responded to a lower concentration of bradykinin (3 nM), it did increase the concentration of free intracellular Ca2+ evoked by 3 nM bradykinin. Both results were consistent with a leftward shift in the stimulus-response relationship for bradykinin following pretreatment with PGE2. Small- and intermediate-size neurons that responded to bradykinin also differed in their expression of immunoreactivity for substance P. Furthermore, intermediate-size neurons that expressed immunoreactivity for substance P were more likely to respond to bradykinin after treatment with prostaglandin E2. These results support the hypothesis that prostaglandin E2 sensitizes some normally unresponsive primary afferent neurons to chemical stimuli. One population of neurons which becomes responsive to bradykinin after treatment with prostaglandin E2 can be defined based on cell size, and furthermore, these neurons are likely to express substance P. During inflammation, recruitment of primary afferent neurons that are immunoreactive for substance P would enhance the participation of substance P in central mechanisms that contribute to hyperalgesia.     

The role of calcium in the desensitization of capsaicin responses in rat dorsal root ganglion neurons

Capsaicin (Cap) is a pungent extract of the Capsicum pepper family, which activates nociceptive primary sensory neurons. Inward current and membrane potential responses of cultured neonatal rat dorsal root ganglion neurons to capsaicin were examined using whole-cell and perforated patch recording methods. The responses exhibited strong desensitization operationally classified as acute (diminished response during constant Cap exposure) and tachyphylaxis (diminished response to successive applications of Cap). Both acute desensitization and tachyphylaxis were greatly diminished by reductions in external Ca2+ concentration. Furthermore, chelation of intracellular Ca2+ by addition of either EGTA or bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid to the patch pipette attenuated both forms of desensitization even in normal Ca2+. Release of intracellular Ca2+ by caffeine triggered acute desensitization in the absence of extracellular Ca2+, and barium was found to effectively substitute for calcium in supporting desensitization. Cap activated inward current at an ED50 of 728 nM, exhibiting cooperativity (Hill coefficient, 2.2); however, both forms of desensitization were only weakly dependent on [Cap], suggesting a dissociation between activation of Cap-sensitive channels and desensitization. Removal of ATP and GTP from the intracellular solutions resulted in nearly complete tachyphylaxis even with intracellular Ca2+ buffered to low levels, whereas changes in nucleotide levels did not significantly alter the acute form of desensitization. These data suggest a key role for intracellular Ca2+ in desensitization of Cap responses, perhaps through Ca2+-dependent dephosphorylation at a locus that normally sustains Cap responsiveness via ATP-dependent phosphorylation. It also seems that the signaling mechanisms underlying the two forms of desensitization are not identical in detail.     

Puerarin inhibits tetrodotoxin-resistant sodium current in rat dorsal root ganglion neurons

The effect of 7-nitroindazole (7-NI), an inhibitor of neuronal nitric oxide synthase (nNOS) on the dimethylphenylpiperazinium(DMPP)-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices was studied. The effect of DMPP (20 microM) to increase the basal release of [3H]NA was significantly potentiated by 7-NI (40 microM). In our previous study we showed that the response to DMPP has two components, a nicotinic receptor-mediated, [Ca2+]-dependent exocytosis followed by a [Ca2+]-independent, uptake blocker-sensitive carrier-mediated release. To clarify which part of the response was affected by the inhibition of nNOS, we investigated the effect of 7-NI on the nicotine-evoked NA release (nicotine has only receptor-mediated effect) and on the DMPP-evoked NA release in Ca(2+)-free medium where the receptor-mediated component is abolished. Nicotine (100 microM) significantly increased the basal release of [3H]NA but this release was not affected, whereas in Ca(2+)-free medium the response to DMPP (20 microM) was still potentiated by 7-NI (40 microM). In the presence of the NA uptake blocker desipramine (10 microM) DMPP (20 microM) was unable to provoke NA release independently from the presence or absence of 7-NI (40 microM). Our data show that 7-NI influences the carrier-mediated component of DMPP-evoked [3H]NA release, which indicates that nitric oxide produced by nNOS may play a role in the regulation of the NA uptake carrier.     

