Distinct inhibition of voltage-activated Ca2+ channels by delta-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents

Both mu- and delta-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca(2+) channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of delta-opioid agonists on voltage-activated Ca(2+) channel currents (I(Ca)) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B(4) (IB(4)). The delta-opioid agonists [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage-activated I(Ca) in IB(4)-negative than IB(4)-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type I(Ca) in IB(4)-negative than IB(4)-positive neurons. However, DPDPE had no significant effect on the R-type I(Ca) in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage-activated I(Ca) in all the DRG neurons with T-type I(Ca). Double immunofluorescence labeling showed that the majority of the delta-opioid receptor-immunoreactive DRG neurons had IB(4) labeling, while all DRG neurons immunoreactive to delta-opioid receptors exhibited Cav(3.2) immunoreactivity. Additionally, DPDPE significantly inhibited high voltage-activated I(Ca) in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that delta-opioid agonists have a distinct effect on voltage-activated Ca(2+) channels in different phenotypes of primary sensory neurons. High voltage-activated Ca(2+) channels are more sensitive to inhibition by delta-opioid agonists in IB(4)-negative than IB(4)-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca(2+) currents.     

A-type voltage-gated K+ currents influence firing properties of isolectin B4-positive but not isolectin B4-negative primary sensory neurons

Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.     

Differential response properties of IB(4)-positive and -negative unmyelinated sensory neurons to protons and capsaicin

Activation of unmyelinated (C-fiber) nociceptors by noxious chemicals plays a critical role in the initiation and maintenance of injury-induced pain. C-fiber nociceptors can be divided into two groups in which one class depends on nerve growth factor during postnatal development and contains neuropeptides, and the second class depends on glial cell line-derived neurotrophic factor during postnatal development and contains few neuropeptides but binds isolectin B(4) (IB(4)). We determined the sensitivity of these two populations to protons and capsaicin using whole cell recordings of dorsal root ganglion neurons from adult mouse. IB(4)-negative unmyelinated neurons were significantly more responsive to protons than IB(4)-positive neurons in a concentration-dependent manner. Approximately 86% of IB(4)-negative neurons responded to pH 5.0 with an inward current compared with only 33% of IB(4)-positive neurons. The subtypes of proton-evoked currents in IB(4)-negative unmyelinated neurons were also more diverse. Many IB(4)-negative neurons exhibited transient, rapidly inactivating proton currents as well as sustained proton currents. In contrast, IB(4)-positive neurons never displayed transient proton currents and responded to protons only with sustained, slowly inactivating inward currents. The two classes of neurons also responded differently to capsaicin. Twice as many na?ve IB(4)-negative unmyelinated neurons responded to 1 microM capsaicin as IB(4)-positive neurons, and the capsaicin-evoked currents in IB(4)-negative neurons were approximately fourfold larger than those in IB(4)-positive neurons. Interestingly, proton exposure altered the capsaicin responsiveness of the two classes of neurons in opposite ways. Brief preexposure to protons increased the number of capsaicin-responsive IB(4)-positive neurons by twofold and increased the capsaicin-evoked currents by threefold. Conversely, proton exposure decreased the number of capsaicin-responsive IB(4)-negative neurons by 50%. These data suggest that IB(4)-negative unmyelinated nociceptors are initially the primary responders to both protons and capsaicin, but IB(4)-positive nociceptors have a unique capacity to be sensitized by protons to capsaicin-receptor agonists.     

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.     

Effects of familial hemiplegic migraine type 1 mutation T666M on voltage-gated calcium channel activities in trigeminal ganglion neurons

