Nociceptin is a potent inhibitor of N-type Ca(2+) channels in rat sympathetic ganglion neurons

Nociceptin, an endogenous agonist of the opioid receptor-like(1) (ORL(1)) receptor, is implicated in a wide range of physiological functions including cardiovascular control. However, the effect of nociceptin on peripheral sympathetic ganglion neurons has not been studied. Whole-cell voltage clamp was used to study Ca(2+) currents on freshly dissociated sympathetic superior cervical ganglion neurons from juvenile rats. Nociceptin (1 microM) caused a fast inhibition of the peak currents by 69+/-3% in all neurons. Strong positive prepulses counteracted the inhibition of the peak current by 64% and no effect of nociceptin was observed when the cells were pre-incubated with Pertussis toxin. The inhibition was reversible and dose-dependent with an EC(50) of 508+/-50 pM. Blockade of N-type channels by 1 microM omega-conotoxin GVIA reduced the peak currents by 83+/-1% and abolished the action of nociceptin. Naloxone could not prevent the inhibition by nociceptin and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin (DAMGO) only depressed a small proportion of the current in 1/7 neurons. These data suggests that nociceptin inhibits transmitter release from sympathetic neurons by a selective blockade of N-type channels, which may be of importance for its depressive effect on the cardiovascular system.     

Modulation of N-type Ca2+ channel current kinetics by PMA in rat sympathetic neurons

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) has been used extensively in studies of G protein modulation of Ca2+ channels. PMA has been shown to be a powerful tool for inducing phosphorylation and interrupting G-protein-mediated signaling pathways. Here we re-examine the effects of PMA on whole-cell N-type Ca2+-channel currents in rat sympathetic neurons. We found that, along with an increase in the current amplitude previously reported by others, PMA pretreatment leads to alterations in current activation and inactivation kinetics. These alterations in current kinetics are voltage-dependent and are not reproduced by internal dialysis with the G protein inhibitor GDPbetaS. Alterations in current kinetics by PMA may therefore indicate the existence of a modulated state, presumably phosphorylated, of N-type Ca2+ channels. We propose that the increase in current amplitude is due primarily to alterations in current kinetics rather than to removal of tonic inhibition.     

Localization of M(2) muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus

The muscarinic receptor family (M(1)-M(4)) mediates cholinergic modulation of hippocampal transmission. Pharmacological and physiological studies have indicated that a presynaptic receptor on cholinergic terminals plays a key role in regulating ACh release, although the molecular identity of this subtype is uncertain. In this study, the localization of the M(2) receptor is described in detail for the pyramidal cell layer in the CAl region of the hippocampus. Electron microscopic analysis of M(2) immunoreactivity in this area revealed mainly presynaptic expression of this subtype. Double-labeling experiments using antibodies to M(2) and to the vesicular acetylcholine transporter, a novel, specific marker of cholinergic terminals, were used to investigate the nature of these presynaptic receptors. These studies have revealed that M(2) is located in cholinergic and non-cholinergic terminals. This is the first direct anatomical evidence that suggests that M(2) may indeed function as a cholinergic autoreceptor in the hippocampus. The distribution of the M(2) receptor in non-cholinergic terminals also suggests functional roles for M(2) as a presynaptic heteroreceptor.     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the Ca(2+)-activated K+ channel in rat hippocampal neurons

The kinetics of the large-conductance Ca(2+)-activated K+ channel (235 pS in symmetrical 150 mM K+) were examined in the inside-out mode of the patch clamp technique. The open probability of the channel increased when [Ca2+]i, [Sr2+]i, or [Ba2+]i was increased. The [Ca2+]i-response relation was fitted with a Hill coefficient of 2 and half-maximum concentrations of 185, 80, 14.5, and 5.5 microM at -40, -20, +20, and +40 mV, respectively. The channel was blocked by TEA or Ba2+. The open-time histogram showed a single exponential component and the closed-time histogram showed at least two exponential components at various [Ca2+]i. Increasing [Ca2+]i decreased the time constant of the slow component of the closed-time histogram. Cell-attached patch recording revealed activation of the large-conductance Ca(2+)-activated K+ channel (BK channel) during the action potential. The deactivation time course was consistent with the fast after-hyperpolarization. A minimum model of the channel, close(2)-close(1)-open, where the transition from close(2) to close(1) requires the binding of 2 Ca2+, reconstructed quick activation of the channel if [Ca2+]i of 40 microM was assumed.     

Developmental changes in heteromeric P2X(2/3) receptor expression in rat sympathetic ganglion neurons.