Actions of capsaicin on mouse dorsal root ganglion cells in vitro

The effects of capsaicin were investigated on different populations of dorsal root ganglion cells in the in vitro mouse spinal cord-dorsal root ganglion preparation using intracellular electrodes. Dorsal root ganglion cells were characterised by the conduction velocity of their propagated action potential evoked by electrical stimulation of the dorsal root, and by the shape of their action potential. All cells with C-fiber characteristics (conduction velocity < 0.6 m/s; broad action potential with shoulder on the descending slope) were depolarised and generated action potentials when capsaicin (100-700 nM) was added to the bathing solution for 30 s. At these concentrations the membrane potential of DRG cells with myelinated fibers (conduction velocity > 2.0 m/s) was unaffected. Concentrations of capsaicin of 1.0-5.0 microM depolarised 50% of cells with conduction velocity > 10 m/s. During the depolarization of the membrane no action potentials were generated. In 50% of the capsaicin-sensitive neurons with conduction velocity faster than 10 m/s there was an initial hyperpolarization. Electrical stimulation of the dorsal root failed to evoke action potentials during the depolarization in 38% of the DRG cells with myelinated fibers and in all C-fibers tested within 10 min of the onset of the capsaicin effect. Passive depolarization of the membrane by intrasomal current injection mimicked the conduction block in neurons with large myelinated fibers. These observations confirm that capsaicin applied directly to the dorsal root ganglion affects, in a dose-dependent manner, both myelinated and unmyelinated primary afferents with a higher potency for C-neurons. Capsaicin evoked action potentials in C-neurons but not in neurons with myelinated fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin receptors in cultured rat dorsal root ganglion cells: influence of length of time in culture

The endogenous nonapeptide bradykinin is a powerful substance which activates nociceptors, resulting in the sensation of pain in man. We used a newly developed non-radioactive method to detect bradykinin binding sites in isolated dorsal root ganglion cells with gold-labelled bradykinin. In a subpopulation of cells, gold-labelled bradykinin was bound in different quantities. The proportion of somata with bradykinin binding markedly depended on the length of time in culture. After 0.75 days, bradykinin was bound to 43% of somata. This proportion increased to 85% after 1.75 days and then decreased to 27% after 5.75 days. Bradykinin was bound to cells of all sizes, ranging from 40 to 2000 microns2 with a maximum of 200-300 microns2. In some cells, binding was also seen along the processes. No correlation was found between the soma size and the density of bradykinin binding. Blocking the bradykinin binding at the B1 receptor with (Des-Arg10)-Lys-bradykinin and at the B2 receptor with D-Arg(Hyp3-Thi5.8-D-Phe7)-bradykinin, respectively, revealed that in 0.75-day-old cultures no or only a very small amount of B1 receptors are present. In 1.75-day-old cultures, the marked increase in the proportion of cells with positive bradykinin binding is due to a de novo expression of the B1 receptor subtype and an up-regulation of the B2 receptor subtype. The selective or combined addition of specific B1 and B2 receptor ligands revealed that both receptor subtypes are co-localized. These data show that cultured sensory neurons express not only B2, but during a short period of time in culture also B1 receptors. The data allow us to hypothesize that a transient increase in bradykinin receptor expression might be caused by cell injury due to disruption of the axon. The injury-induced up-regulation of the receptor in vivo could cause physiological reactions.     

Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons

Whole cell voltage- and current-clamp recordings were carried out to investigate the effects of clonidine, an alpha 2-adrenoceptor agonist, in L4 and L5 dorsal root ganglion (DRG) neurons of the rat. In voltage-clamp mode, application of 20 microM clonidine reversibly reduced the inward current evoked by hyperpolarizing voltage steps. The "clonidine-sensitive current" was obtained by subtracting the current during clonidine application from the control current, and its properties were as follows. 1) It was a slowly activating inward current evoked by hyperpolarization. 2) The reversal potential in the standard extracellular solution ([K+]o = 5 mM, [Na+]o = 151 mM) was -38.3 mV, and reduction of [Na+]o shifted it to a more negative potential, whereas an increase of [K+]o shifted it to a more positive potential, indicating that the current was carried by Na+ and K+ (PNa/PK = 0.22). 3) The relationship between the chord conductance underlying the clonidine-sensitive current and voltage could be fitted by a Boltzmann equation. These results indicate that the clonidine-sensitive current corresponds to a hyperpolarization-activated current (Ih), i.e., clonidine inhibits Ih in rat DRG neurons. DRG neurons were classified as small (15.9-32.9 microns diam), medium-sized (33-42.9 microns), and large (43-63.6 microns), and 7 of 19, 24 of 25, and 22 of 22 of these types exhibited Ih with mean +/- SE clonidine-induced inhibition values of 36.1 +/- 3.5% (n = 7), 43.1 +/- 3.7% (n = 24), and 35.1 +/- 2.7% (n = 22), respectively. Clonidine application to L4 and L5 DRG neurons excised from rats the sciatic nerves of which had been transected 14-35 days previously (transected DRG neurons) also reduced Ih. In current-clamp mode, 9 of 13 intact and 4 of 6 transected medium-sized DRG neurons that exhibited Ih responded to clonidine with hyperpolarization (> 2 mV). Some medium-sized DRG neurons exhibited repetitive action potentials in response to a depolarizing current pulse, and clonidine reduced the firing discharge frequencies in 8 of 11 intact and 3 of 4 transected neurons tested. Injection of a hyperpolarizing current pulse produced time-dependent rectification in DRG neurons that exhibited Ih, and clonidine blocked this rectification in all intact and transected neurons tested. These results suggest that inhibition of Ih due to alpha 2-adrenoceptor activation contributes to modulation of DRG neuronal activity in rats. On the basis of our findings, we discuss the possible mechanisms whereby sympathetically released norepinephrine modulates the abnormal activity of DRG neuronal cell bodies after nerve injury.     

Developmental regulation of apoptosis in dorsal root ganglion neurons

The survival of dorsal root ganglion (DRG) neurons, both in vivo and in vitro, is dependent on the availability of nerve growth factor (NGF) for a transient period early in development after which these neurons become independent of NGF for survival. The precise molecular mechanism by which developing DRG neurons gain independence from NGF has not been determined. We used an in vitro model of DRG neuronal development to test hypotheses that independence from NGF in mature DRG neurons could be caused by developmental regulation of either elements of the NGF withdrawal signal transduction pathway or of proteins important for activation of the apoptosis output pathway. Interruption of phosphotidylinositol-3 kinase activation, by treatment with the specific inhibitor LY294002, resulted in apoptosis in immature but not mature DRG neurons in a manner similar to that observed with NGF withdrawal. Further downstream along the signal transduction pathway, c-JUN phosphorylation occurred in both immature and mature DRG neurons after NGF withdrawal or treatment with LY294002, despite the fact that the older neurons did not undergo apoptosis. In contrast, the ratio of expression of the proapoptotic gene bax to antiapoptotic gene bcl-xL was many times higher in immature than mature neurons, both in vivo and in vitro. Taken together, these results strongly suggest that developmental regulation of NGF withdrawal-induced apoptosis in DRG occurs via control of the relative level of expression of members of the bcl-2 gene family.     

Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes

The [3H]resiniferatoxin (RTX) binding assay using membrane preparations has been used to identify and characterize the vanilloid receptors in the central and peripheral nervous system of different species. In the present study, using cultured adult rat dorsal root ganglion neurons either in suspension or attached to the tissue culture plates, we developed an assay to measure specific [3H]RTX binding by the intact cells. We were able to characterize the vanilloid binding characteristics of the neurons and compared those to the properties of vanilloid binding sites present in rat dorsal root ganglia membrane preparations. We found that [3H]RTX bound with similar affinity and positive cooperativity to attached neurons (cultured for 5 days before being assayed), neurons in suspension (using a filtration assay) and dorsal root ganglion membrane preparations. Dissociation constants obtained in the three assays were 47.6 +/- 3.5 pM, 38.4 +/- 3.1 pM and 42.6 +/- 3.1 pM, respectively. The cooperativity indexes determined by fitting the data to the Hill equation were 1.73 +/- 0.11, 1.78 +/- 0.12 and 1.78 +/- 0.09, respectively. The maximal binding capacity was 0.218 +/- 0.026 fmol/10(3) cells and 0.196 +/- 0.021 fmol/10(3) cells in the case of the attached cells and cells in suspension, respectively. Nonradioactive RTX, capsaicin, capsazepine and resiniferonol 20-homovanillylamide fully displaced specifically bound [3H]RTX from cells in suspension with Ki and Hill coefficient values of 42.5 +/- 5.3 pM, 2.06 +/- 0.16 microM, 3.16 +/- 0.21 microM and 32.4 +/- 4.1 nM and 1.79 +/- 0.17, 1.68 +/- 0.06, 1.72 +/- 0.11 and 1.81 +/- 0.12, respectively. Structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and inducing 45Ca uptake in rat dorsal root ganglion neurons. Affinities for receptor binding and stimulation of 45Ca uptake of RTX, resiniferonol 20-homovanillylamide, RTX-thiourea, tinyatoxin, phorbol 12,13-dibenzoate 20-homovanillylamide and capsaicin were 38.5 +/- 2.9 pM, 25.7 +/- 3.0 nM, 68.5 +/- 3.8 nM, 173 +/- 25 pM, 7.98 +/- 0.83 microM and 4.93 +/- 0.35 microM as compared to 0.94 +/- 0.12 nM, 26.5 +/- 3.5 nM, 149 +/- 30 nM, 1.46 +/- 0.25 nM, 1.41 +/- 0.48 microM and 340 +/- 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperativity of receptor binding; however, stimulation of 45Ca uptake appeared to follow a non-cooperative mechanism of action. The competitive capsaicin antagonist capsazepine inhibited specific binding of [3H]RTX by rat dorsal root ganglion membrane preparations with Ki and Hill coefficient values of 3.89 +/- 0.38 microM and 1.74 +/- 0.11. On the other hand it inhibited the induction of 45Ca uptake into the cells induced by capsaicin and RTX in a non-cooperative fashion with Ki values of 271 +/- 29 nM and 325 +/- 47 nM. Our results show that the membrane binding assay relates to the reality of receptor function in the intact, cultured neurons, both in terms of affinity and positive cooperativity. However the different vanilloid derivatives displayed markedly distinct structure-activity relations for high affinity receptor binding and stimulation of 45Ca uptake into rat dorsal root ganglion neurons. Among various explanations for this discrepancy, we favor the possibility that the two assays detect distinct classes of the vanilloid (capsaicin) receptor present in primary sensory neurons.     

OFQ reverses the kappa-opioid receptor-mediated depression of calcium current in rat dorsal root ganglion neurons

Since the characterization of orphanin FQ (OFQ), the endogenous ligand of ORL1 receptor, much work has focused on its physiological functions. OFQ was reported to antagonize the effect of opioid-induced antinociception, although its mechanism remains obscure. In the present study, whole-cell patch clamp recording technique was used to observe if OFQ can reverse the inhibition of calcium current produced by the kappa-opioid agonist U50,488H (U50) in acutely dissociated rat DRG neurons. The concentrations of OFQ and U50 were 50 nM and 10 microM, respectively. Among 49 cells recorded, the calcium channel currents of 37 (75.5%) cells were inhibited by U50, of which 30 (81.1%) cells could be reversed by OFQ. It was interesting to note the similarity between OFQ and the well characterized anti-opioid peptide CCK-8 in that it reversed kappa-opioid receptor agonist induced suppression on calcium channel current, while by itself showed a calcium channel suppressive effect. Thus OFQ may be regarded as another anti-opioid peptide.     