Familial hemiplegic migraine type 1 (FHM-1), a rare hereditary form of migraine with aura and hemiparesis, serves as a good model for exploring migraine pathophysiology. The FHM-1 gene encodes the pore-forming Ca(V)2.1 subunit of human P/Q-type voltage-gated Ca(2+) channels (VGCCs). Some FHM-1 mutations result in a decrease of whole cell P/Q-type current density in transfected cells/neurons. Questions remain as to whether and how these mutations may increase the gain of the trigeminal nociceptive pathway underlying migraine headache. Here, we investigated the effects of T666M, the most frequently occurring FHM-1 mutation, on VGCC currents and neuronal excitability in trigeminal ganglion (TG) neurons. We expressed human wild-type and T666M Ca(V)2.1 subunits in cultured TG neurons from Ca(V)2.1 knockout mice and recorded whole cell VGCC currents in transfected neurons. Currents mediated by individual VGCC subtypes were dissected according to their pharmacological and biophysical properties. TG neurons were sorted into three subpopulations based on their soma size and their affinity to isolectin B4 (IB4). We found that the T666M mutation did not affect total or surface expression of Ca(V)2.1 proteins but caused a profound reduction of P/Q-type current in all subtypes of TG neurons. Interestingly, a compensatory increase in Ca(V)3.2-mediated low-voltage-activated T-type currents only occurred in small IB4-negative (IB4(-)) TG neurons expressing T666M subunits. Current-clamp recordings showed that the T666M mutation resulted in hyperexcitability of the small IB4(-) TG population. Taken together, our results suggest a possible scenario through which FHM-1 mutations might increase the gain of the trigeminal nociceptive pathway.     

Differential pH and capsaicin responses of Griffonia simplicifolia IB4 (IB4)-positive and IB4-negative small sensory neurons

Protons play a key role in nociception caused by inflammation and ischaemia, but little is known about the relative sensitivities of different dorsal root ganglion (DRG) neurons. We have therefore examined the responses in vitro of rat DRG cells classified according to whether or not they bind Griffonia simplicifolia IB4 (IB4), a lectin which is widely used to distinguish between two major populations of small diameter neurons. Under voltage-clamp conditions, proton-activated inward currents were found in approximately 90% of small DRG neurons and showed one of three waveforms: transient, sustained or mixed. The majority of IB4-positive (IB4+) neurons (63%) gave rise to sustained inward currents that were sensitive to capsazepine. In contrast, the most prevalent waveform in small IB4-negative (IB4-) neurons (69%) was a mixed response containing transient and sustained components. The transient component was inhibited by amiloride whilst the sustained component showed a variable sensitivity to capsazepine. We also found that more IB4+ cells responded to capsaicin and, on average, gave rise to a larger magnitude of response than small IB4- neurons, consistent with their higher prevalence and greater amplitude of vanilloid receptor 1 (TRPV1)-like acid responses. The increase in intracellular Ca(2+) induced by capsaicin was also slightly greater in IB4+ neurons and in these cells its magnitude correlated with the level of TRPV1 immunoreactivity. Our data suggest that acid-sensing ion channels (ASICs) and TRPV1 are the major acid-sensitive receptors in small IB4- neurons, whilst TRPV1 is the predominant one in IB4+ neurons. Because ASIC-like responses were approximately 10-fold more sensitive to changes in H(+) than TRPV1-like responses, we speculate that small IB4- rather than IB4+ neurons play an essential role in sensing acid. Our results also highlight differences in capsaicin responses between IB4+ and IB4- small neurons and reveal the close link between capsaicin responses and levels of TRPV1 expression.     

Characteristics of calcium currents in rat geniculate ganglion neurons

Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract (rNST). We used whole cell recording to study the characteristics of the Ca(2+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. PA neurons were significantly larger than CT and GSP neurons, and CT neurons could be further subdivided based on soma diameter. Although all GG neurons possess both low voltage-activated (LVA) "T-type" and high voltage-activated (HVA) Ca(2+) currents, CT, GSP, and PA neurons have distinctly different Ca(2+) current expression patterns. Of GG neurons that express T-type currents, the CT and GSP neurons had moderate and PA neurons had larger amplitude T-type currents. HVA Ca(2+) currents in the GG neurons were separated into several groups using specific Ca(2+) channel blockers. Sequential applications of L, N, and P/Q-type channel antagonists inhibited portions of Ca(2+) current in all CT, GSP, and PA neurons to a different extent in each neuron group. No difference was observed in the percentage of L- and N-type Ca(2+) currents reduced by the antagonists in CT, GSP, and PA neurons. Action potentials in GG neurons are followed by a Ca(2+) current initiated after depolarization (ADP) that may influence intrinsic firing patterns. These results show that based on Ca(2+) channel expression the GG contains a heterogeneous population of sensory neurons possibly related to the type of sensory information they relay to the rNST.     

Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder.

The effect of a nitric oxide (NO) donor on high-voltage-activated Ca(2+) channel currents (I(Ca)) was examined using the whole cell patch-clamp technique in L(6)-S(1) dorsal root ganglion (DRG) neurons innervating the urinary bladder. The neurons were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall. Approximately 70% of bladder afferent neurons exhibited tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these neurons were sensitive to capsaicin, while the remaining neurons had TTX-sensitive spikes and were insensitive to capsaicin. The peak current density of nimodipine-sensitive L-type Ca(2+) channels activated by depolarizing pulses (0 mV) from a holding potential of -60 mV was greater in bladder afferent neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of omega-conotoxin GVIA-sensitive N-type Ca(2+) channels was similar (43-45 pA/pF) in both types of neurons. In both types of neurons, the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) (500 microM), reversibly reduced (23.4-26.6%) the amplitude of I(Ca) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV. SNAP-induced inhibition of I(Ca) was reduced by 90% in the presence of omega-conotoxin GVIA but was unaffected in the presence of nimodipine, indicating that NO-induced inhibition of I(Ca) is mainly confined to N-type Ca(2+) channels. Exposure of the neurons for 30 min to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), an inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced reduction in I(Ca). Extracellular application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by reducing the peak amplitude of I(Ca) (28.6% of reduction). Action potential configuration and firing frequency during depolarizing current pulses were not altered by the application of SNAP (500 microM) in bladder afferent neurons with TTX-resistant and -sensitive APs. These results indicate that NO acting via a cGMP signaling pathway can modulate N-type Ca(2+) channels in DRG neurons innervating the urinary bladder.     

Agonist-independent modulation of N-type calcium channels by ORL1 receptors

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel-ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.     

Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein expression in large versus small diameter dorsal root ganglion neurons: correlation with hyperpolarization-activated current gating

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons. We studied HCN1-4 channel mRNA and protein expression and correlated these findings with I(h) functional properties in rat DRG neurons of different size. Quantitative RT-PCR (TaqMan) analysis demonstrated that HCN2 and HCN1 mRNAs were more abundantly expressed in large diameter (55-80 microm) neurons, while HCN3 mRNA was preferentially expressed in small diameter (20-30 microm) neurons. HCN4 mRNA expression was very low in neurons of all sizes. At the protein level, subunit-selective polyclonal antibodies and immunofluorescence indicated that HCN1 and HCN3 are present in large diameter neurons and small diameter neurons. Staining in small diameter neurons was in IB4-positive (non-peptidergic) and IB4-negative (peptidergic) cells. HCN2 immunofluorescent staining was heterogeneous and predominantly in large diameter neurons and in small diameter IB4-negative neurons. HCN4 was poorly expressed in all neurons. Functionally, I(h) amplitude and density were significantly larger, and activation kinetics faster, in large diameter neurons when compared with small neurons. I(h) activation rates in small and large diameter DRG neurons were consistent with the relative abundance of HCN subunits in the respective cell type, considering the reported HCN channel activation rates in heterologous systems (HCN1>HCN2 approximately HCN3>HCN4), suggesting exclusivity of roles of different HCN subunits contributing to the excitability of DRG neurons of different size. Additionally, a functional role of I(h) in small DRG neuron excitability was evaluated using a computational model.     