We have used whole cell patch clamp recording and immunohistochemistry to investigate the expression of P2X(2/3) receptors in rat superior cervical ganglion neurons during late embryonic and early post-natal development. Neurons from E18 and P1 animals responded to the nicotinic agonist dimethylphenylpiperazinium (DMPP), and the purinoceptor agonists ATP and alpha,beta-meATP with sustained inward currents. Responsiveness to DMPP was maintained at P 17, while that to ATP declined dramatically, and responses to alpha,beta-meATP were rarely detected. Immunohistochemistry for the P2X(3) subunit revealed widespread staining in superior cervical ganglia from P1 rats, but little immunoreactivity in ganglia from P17 animals. In neurons from P1 animals, the response to alpha,beta-meATP exhibited pharmacological properties of the heteromeric P2X(2/3) receptor. In conclusion, sympathetic neurons of the rat superior cervical ganglion are more responsive to ATP and alpha,beta-meATP at birth and during the early post-natal period, due largely to the expression of the P2X(3) subunit, but these responses are much reduced in mature rats.     

Electrophysiological and immunofluorescence characterization of Ca(2+) channels of acutely isolated rat sphenopalatine ganglion neurons

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.     

Ca2+ enhances U-type inactivation of N-type (CaV2.2) calcium current in rat sympathetic neurons

Ca2+ -dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (tau approximately 150 ms) and slow (tau approximately 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5-30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.     

Effect of high Ba(2+) on norepinephrine-induced inhibition of N-type calcium current in bullfrog sympathetic neurons

The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba(2+). Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba(2+) external solution without an effect on maximum inhibition. The IC(50) was 0.6 and 3 microM in 2 and 100 mM Ba(2+), respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba(2+). The inhibition induced by 1 microM NE was reduced in 100 mM Ba(2+) compared with that in 2 mM Ba(2+). However, the response to 100 microM NE was identical between high and low Ba(2+). Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba(2+), the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration.     

High voltage-activated Ca(2+) channel currents in isolectin B(4)-positive and -negative small dorsal root ganglion neurons of rats

Voltage-gated Ca(2+) channels in the primary sensory neurons are important for neurotransmitter release and regulation of nociceptive transmission. Although multiple classes of Ca(2+) channels are expressed in the dorsal root ganglion (DRG) neurons, little is known about the difference in the specific channel subtypes among the different types of DRG neurons. In this study, we determined the possible difference in high voltage-activated Ca(2+) channel currents between isolectin B(4) (IB(4))-positive and IB(4)-negative small-sized (15-30 microm) DRG neurons. Rat DRG neurons were acutely isolated and labeled with IB(4) conjugated to a fluorescent dye. Whole-cell patch clamp recordings of barium currents flowing through calcium channels were performed on neurons with and without IB(4). The peak current density of voltage-gated Ca(2+) currents was not significantly different between IB(4)-positive and IB(4)-negative neurons. Also, both nimodipine and omega-agatoxin IVA produced similar inhibitory effects on Ca(2+) currents in these two types of neurons. However, block of N-type Ca(2+) channels with omega-conotoxin GVIA produced a significantly greater reduction of Ca(2+) currents in IB(4)-positive than IB(4)-negative neurons. Furthermore, the IB(4)-positive neurons had a significantly smaller residual Ca(2+) currents than IB(4)-negative neurons. These data suggest that a higher density of N-type Ca(2+) channels is present in IB(4)-positive than IB(4)-negative small-sized DRG neurons. This differential expression of the subtypes of high voltage-activated Ca(2+) channels may contribute to the different function of these two classes of nociceptive neurons.     

Fulfenamic acid sensitive,Ca(2+)-dependent inward current induced by nicotinic acetylcholine receptors in dopamine neurons

Nicotinic acetylcholine receptors (nAChRs) exhibit high Ca(2+) permeabilities and the Ca(2+)-influx through the nAChRs may be involved in regulation of a variety of signal processing in the postsynaptic neurons. The mesencephalic dopamine (DA) neurons receive cholinergic inputs from the brainstem and express abundant nAChRs. Here we report that the Ca(2+)-influx induced by a transient pressure application of ACh activates an inward current mediated by nAChRs and subsequently an inward current component that is sensitive to fulfenamic acid (FFA) and phenytoin, presumably a Ca(2+)-activated nonselective cation current in the DA neurons in the midbrain slices of the rat. The FFA- and phenytoin-sensitive current exhibits a negative slope conductance below -40 mV, suggesting its role in significant enhancement of depolarizing responses. In the current clamp recordings with perforated patch clamp configuration, bath application of carbachol markedly enhanced the glutamate-induced depolarization, which led to a long-lasting depolarizing hump. Activation of nAChRs is involved in this process, in cooperation with muscarinic receptors that suppress afterhyperpolarization caused by Ca(2+)-activated K(+)-channels. The long-lasting depolarizing hump was suppressed by FFA. All these results suggested a potential role of the FFA-sensitive current triggered by nAChR activation in marked enhancement of the excitatory synaptic response in DA neurons.     

