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.     

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.     

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.     

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.     

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.     

Differential effects of combined trk receptor mutations on dorsal root ganglion and inner ear sensory neurons

We have generated double mutant mice deficient in pairs of two different Trk receptors and have analysed the effects on survival and differentiation of dorsal root ganglion (DRG), inner ear cochlear and vestibular sensory neurons. In most combinations of mutant trk alleles, the defects observed in double compared to single mutant mice were additive. However, double homozygous trkA-/-;trkB-/- DRG and trkB-/-;trkC-/- vestibular neurons showed the same degree of survival as single trkA-/- and trkB-/- mice, respectively, suggesting that those neurons required both Trk signaling pathways for survival. In situ hybridisation analysis of DRG neurons of double mutant mice revealed differential expression of excitatory neuropeptides. Whereas calcitonin-gene-related peptide expression correlated with the trkA phenotype, substance P expression was detected in all combinations of double mutant mice. In the inner ear, TrkB- and TrkC-dependent neurons were shown to at least partially depend on each other for survival, most likely indirectly due to abnormal development of their common targets. This effect was not observed in DRGs, where neurons depending on different Trk receptors generally innervate different targets.     

Myelin-stimulated macrophages release neurotrophic factors for adult dorsal root ganglion neurons in culture

1. Our previous study demonstrated that cultured macrophages release neurotrophic factors spontaneously. In a histological study of Wallerian degeneration, macrophages phagocytosed myelin debris and expressed activated markers. 2. To investigate the role of myelin-stimulated macrophages on neurite regeneration, we prepared conditioned media from cultured mouse peritoneal macrophages which had phagocytosed a myelin fraction. This conditioned media enhanced both neurone survival and neurite regeneration of adult dorsal root ganglia (DRG) neurons compare to conditioned media from macrophage cultures without myelin. 3. The production of the neurotrophic supernatant was dose-dependent on myelin fraction and specific for myelin because supernatants from macrophages incubated with LPS (lipoplysaccharide), MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) or latex beads were not neurotrophic. 4. The neurotrophic factors from myelin-stimulated macrophages were different from spontaneously released macrophage factors as they differed in heat-sensitivity. 5. These results suggest that myelin-stimulated macrophages contribute to axon regeneration after Wallerian degeneration.     

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.     

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.     

Suppression of substance P biosynthesis in sensory neurons of dorsal root ganglion by prodrug esters of potent peptidylglycine alpha-amidating monooxygenase inhibitors

Substance P as well as many other neuropeptides are synthesized as glycine-extended precursors and converted to the biologically active C-terminal amides by posttranslational modification. The final step of posttranslational processing is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM). In a previous study, N-substituted homocysteine analogs were found to be potent inhibitors of PAM partially purified from conditioned medium of cultured rat medullary thyroid carcinoma CA-77 cells. These compounds, however, were only modest inhibitors of substance P production in cultured dorsal root ganglion cells, possibly because of poor cell penetration. Several ester derivatives of hydrocinnamoyl-phenylalanyl-homocysteine, one of the most potent PAM inhibitors, were prepared to increase the intracellular accessibility of these compounds. Hydrocinnamoyl-phenylalanyl-(S-benzoyl-homocysteine) benzyl ester was identified as the most potent compound, inhibiting substance P biosynthesis in dorsal root ganglion cells with an IC50 of 2 microM. Inhibition of PAM resulted in a concomitant increase in the glycine-extended substance p (substance P-Gly) precursor peptide. In the presence of 3 microM benzyl ester derivative, the intracellular substance P-Gly level was 2.4-fold higher while the substance P level was 2.1-fold lower than the corresponding peptides in control cells. These results suggest that PAM inhibition represents an effective method for suppression of substance P biosynthesis and, therefore, may have therapeutic utility in conditions associated with elevated substance P levels. Furthermore, PAM inhibition may also prove useful in decreasing other amidated peptides.     

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.     

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.     

Use-dependent block of Ih in mouse dorsal root ganglion neurons by sinus node inhibitors

We have used ethylation protection experiments and modification interference using phosphorothioate nucleosides to identify phosphate groups involved in the magnesium-dependent tertiary structure and function of the VS ribozyme, a small, self-cleaving RNA. Phosphorothioate interference-rescue experiments in the presence of the thiophilic manganese ion implicate four phosphate groups in direct metal ion binding. Phosphorothioate substitution also creates a new manganese binding site that increases the cis cleavage rate of the ribozyme, possibly by disrupting an inhibitory structure. Interpreting these data in the context of a recently developed structural model shows that almost all of the important phosphate groups are located in the central core of the ribozyme. The model suggests roles for certain phosphate groups in particular steps of RNA folding and identifies a candidate region for the active site of the ribozyme.     

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.     

Expression of the trk family of neurotrophin receptors in developing and adult dorsal root ganglion neurons

Expression of trk receptors is a major determinant of neurotrophin responsiveness of sensory neurons. Although it has been apparent for some time that subpopulations of dorsal root and trigeminal ganglion neurons respond in vitro to each of the members of the neurotrophin family, the extent to which functionally distinct subclasses of sensory neurons are dependent on the actions of different neurotrophins for their development and function remains an active area of investigation. One step towards elucidating the role of various neurotrophins in development and function of sensory neurons has been to examine the distribution of trk receptors on sensory neurons. These studies have clearly revealed that members of the trk family are differentially expressed in functionally distinct populations of both developing and mature sensory neurons and, further, have provided evidence consistent with a shift in neurotrophin responsiveness during the development of sensory neurons.     

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43 degrees C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca2+ concentration ([Ca2+]i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca2+]i from 52.5 +/- 9.5 nM (mean +/- S.D., n = 18) to 171.0 +/- 15.6 nM in cells between 15 and 25 microns in diameter. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. The Na(+)-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG 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.