Axotomy reduces the effect of analgesic opioids yet increases the effect of nociceptin on dorsal root ganglion neurons

There is some doubt as to the effectiveness of opioids in the management of neuropathic pain. We therefore examined the actions of morphine and the opioid-like peptide nociceptin (both 1 mu) on dorsal root ganglion (DRG) neurons that were isolated from control or from nerve-injured rats. Both substances reduced omega-conotoxin (CTX) GVIA-sensitive, N-type Ca2+ channel current and small persistent nifedipine/ CTX-insensitive (non-N, non-L type) current. Nifedipine-sensitive L-type current was unaffected. The effect of nociceptin was antagonized by naloxone benzoylhydrazone (nalbzoh) but not by naloxone. Sciatic nerve section (axotomy) profoundly reduced the effects of morphine and the mu-receptor agonist D-ala2, N-Me-Phe4,Gly-ol5 enkephalin (DAMGO). The effect of the kappa-agonist [(+)-(5alpha,7alpha, 8beta)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)-benzeneacetamide] (U69593) was unchanged, whereas the effect of nociceptin was increased. All agonists produced their strongest effects on the small, putative nociceptive cells and their weakest effects on the largest cells. The delta-receptor agonist, enkephalin D-pen2,5 (DPDPE), was without effect on control or on axotomized cells. These and other data suggest that the functional downregulation of mu-opioid receptors on sensory nerves contributes to the poor efficacy of opioids in neuropathic pain. Also, the increased effectiveness of nociceptin after axotomy supports the hypothesis that its actions are mediated via a "non-opioid" receptor. Pronounced suppression of Ca2+ channel current in axotomized DRG neurons by nociceptin led to a reduction in Ca2+-dependent K+ conductance and a marked increase in excitability. Despite this, the spinal administration of nociceptin or agonists that activate ORL1 (opioid-like orphan receptor) may prove to be of clinical interest in the management of neuropathic pain.     

SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model

In contrast to conventional T cells, natural killer (NK) 1.1+ T cell receptor (TCR)-alpha/beta+ (NK1+T) cells, NK cells, and intestinal intraepithelial lymphocytes (IELs) bearing CD8-alpha/alpha chains constitutively express the interleukin (IL)-2 receptor (R)beta/15Rbeta chain. Recent studies have indicated that IL-2Rbeta/15Rbeta chain is required for the development of these lymphocyte subsets, outlining the importance of IL-15. In this study, we investigated the development of these lymphocyte subsets in interferon regulatory factor 1-deficient (IRF-1-/-) mice. Surprisingly, all of these lymphocyte subsets were severely reduced in IRF-1-/- mice. Within CD8-alpha/alpha+ intestinal IEL subset, TCR-gamma/delta+ cells and TCR-alpha/beta+ cells were equally affected by IRF gene disruption. In contrast to intestinal TCR-gamma/delta+ cells, thymic TCR-gamma/delta+ cells developed normally in IRF-1-/- mice. Northern blot analysis further revealed that the induction of IL-15 messenger RNA was impaired in IRF-1-/- bone marrow cells, and the recovery of these lymphocyte subsets was observed when IRF-1-/- cells were cultured with IL-15 in vitro. These data indicate that IRF-1 regulates IL-15 gene expression, which may control the development of NK1+T cells, NK cells, and CD8-alpha/alpha+ IELs.     