Ca(2+)-independent but voltage-dependent secretion in mammalian dorsal root ganglion neurons

We have investigated the Ca(2+) dependence of vesicular secretion from the soma of dorsal root ganglion (DRG) neurons, which secrete neuropeptides by exocytosis of dense-core vesicles. In patch-clamped somata of rat DRG neurons, we found a depolarization-induced membrane capacitance increase (DeltaC(m)) in the absence of extracellular Ca(2+) and in the presence of a Ca(2+) chelator (BAPTA) in the intracellular solution. Depletion of internal Ca(2+) stores by thapsigargin in the Ca(2+)-free bath also did not block the DeltaC(m), indicating that Ca(2+) release from internal Ca(2+) stores may not have been involved. Furthermore, the Ca(2+)-independent DeltaC(m) was blocked by whole-cell dialysis with tetanus toxin and was accompanied by pulsatile secretion of false transmitters, as detected by amperometric measurements. These results indicate the existence of Ca(2+)-independent but voltage-dependent vesicular secretion (CIVDS) in a mammalian sensory neuron.     

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.     

Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice

Blockade of cholinergic neurotransmission by muscarinic receptor antagonists produces profound deficits in attention and memory. However, the antagonists used in previous studies bind to more than one of the five muscarinic receptor subtypes. Here we examined memory in mice with a null mutation of the gene coding the M1 receptor, the most densely distributed muscarinic receptor in the hippocampus and forebrain. In contrast with previous studies using nonselective pharmacological antagonists, the M1 receptor deletion produced a selective phenotype that included both enhancements and deficits in memory. Long-term potentiation (LTP) in response to theta burst stimulation in the hippocampus was also reduced in mutant mice. M1 null mutant mice showed normal or enhanced memory for tasks that involved matching-to-sample problems, but they were severely impaired in non-matching-to-sample working memory as well as consolidation. Our results suggest that the M1 receptor is specifically involved in memory processes for which the cortex and hippocampus interact.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

Differential modulation of nicotinic acetylcholine receptor subtypes and synaptic transmission in chick sympathetic ganglia by PGE(2)

The diversity of neuronal nicotinic acetylcholine receptors (nAChRs) is likely an important factor in the modulation of synaptic transmission by acetylcholine and nicotine. We have tested whether postsynaptic nAChRs are modulated in a subtype-specific manner by prostaglandin E(2) (PGE(2)), a regulator of neuronal excitability in both the central and peripheral nervous systems, and examined the effects of PGE(2) on nicotinic transmission. Somatodendritic nAChRs in chick lumbar sympathetic ganglia include four nAChR subtypes distinguished on the basis of conductance and kinetic profile. Nanomolar PGE(2) applied to the extrapatch membrane differentially regulates opening probability (Po), frequency and the opening duration of each nAChR channel subtype in cell-attached patches. PGE(2) decreases the Po of the predominant nAChR subtype (36 pS) and significantly increases Po and open duration of the 23 pS subtype. The 23 pS subtype is gated by the alpha 7-selective agonist choline, and choline-gated currents are inhibited by alpha-bungarotoxin. To examine whether PGE(2) modulates nAChRs at synaptic sites, we studied the effects of PGE(2) on amplitude and decay of synaptic currents in visceral motoneuron-sympathetic neuron co-cultures. PGE(2) significantly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), consistent with the predominant inhibition by PGE(2) of all but the 23 pS subtype. The time constant of mEPSCs at PGE(2)-treated synapses is prolonged, which is also consistent with an increased contribution of the longer open duration of the 23 pS nAChR subtype with PGE(2) treatment. To examine the presynaptic effect of PGE(2), nanomolar nicotine was used. Nicotine induces facilitation of synaptic transmission by increasing mEPSC frequency, an action thought to involve presynaptic, alpha 7-containing nAChRs. In the presence of PGE(2), nicotine-induced synaptic facilitation persists. Thus the net effect of PGE(2) is to alter the profile of nAChRs contributing to synaptic transmission from larger conductance, briefer opening channels to smaller conductance, longer opening events. This subtype-specific modulation of nAChRs by PGE(2) may provide a mechanism for selective activation and suppression of synaptic pathways mediated by different nAChR subtype(s) at both pre- and postsynaptic sites.     

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.     

Presynaptic M1 muscarinic receptor modulates spontaneous release of acetylcholine from rat basal forebrain slices

Spontaneous release of acetylcholine (ACh) from rat basal forebrain slices in the presence of cholinesterase inhibitor was directly determined using a specific radioimmunoassay for ACh. The release was calcium dependent. A consistent amount of ACh release was observed throughout the experiment. Atropine (10(-8) to 10(-5) M) and pirenzepine (10(-7) to 10(-5) M) enhanced spontaneous ACh release. These findings indicate the presence of an M1 muscarinic autoreceptor that modulates spontaneous release of ACh in the rat basal forebrain.