Distribution patterns of different P2x receptor phenotypes in acutely dissociated dorsal root ganglion neurons of adult rats

P2x receptors may be used to detect ATP release from tissues during physiological and pathological conditions. We used whole-cell patch clamp recordings to study the expression of P2x receptor phenotypes, their distribution patterns, and their sensitivity to alphabetamATP and suramin in dorsal root ganglion (DRG) neurons acutely dissociated from adult rats. Based on the onset and decay rates of 10 microM ATP-evoked currents, we showed three types of P2x currents: fast, slow, and mixed. Each of these P2x receptor phenotypes had a distinct distribution pattern among DRG neurons. The fast P2x currents were predominantly expressed in small-diameter, isolectin-B4 (IB4)-positive, and capsaicin-sensitive DRG neurons. The slow P2x currents were expressed in both small and medium DRG neurons, and about half of them were IB4 positive. The mixed P2x currents were also expressed in both small and medium-sized DRG neurons, and most of these neurons were IB4-positive neurons. The slow and mixed P2x current groups had both capsaicin-sensitive and -insensitive DRG neurons. All phenotypes revealed with 10 microM ATP could be inhibited by 30 microM suramin. All DRG neurons with fast or mixed P2x currents were also sensitive to 10 microM alphabetamATP, and alphabetamATP evoked currents similar to those induced by ATP. The group expressing slow P2x currents could be further divided into alphabetamATP-sensitive and -insensitive groups. Thus, the relationships among P2x receptor phenotypes, cell sizes, IB4 positivity, and capsaicin sensitivity are more complicated than previously thought, and different P2x receptors may be involved in both nociceptive and non-nociceptive functions.     

Inhibitory effect of pranidipine on N-type voltage-dependent Ca2+ channels in mice

We investigated the N-type voltage-dependent calcium channel blocking action of pranidipine, a novel dihydropyridine (DHP) derivative. Pranidipine significantly suppressed KCl-induced intracellular calcium changes ([Ca(2+)](i)) in a dose-dependent fashion in dorsal root ganglion neurons. A patch-clamp investigation revealed a dose-dependent blocking effect on N-type currents. Intrathecal injection of pranidipine significantly shortened the licking time in the late phase of the formalin test, as occurs with cilnidipine and amlodipine, which act on L- and N-type channels. Conversely, nicardipine, which acts exclusively on L-type channels, had no antinociceptive effect. Our results indicate that pranidipine inhibits N-type calcium channels. Furthermore, it exerts an antinociceptive effect, which might be related to an attenuation of synaptic transmission by nociceptive neurons due to the blocking effect of pranidipine on N-type calcium channels in primary nociceptive afferent fibers.     

M(1) and M(2) muscarinic acetylcholine receptor subtypes mediate Ca(2+) channel current inhibition in rat sympathetic stellate ganglion neurons

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca(2+) channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca(2+) channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca(2+) channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca(2+) channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca(2+) currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M(1) receptor blocker, M(1)-toxin, resulted in VD inhibition of Ca(2+) currents after Oxo-M application. On the other hand, VI modulation of Ca(2+) currents was observed after pretreatment of cells with methoctramine (M(2) mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M(1) and M(2) mAChR blockers but was unaltered when SG neurons were exposed to the M(4) mAChR toxin, M(4)-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M(1) and M(2) receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M(1) and M(2) mAChR, and activation of these receptors leads to inhibition of Ca(2+) channel currents through VI and VD pathways, respectively.     

Repertoire of high voltage-activated Ca2+ channels in the lateral superior olive: functional analysis in wild-type, Ca(v)1.3(-/-), and Ca(v)1.2DHP(-/-) mice

Voltage-gated Ca(2+) (Ca(v))1.3 α-subunits of high voltage-activated Ca(2+) channels (HVACCs) are essential for Ca(2+) influx and transmitter release in cochlear inner hair cells and therefore for signal transmission into the central auditory pathway. Their absence leads to deafness and to striking structural changes in the auditory brain stem, particularly in the lateral superior olive (LSO). Here, we analyzed the contribution of various types of HVACCs to the total Ca(2+) current (I(Ca)) in developing mouse LSO neurons to address several questions: do LSO neurons express functional Ca(v)1.3 channels? What other types of HVACCs are expressed? Are there developmental changes? Do LSO neurons of Ca(v)1.3(-/-) mice show any compensatory responses, namely, upregulation of other HVACCs? Our electrophysiological and pharmacological results showed the presence of functional Ca(v)1.3 and Ca(v)1.2 channels at both postnatal days 4 and 12. Aside from these L-type channels, LSO neurons also expressed functional P/Q-type, N-type, and, most likely, R-type channels. The relative contribution of the four different subtypes to I(Ca) appeared to be 45%, 29%, 22%, and 4% at postnatal day 12, respectively. The physiological results were flanked and extended by quantitative RT-PCR data. Altogether, LSO neurons displayed a broad repertoire of HVACC subtypes. Genetic ablation of Ca(v)1.3 resulted in functional reorganization of some other HVACCs but did not restore normal I(Ca) properties. Together, our results suggest that several types of HVACCs are of functional relevance for the developing LSO. Whether on-site loss of Ca(v)1.3, i.e., in LSO neurons, contributes to the recently described malformation of the LSO needs to be determined by using tissue-specific Ca(v)1.3(-/-) animals.     