Voltage-operated potassium currents in the somatic membrane of rat dorsal root ganglion neurons: ontogenetic aspects

Whole-cell transmembrane potassium currents were studied in somatic membrane of freshly isolated rat dorsal root ganglion neurons. We defined three types of potassium currents, which were separated on the basis of their different potential dependence of activation and sensitivity to external tetraethylammonium and 4-aminopyridine. The potential dependence of kinetic and steady-state properties of a fast inactivating potassium current, a slow inactivating potassium current and a non-inactivating delayed rectifier current were described by the Hodgkin-Huxley equations. A transient fast inactivating potassium current was activated at the most negative membrane potentials and was not reduced in the presence of 10 mM tetraethylammonium in the external solution. 4-Aminopyridine (2 mM) caused an 80% inhibition of this current. The activation of the fast inactivating potassium current was properly described by fitting a single exponent raised to the fourth power. The time constant of activation changed from 4 to 1 ms in the voltage range between -30 and +40 mV. The time constant of inactivation decreased from 35 to 15 ms over the same range of potentials. Parameters for the fit of a Boltzmann equation to mean values for steady-state activation were V1/2=-20mV, k=11.8mV, and for steady-state inactivation V1/2= -85 mV, k=-9.8 mV. A transient slow inactivating potassium current had an activation threshold between -40 and -30 mV. At 2 mM 4-aminopyridine, the depression of the slow potassium current was 55%. The extracellular application of 10 mM tetraethylammonium was less effective and evoked a 40% reduction. The activation of the slow inactivating potassium current was also described by a single exponential function raised to the fourth power. The time constant of activation decreased from 12 ms at a membrane potential of -10 mV to 4 ms at the potential of 60 mV. The inactivation of slow inactivating potassium current was described by two exponents. The time constant for the fast exponent ranged from 300 ms at -20 mV to 160 ms at +60 mV. The slower exponent was also potential dependent and its time constant ranged from approximately 2600 to 1600 ms over the same potentials. Parameters for the Boltzmann equation fittings to mean values were V1/2= -12.8 mV, k=13.4 mV and V1/2= -54.6 mV, k= -12 mV for steady-state activation and inactivation, respectively. A non-inactivating delayed rectifier potassium current was activated at the most positive membrane potentials. This non-inactivating current did not change in the presence of 4-aminopyridine. Extracellular tetraethylammonium (10 mM) caused a 70% reduction of this current. The activation of the non-inactivating potassium current was described by one exponent raised to the fourth power. The time constant for activation ranged from 85 ms at -5 mV to 30 ms at 45 mV. No time-dependent inactivation was observed during 15-s testing potentials in the voltage range between 10 and +60 mV. The activation behavior was characterized by V1/2=15.3 mV, k=12.5 mV. The densities of these potassium currents were studied for three groups of animals: one, five to six and 14-15 days of postnatal development. Fifty cells were examined in each age group. All three types of potassium currents were found in each investigated neuron. The mean densities of slow and fast inactivating potassium currents increased during ontogenetic development. The densities of non-inactivating delayed rectifier potassium current decreased in the first week of ontogenetic development and did not change thereafter.     

Electrophysiological studies on rat dorsal root ganglion neurons after peripheral axotomy: changes in responses to neuropeptides

The effect of three peptides, galanin, sulfated cholecystokinin octapeptide, and neurotensin (NT), was studied on acutely extirpated rat dorsal root ganglia (DRGs) in vitro with intracellular recording techniques. Both normal and peripherally axotomized DRGs were analyzed, and recordings were made from C-type (small) and A-type (large) neurons. Galanin and sulfated cholecystokinin octapeptide, with one exception, had no effect on normal C- and A-type neurons but caused an inward current in both types of neurons after sciatic nerve cut. In normal rats, NT caused an outward current in C-type neurons and an inward current in A-type neurons. After sciatic nerve cut, NT only caused an inward current in both C- and A-type neurons. These results suggest that (i) normal DRG neurons express receptors on their soma for some but not all peptides studied, (ii) C- and A-type neurons can have different types of receptors, and (iii) peripheral nerve injury can change the receptor phenotype of both C- and A-type neurons and may have differential effects on these neuron types.     

Evidence for the existence of substance P autoreceptor in the membrane of rat dorsal root ganglion neurons

Substance P, a putative peptide neurotransmitter contained in primary sensory neurons, is suggested to play a major role in nociceptive transmission. In the present study, the existence of substance P autoreceptor in dorsal root ganglion neurons was identified with a method we developed recently and substance P-activated inward current in the dorsal root ganglion neurons and its ionic mechanism were also explored preliminarily. The majority of the cells examined (68/76, 89.5%) were sensitive to external application of substance P (0.01-10 microM) with a concentration-dependent inward current. This current was found to result from the opening of nonselective ion channel, preferring the Na+ channel. The substance P-activated current can be suppressed by Cd2+ (0.05 microM), which suggested Ca2+ may also be involved. Soon after the neurons had been identified to be endowed with substance P receptor with whole-cell patch-clamp technique, 17 cells were chosen for immunocytochemical staining to detect substance P-immunoreactivity. Seven neurons which were classified into small and intermediate size were found to reveal substance P-immunoreactivity. Using this method we have identified the existence of substance P autoreceptor in rat DRG neurons.     

Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons

Adrenalectomy (ADX) is known to block the acquisition of intravenous cocaine self-administration. A previous study therefore examined whether ADX decreases sensitivity of the 'brain reward system' in general, or its response to cocaine in particular, by measuring thresholds for intracranial self-stimulation with and without concurrent cocaine administration. ADX had no effect on thresholds for lateral hypothalamic self-stimulation (LHSS) and did not alter the cocaine dose-response curve for lowering the LHSS threshold. This result suggested that ADX does not affect sensitivity of the brain reward system. However, medial prefrontal cortex (MPFC) appears to be an important site in the mediation of cocaine reinforcing effects, and MPFC self-stimulation (MPFCSS) is mediated by a neural substrate that is largely independent of that which mediates LHSS. The present study therefore assessed whether ADX diminishes cocaine facilitation of MPFCSS. It was found that the threshold-lowering effect of cocaine (5.0, 10.0 and 20.0 mg/kg, i.p. ) did not differ between ADX rats maintained on 0.7% saline, ADX rats maintained on corticosterone (50 microg/ml) in 0.7% saline, and sham-operated controls. However, there was a trend toward desensitization of MPFCSS, itself, following ADX in the group that did not receive corticosterone supplementation. Based on this observation, and the similar responses of MPFCSS and cocaine self-administration to noncontingent priming stimulation, stress, and NMDA receptor antagonism, it is speculated that acquisition of MPFCSS and cocaine self-administration may be dependent upon a common sensitization process that is regulated by corticosterone.     

Peripheral and central target requirements for survival of embryonic rat dorsal root ganglion neurons in slice cultures

Developmental cell death in the nervous system usually is controlled by the availability of target-derived trophic factors. It is well established that dorsal root ganglia (DRG) neurons require the presence of their peripheral target for survival, but because of their central projections, it is possible that the spinal cord also may be required. Before examining this possibility in rat embryos, we first used terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to determine that thoracic DRG cell death occurred from embryonic day 15 (E15) to E18. To determine the target requirements of DRG neurons, we used organotypic slice cultures of E15 thoracic trunk segments. After peripheral target removal, essentially all DRG neurons disappeared within 5 d. In contrast, after removal of the spinal cord, approximately half of the DRG neurons survived for at least 8 d. Hence, some E15 DRG neurons could survive without the spinal cord. However, those DRG neurons that died after spinal cord ablation apparently required trophic factors from both central and peripheral targets, because the presence of only one of these tissues was not adequate by itself to support this cell group. Addition of neurotrophin-3 (NT-3) to the culture medium rescued some DRG neurons after CNS removal, suggesting a possible role for NT-3 in vivo. In other experiments, cultures were established from older (E16) embryos, and essentially all neurons survived after spinal cord ablation, even without added factors. These and other experiments indicated that approximately 65% of DRG neurons are transiently dependent on the CNS early in development.     