Immunohistochemical demonstration of the calcium channel alpha2 subunit in the chicken dorsal root ganglion and spinal cord: a special reference to colocalization with calbindin-D28k in dorsal root ganglion neurons

We used immunohistochemical methods to examine the distribution of the calcium channel alpha2 (CCalpha2) subunit in the chicken spinal cord and dorsal root ganglion (DRG) neurons and determine its relationship with calbindin-D28k (CB) in the DRG neurons. In the spinal cord, CCalpha2 subunit was detected in nerve terminals, which were observed as dot-like structures, and in laminae I, II, III and Lissauer's tract in the dorsal horn. In the DRG neurons, approximately 65% of the total neurons were CCalpha2 subunit positive, and most (86%) of these neurons were small to medium sized, suggesting that the CCalpha2 subunit and/or a complex of the CCalpha2 and delta subunits is possibly localized in a number of nociceptive neurons. A majority (77%) of the positive neurons showed CB immunoreactivity and most (88%) of these neurons were small to medium sized. This may indicate a close correlation between the CCalpha2 subunit and CB in the nociceptive neurons. Thus, it is postulated that the mode of nociceptive transmission may involve a cellular Ca(2+)-regulating system that consists of both Ca(2+) entry via calcium channels with the alpha2delta subunit and intracellular Ca(2+)-binding activity of CB in the nociceptive neurons of the DRG.     

Pharmacological and biophysical properties of Ca2+ channels and subtype distributions in human adrenal chromaffin cells

In this study, we explored the pharmacological and biophysical properties of voltage-activated Ca(2+) channels in human chromaffin cells using the perforated-patch configuration of the patch-clamp technique. According to their pharmacological sensitivity to Ca(2+) channel blockers, cells could be sorted into two groups of similar size showing the predominance of either N- or P/Q-type Ca(2+) channels. R-type Ca(2+) channels, blocked by 77% with 20 muM Cd(2+) and not affected by 50 muM Ni(2+), were detected for the first time in human chromaffin cells. Immunocytochemical experiments revealed an even distribution of alpha (1E) Ca(2+) channels in these cells. With regard to their biophysical properties, L- and R-type channels were activated at membrane potentials that were 15-20 mV more negative than P/Q- and N-type channels. Activation time constants showed no variation with voltage for the L-type channels, decreased with increasing potentials for the R- and P/Q-type channels, and displayed a bell shape with a maximum at 0 mV for the N-type channels. R-type channels were also the most inactivated channels. We thus show here that human chromaffin cells possess all the Ca(2+) channel types described in neurons, L, N, P/Q, and R channels, but the relative contributions of N and P/Q channels differ among cells. Given that N- and P/Q-type Ca(2+) channel types can be differentially modulated, these findings suggest the possibility of cell-specific regulation in human chromaffin cells.     

Inhibition of rapid heat responses in nociceptive primary sensory neurons of rats by vanilloid receptor antagonists

Recent studies demonstrated that heat-sensitive nociceptive primary sensory neurons respond to the vanilloid receptor (VR) agonist capsaicin, and the first cloned VR is a heat-sensitive ion channel. Therefore we studied to what extent heat-evoked currents in nociceptive dorsal root ganglion (DRG) neurons can be attributed to the activation of native vanilloid receptors. Heat-evoked currents were investigated in 89 neurons acutely dissociated from adult rat DRGs as models for their own terminals using the whole cell patch-clamp technique. Locally applied heated extracellular solution (effective temperature approximately 53 degrees C) rapidly activated reversible and reproducible inward currents in 80% (62/80) of small neurons (< or = 32.5 microm), but in none of nine large neurons (P < 0.001, chi(2) test). Heat and capsaicin sensitivity were significantly coexpressed in this subpopulation of small DRG neurons (P < 0.001, chi(2) test). Heat-evoked currents were accompanied by an increase of membrane conductance (320 +/- 115%; mean +/- SE, n = 7), had a reversal potential of 5 +/- 2 mV (n = 5), which did not differ from that of capsaicin-induced currents in the same neurons (4 +/- 3 mV), and were carried at least by Na(+) and Ca(2+) (pCa(2+) > pNa(+)). These observations are consistent with the opening of temperature-operated nonselective cation channels. The duration of action potentials was significantly higher in heat-sensitive (10-90% decay time: 4.45 +/- 0.39 ms, n = 12) compared with heat-insensitive neurons (2.18 +/- 0.19 ms, n = 6; P < 0.005, Student's t-test), due to an inflection in the repolarizing phase. This property as well as capsaicin sensitivity and small cell size are characteristics of nociceptive DRG neurons. When coadministered with heat stimuli, the competitive VR antagonist capsazepine (1 microM to 1 mM) significantly reduced heat-evoked currents in a dose-dependent manner (IC(50) 13 microM, Hill slope -0.58, maximum effect 75%). Preincubation for 12-15 s shifted the IC(50) by approximately 0.5 log(10) units to an estimated IC(50) of approximately 4 microM. The noncompetitive VR antagonist ruthenium red (5 microM) significantly reduced heat-evoked currents by 33 +/- 6%. The effects of both VR antagonists were rapidly reversible. Our results provide evidence for a specific activation of native VRs in nociceptive primary sensory neurons by noxious heat. The major proportion of the rapid heat-evoked currents can be attributed to the activation of these temperature-operated channels, and noxious heat may be the signal detected by VRs under physiological conditions.     

Characterization of Ca(2+) channels in rat subthalamic nucleus neurons

The subthalamic nucleus (STN) plays a key role in motor control. Although previous studies have suggested that Ca(2+) conductances may be involved in regulating the activity of STN neurons, Ca(2+) channels in this region have not yet been characterized. We have therefore investigated the subtypes and functional characteristics of Ca(2+) conductances in STN neurons, in both acutely isolated and slice preparations. Acutely isolated STN cells were identified by retrograde filling with the fluorescent dye, Fluoro-Gold. In acutely isolated STN neurons, Cd(2+)-sensitive, depolarization-activated Ba(2+) currents were observed in all cells studied. The current-voltage relationship and current kinetics were characteristic of high-voltage-activated Ca(2+) channels. The steady-state voltage-dependent activation curves and inactivation curves could both be fitted with a single Boltzmann function. Currents evoked with a prolonged pulse, however, inactivated with multiple time constants, suggesting either the presence of more than one Ca(2+) channel subtype or multiple inactivation processes with a single channel type in STN neurons. Experiments using organic Ca(2+) channel blockers revealed that on average, 21% of the current was nifedipine sensitive, 52% was sensitive to omega-conotoxin GVIA, 16% was blocked by a high concentration of omega-agatoxin IVA (200 nM), and the remainder of the current (9%) was resistant to the co-application of all blockers. These currents had similar voltage dependencies, but the nifedipine-sensitive current and the resistant current activated at slightly lower voltages. omega-Agatoxin IVA at 20 nM was ineffective in blocking the current. Together, the above results suggest that acutely isolated STN neurons have all subtypes of high-voltage-activated Ca(2+) channels except for P-type, but have no low-voltage-activated channels. Although acutely isolated neurons provide a good preparation for whole cell voltage-clamp study, dendritic processes are lost during dissociation. To gain information on Ca(2+) channels in dendrites, we thus studied Ca(2+) channels of STN neurons in a slice preparation, focusing on low-voltage-activated channels. In current-clamp recordings, a slow spike was always observed following termination of an injected hyperpolarizing current. The slow spike occurred at resting membrane potentials and was sensitive to micromolar concentrations of Ni(2+), suggesting that it is a low-threshold Ca(2+) spike. Together, our results suggest that STN neurons express low-voltage-activated Ca(2+) channels and several high-voltage-activated subtypes. Our results also suggest the possibility that the low-voltage-activated channels have a preferential distribution to the dendritic processes.     

Age-related functional changes of high-voltage-activated calcium channels in different neuronal subtypes of mouse striatum

By means of whole-cell patch-clamp recordings, we characterized the developmental profile of high-voltage-activated (HVA) calcium (Ca(2+)) channel subtypes in distinct neuronal populations of mouse striatum. Acutely dissociated medium spiny neurons (MSNs) and cholinergic interneurons (ChIs) were recorded from mice at five developmental stages: postnatal-days (PD) 14, 23, 40, 150 and 270. During ageing, total HVA Ca(2+) current recorded from both MSNs and ChIs was unchanged. However, the pharmacological analysis of the differential contribution of HVA Ca(2+) channel subtypes showed a significant rearrangement of each component. In both neuronal subtypes, a large fraction of the total HVA current recorded from PD14 mice was inhibited by the L-type HVA channel blocker nifedipine. This dihydropyridine-sensitive component accounted for nearly 50%, in MSNs, and 35%, in ChIs, of total current at PD14, but its contribution was down-regulated up to 20-25% at 9 months. Likewise, the N-type, omega-conotoxin GVIA-sensitive component decreased from 35% to 40% to about 25% in MSNs and 15% in ChIs. The P-type, omega-agatoxin-sensitive fraction did not show significant changes in both neuronal subtypes, whereas the Q-type, omega-conotoxin MVIIC-sensitive channels did show a significant up-regulation at 9 months. As compared with striatal neurons, we recorded pyramidal neurons dissociated from cortical layers IV-V and found no significant developmental change in the different components of HVA Ca(2+) currents. In conclusion, our data demonstrate a functional reconfiguration of HVA Ca(2+) channels in striatal but not cortical pyramidal neurons during mouse development. Such changes might have profound implications for physiological and pathophysiological processes of the striatum.     

Sulfur dioxide derivatives modulation of high-threshold calcium currents in rat dorsal root ganglion neurons

This study addressed the effect of sulfur dioxide (SO(2)) derivatives on high-voltage-activated calcium currents (HVA-I(Ca)) in somatic membrane of freshly isolated rat dorsal root ganglion (DRG) neurons by using the whole-cell configuration of patch-clamp technique. High-threshold Ca(2+) channels are highly expressed in small dorsal root ganglion neurons. SO(2) derivatives increased the amplitudes of calcium currents in a concentration-dependent and voltage-dependent manner. The 50% enhancement concentrations (EC(50)) of SO(2) derivatives on HVA-I(Ca) was about 0.4 microM. In addition, SO(2) derivatives significantly shifted the activation and inactivation curve in the depolarizing direction. Parameters for the fit of a Boltzmann equation to mean values for the activation were V(1/2)=-17.9+/-1.3 mV before and -12.5+/-1.1 mV after application 0.5 microM SO(2) derivatives 2 min (P<0.05). The half inactivation of HVA-I(Ca) was shifted 9.7 mV to positive direction (P<0.05). Furthermore, SO(2) derivatives significantly prolonged the slow constant of inactivation, slowed the fast recovery but markedly accelerated the slow recovery of HVA-I(Ca) from inactivation. From HP of -60 mV 0.5 microM SO(2) derivatives increased the amplitude of HVA-I(Ca) with a depolarizing voltage step to -10 mV about 54.0% in small DRG neurons but 33.3% in large DRG neurons. These results indicated a possible correlation between the change of calcium channels and SO(2) inhalation toxicity, which might cause periphery neurons abnormal regulation of nociceptive transmission via calcium channels.