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5-8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.     

Down-regulation of mu-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy

To understand the role of opioids and their receptors in chronic pain following peripheral nerve injury, we have studied the mu-opioid receptor in rat and monkey lumbar 4 and 5 dorsal root ganglion neurons and the superficial dorsal horn of the spinal cord under normal circumstances and after peripheral axotomy. Our results show that many small neurons in rat and monkey dorsal root ganglia, and some medium-sized and large neurons in rat dorsal root ganglia, express mu-opioid receptor-like immunoreactivity. Most of these neurons contain calcitonin gene-related peptide. The mu-opioid receptor was closely associated with the somatic plasmalemma of the dorsal root ganglion neurons. Both mu-opioid receptor-immunoreactive nerve fibers and cell bodies were observed in lamina II of the dorsal horn. The highest intensity of mu-opioid receptor-like immunoreactivity was observed in the deep part of lamina II. Most mu-opioid receptor-like immunoreactivity in the dorsal horn originated from spinal neurons. A few mu-opioid receptor-positive peripheral afferent terminals in the rat and monkey dorsal horn were calcitonin gene-related peptide-immunoreactive. In addition to pre- and post-junctional receptors in rat and monkey dorsal horn neurons, mu-opioid receptors were localized on the presynaptic membrane of some synapses of primary afferent terminals in the monkey dorsal horn. Peripheral axotomy caused a reduction in the number and intensity of mu-opioid receptor-positive neurons in the rat and monkey dorsal root ganglia, and of mu-opioid receptor-like immunoreactivity in the dorsal horn of the spinal cord. The decrease in mu-opioid receptor-like immunoreactivity was more pronounced in the monkey than in the rat dorsal root ganglia and spinal cord. It is probable that there was a parallel trans-synaptic down-regulation of mu-opioid-like immunoreactivity in local dorsal horn neurons of the monkey. These data suggest that one factor underlying the well known insensitivity of neuropathic pain to opioid analgesics could be due to a marked reduction in the number of mu-opioid receptors in the axotomized sensory neurons and in interneurons in the dorsal horn of the spinal cord.     

Responses of cultured adult monkey trigeminal ganglion neurons to capsaicin

The sensitivity of adult primate (Macaca mulatta) trigeminal ganglion neurons to capsaicin was studied using whole-cell recording techniques. Neurons responding to capsaicin (9 out of 14) generated inward currents of up to 3.0 nA (median = 0.23 nA; interquartile range = 1.19 nA) upon drug application measured at -60 mV. Capsaicin-sensitive neurons had longer action potential (AP) durations than capsaicin-insensitive neurons.     

c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy

The response of the mature central nervous system (CNS) to injury differs significantly from the response of the peripheral nervous system (PNS). Axotomized PNS neurons generally regenerate following injury, while CNS neurons do not. The mechanisms that are responsible for these differences are not completely known, but both intrinsic neuronal and extrinsic environmental influences are likely to contribute to regenerative success or failure. One intrinsic factor that may contribute to successful axonal regeneration is the induction of specific genes in the injured neurons. In the present study, we have evaluated the hypothesis that expression of the immediate early gene c-jun is involved in a successful regenerative response. We have compared c-Jun expression in dorsal root ganglion (DRG) neurons following central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, several dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but following a central axotomy, only 18% of the neurons expressed c-Jun. Following dorsal rhizotomy and transplantation, however, c-Jun expression was upregulated dramatically; under those experimental conditions, 63% of the DRG neurons were c-Jun-positive. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions.