Muscarinic Receptors, from Synaptic Plasticity to its Role in Network Activity.

Acetylcholine acting via metabotropic receptors plays a key role in learning and memory by regulating synaptic plasticity and circuit activity. However, a recent overall view of the effects of muscarinic acetylcholine receptors (mAChRs) on excitatory and inhibitory long-term synaptic plasticity and on circuit activity is lacking. This review focusses on specific aspects of the regulation of synaptic plasticity and circuit activity by mAChRs in the hippocampus and cortex. Acetylcholine increases the excitability of pyramidal neurons, facilitating the generation of dendritic Ca2+-spikes, NMDA-spikes and action potential bursts which provide the main source of Ca2+ influx necessary to induce synaptic plasticity. The activation of mAChRs induced Ca2+ release from intracellular IP3-sensitive stores is a major player in the induction of a NMDA independent long-term potentiation (LTP) caused by an increased expression of AMPA receptors in hippocampal pyramidal neuron dendritic spines. In the neocortex, activation of mAChRs also induces a long-term enhancement of excitatory postsynaptic currents. In addition to effects on excitatory synapses, a single brief activation of mAChRs together with short repeated membrane depolarization can induce a long-term enhancement of GABA A type (GABAA) inhibition through an increased expression of GABAA receptors in hippocampal pyramidal neurons. By contrast, a long term depression of GABAA inhibition (iLTD) is induced by muscarinic receptor activation in the absence of postsynaptic depolarizations. This iLTD is caused by an endocannabinoid-mediated presynaptic inhibition that reduces the GABA release probability at the terminals of inhibitory interneurons. This bidirectional long-term plasticity of inhibition may dynamically regulate the excitatory/inhibitory balance depending on the quiescent or active state of the postsynaptic pyramidal neurons. Therefore, acetylcholine can induce varied effects on neuronal activity and circuit behavior that can enhance sensory detection and processing through the modification of circuit activity leading to learning, memory and behavior.

Voltage-gated potassium conductances in Gymnotus electrocytes(AB).

Electrocytes are muscle-derived cells that generate the electric organ discharge (EOD) in most gymnotiform fish. We used an in vitro preparation to determine if the complex EOD of Gymnotus carapo was related to the membrane properties of electrocytes. We discovered that in addition to the three Na(+)-mediated conductances described in a recent paper [Sierra F, Comas V, Buño W, Macadar O (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A 191:1-11] there were four K(+)-dependent conductances. Membrane depolarization activated a delayed rectifier (I(K)) and an A-type (I(A)) current. I(A) displayed fast voltage-dependent activation-inactivation kinetics, was blocked by 4-aminopyridine (1 mM) and played a major role in action potential (AP) repolarization. Its voltage dependence and kinetics shape the brief AP that typifies Gymnotus electrocytes. The I(K) activated by depolarization contributed less to AP repolarization. Membrane hyperpolarization uncovered two inward rectifiers (IR1 and IR2) with voltage dependence and kinetics that correspond to the complex "hyperpolarizing responses" (HRs) described under current-clamp. IR1 shows "instantaneous" activation, is blocked by Ba(2+) and Cs(+) and displays a voltage and time dependent inactivation that matches the hyperpolarizing phase of the HR. The activation of IR2 is slower and at more negative potentials than IR1 and is resistant to Ba(2+) and Cs(+). This current fits the depolarizing phase of the HR. The EOD waveform of Gymnotus carapo is more complex than that of other gymnotiform fish species, the complexity originates in the voltage responses generated through the interactions of three Na(+) and four K(+) voltage- and time-dependent conductances although the innervation pattern also contributes [Trujillo-Cenóz O, Echagüe JA (1989) Waveform generation of the electric organ discharge in Gymnotus carapo. I. Morphology and innervation of the electric organ. J Comp Physiol A 165:343-351].

Presynaptic muscarinic control of glutamatergic synaptic transmission.

The hippocampus receives cholinergic projections from the medial septal nucleus and Broca's diagonal band that terminate in the CA1, CA3, and dentate gyrus regions (Frotscher and Leranth, 1985). Glutamatergic synapses between CA3 and CA1 pyramidal neurons are presynaptically inhibited by acetylcholine (ACh), via activation of muscarinic ACh receptors (mAChRs) at the terminals of Schaffer collaterals (SCs) (Hounsgaard, 1978; Fernández de Sevilla et al., 2002, 2003). There are two types of SC-CA1 pyramidal neuron synapses. One type, called functional synapse, shows postsynaptic alpha- amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-receptor mediated currents at resting potential (Vm) and both AMPA and N-methyl-D-aspartate receptor (NMDAR)-mediated currents when depolarized. The other type, termed silent synapse, only displays postsynaptic NMDAR-mediated currents at depolarized Vms, but does not respond at the resting Vm (Isaac et al., 1995). Using hippocampal slices obtained from young Wistar rats, we examined the effects of activation of cholinergic afferents at the stratum oriens/alveus on excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by stimulation of SCs. We also tested the action of the nonhydrolyzable cholinergic agonist carbamylcholine chloride (CCh) on EPSCs evoked by minimal stimulation of SCs (which activates a single or very few synapses) in functional and silent synapses.

High-frequency stimulation of the subthalamic nucleus silences subthalamic neurons: a possible cellular mechanism in Parkinson's disease.

The subthalamic nucleus participates in the control of movement and is considered a surgical target in the treatment of parkinsonian symptoms. Using the rat brain in vitro slice technique we show that sustained high-frequency (>100 Hz) electrical stimulation (i.e., 'tetanic stimulation') of the nucleus, as used in humans to treat Parkinson's disease, silenced subthalamic neurons. Two main cell types were identified. 'Tonic cells' (68%) showed delayed inward rectification, fired continuously, switched to bursting and stopped firing when strongly depolarized with injected current. Tetanic stimulation of the nucleus induced a steady depolarization (approximately 18 mV) that triggered action potentials at a high rate followed by bursts and finally (approximately 25 s) totally silenced tonic cells. The control tonic activity was recovered rapidly (<10 s) after ending stimulation. 'Phasic cells' (25%) discharged a single initial brief burst of action potentials both when depolarized by prolonged current injection and tetanic stimulation and did not show inward rectification. An infrequent cell type called 'phasic-tonic' (7%) showed a mixed discharge. We suggest that the silencing effect of tetanic stimulation is not a frequency-dependent presynaptic depression and could result from the gradual inactivation of Na+-mediated action potentials. These findings suggest that the remission of parkinsonian symptoms by treatment with high-frequency electrical stimulation of the subthalamic nucleus in humans may primarily reside on its capacity to suppress the action potential activity of subthalamic neurons.

Synaptic regulation of the slow Ca2+-activated K+ current in hippocampal CA1 pyramidal neurons: implication in epileptogenesis.

The slow Ca2+-activated K+ current (sI(AHP)) plays a critical role in regulating neuronal excitability, but its modulation during abnormal bursting activity, as in epilepsy, is unknown. Because synaptic transmission is enhanced during epilepsy, we investigated the synaptically mediated regulation of the sI(AHP) and its control of neuronal excitability during epileptiform activity induced by 4-aminopyridine (4AP) or 4AP+Mg2+-free treatment in rat hippocampal slices. We used electrophysiological and photometric Ca2+ techniques to analyze the sI(AHP) modifications that parallel epileptiform activity. Epileptiform activity was characterized by slow, repetitive, spontaneous depolarizations and action potential bursts and was associated with increased frequency and amplitude of spontaneous excitatory postsynaptic currents and a reduced sI(AHP.) The metabotropic glutamate receptor (mGluR) antagonist (S)-alpha-methyl-4-carboxyphenylglycine did not modify synaptic activity enhancement but did prevent sI(AHP) inhibition and epileptiform discharges. The mGluR-dependent regulation of the sI(AHP) was not caused by modulated intracellular Ca2+ signaling. Histamine, isoproterenol, and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid reduced the sI(AHP) but did not increase synaptic activity and failed to evoke epileptiform activity. We conclude that 4AP or 4AP+Mg-free-induced enhancement of synaptic activity reduced the sI(AHP) via activation of postsynaptic group I/II mGluRs. The increased excitability caused by the lack of negative feedback provided by the sI(AHP) contributes to epileptiform activity, which requires the cooperative action of increased synaptic activity.

Properties and plasticity of synaptic inputs to rat dorsal column neurones recorded in vitro.

1. The mechanisms regulating the flow of sensory signals and their modification by synaptic interactions in the dorsal column nuclei are incompletely understood. Therefore, we examined the interactions between EPSPs evoked by stimulation of dorsal column and corticofugal fibres in the dorsal column nuclei cells using an in vitro slice technique. 2. Dorsal column EPSPs had briefer durations at depolarised membrane potentials than corticofugal EPSPs. Superfusion of the NMDA receptor antagonist 2D(-)-2-amino-5-phosphonovaleric acid (AP5) did not modify dorsal column EPSPs but reduced corticofugal EPSPs. Application of the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) abolished both dorsal column and corticofugal EPSPs in cells held at the resting potential. Therefore, dorsal column EPSPs were mediated by non-NMDA receptors but corticofugal EPSPs revealed both non-NMDA- and NMDA-dependent components. 3. Paired-pulse stimulation of dorsal column fibres elicited a depression of the second EPSP at pulse intervals of < 50 ms; however, paired-pulse stimulation of corticofugal fibres evoked facilitation of the second EPSP at pulse intervals of < 30 ms. When stimulation of the corticofugal fibres preceded stimulation of the dorsal column fibres, facilitation of the dorsal column EPSP was observed at pulse intervals of < 100 ms. This facilitation was blocked at hyperpolarised membrane potentials or in the presence of AP5, suggesting activation of NMDA receptors. There was a depression of corticofugal EPSPs by previous dorsal column stimulation. 4. Dorsal column EPSPs were gradually depressed during stimulation with barrages at frequencies of > 10 Hz, while corticofugal EPSPs were facilitated and summated at frequencies > 30 Hz. Hyperpolarisation and application of AP5 prevented the facilitation of corticofugal EPSPs. High frequency stimulation of the corticofugal input elicited a short-lasting AP5-sensitive facilitation of both corticofugal and dorsal column EPSPs. Depolarising current facilitated dorsal column EPSPs but not corticofugal EPSPs. 5. These results indicate that synaptic interactions include different forms of activity-dependent synaptic plasticity, with the participation of NMDA receptors and probably Ca(2+) inflow through voltage-gated channels. These complex synaptic interactions may represent the cellular substrate of the integrative function of the dorsal column nuclei observed in vivo.

Pharmacological involvement of the calcium channel blocker flunarizine in dopamine transmission at the striatum.

Single intrastriatal microinjections of 25, 50 and 100nmol/microl of flunarizine in normal rats produced a dose-dependent turning behavior toward the injected side when they were challenged with apomorphine (1mg/kg, s.c). This effect was seen at 1, 3 and 7 days following administration of the high dose of flunarizine, but had subsided by 24h after administration of the intermediate dose; the low dose was ineffective. However, intrastriatal injection of the high dose of flunarizine resulted in a local lesion and thereafter this dose was not used. A similar dose-response relationship was determined for nifedipine, an L-type calcium channel antagonist. Injection of this antagonist did not result in apomorphine-elicited rotational behavior, reflecting its lack of antidopaminergic action. Intrastriatal injections of haloperidol (5microg/microl), an antagonist of dopamine D(2) receptors, or the sodium channel blocker lidocaine (40microg/microl), were given in order to compare their effects to those observed with flunarizine. Intracerebral injection of haloperidol produced ipsilateral turning in response to systemic administration of apomorphine given 60min after. The same response was obtained with the injection of apomorphine 10min after the injection of intracerebral lidocaine. This effect was no longer apparent 24h after the microinjection of haloperidol and 60min after the injection of lidocaine. In rats rendered hemiparkinsionian by lesioning the nigrostriatal pathway with 6OHDA, intrastriatal microinjection of flunarizine (50nmol/microl) significantly reduced apomorphine (0.2mg/kg, s.c.)-elicited turning behavior towards the non-lesioned side. These results suggest an antidopaminergic effect of flunarizine mediated by antagonistic action of post-synaptic striatal dopamine receptors. However, an action of the drug on sodium channels may not be ruled out. These studies offer additional supporting evidence for the induction or aggravation of extrapyramidal side-effects in patients receiving flunarizine.

Insulin-like growth factor I potentiates kainate receptors through a phosphatidylinositol 3-kinase dependent pathway.

Neurotrophic factors modulate synaptic plasticity through mechanisms that include regulation of membrane ion channels and neurotransmitter receptors. Recently, it was shown that insulin-like growth factor I (IGF-I) induces depression of AMPA-mediated currents without affecting NMDA-receptor function in neurons. We now report that IGF-I markedly potentiates the kainate-preferring ionotropic glutamate receptor in young cerebellar granule neurons expressing functional kainate-, but not AMPA-mediated currents. Potentiation of kainate responses by IGF-I is blocked by wortmannin, a phosphatidylinositol 3-kinase (P13K) inhibitor, indicating a role for this kinase in the effect of IGF-I. These results reinforce the notion that modulation of ionotropic glutamate receptors are involved in the regulatory actions of IGF-I on neuronal plasticity.

N- and P/Q-type Ca(2+) channels are involved in neurotransmitter release but not in synaptic depression in the spinal cord of the neonatal rat.

This study investigated the involvement of N- and P/Q-type Ca(2+) channels in sensorimotor transmission and synaptic depression in the in vitro neonatal rat spinal cord preparation. Postsynaptic potentials were intracellularly recorded from spinal motoneurones during stimulation of the dorsal roots. We found that omega-agatoxin-IVA (P/Q-type Ca(2+) channels blocker), omega-conotoxin-GVIA (N-type Ca(2+) channel blocker) and omega-conotoxin-MVIIC (N-, P/Q-type Ca(2+) channel blocker) markedly decreased both poly- and monosynaptic potentials. We also found that the frequency-dependent depression which occurred in the monosynaptic response, for stimulus intervals shorter than 30 s, persisted in the presence of the various Ca(2+) channels blockers. Hyperpolarizing the motoneurons significantly reduced depression, suggesting contribution from some additional postsynaptic mechanisms. We conclude that at birth, as in adult central nervous system (CNS) synapses, several types of voltage dependent calcium channels mediate sensorimotor neurotransmission and that synaptic depression, which is characteristic of neonatal sensorimotor transmission, does not involve these Ca(2+) channels.

Sustained GABA-induced regulation of the inactivation of the voltage-dependent Ca2+ current in crustacean muscle fibers.

We describe a new form of gamma-aminobutyric acid (GABA) -mediated regulation of the inactivation and of the recovery from inactivation of the L-type Ca2+ current (I(Ca)) in crayfish muscle. GABA (1 mM) was applied during a 2-min period and the peak I(Ca) was measured using two-electrode voltage-clamp recordings 30 min after returning to the control solution. Prepulse-pulse protocols showed that the GABA-mediated inhibition of I(Ca) decreased (>50%) both with increasing prepulse depolarization and as the delay between prepulse and pulse was reduced. GABA also shifted to depolarized values (>5 mV) the S-shaped plots of the peak I(Ca) evoked by a constant depolarizing pulse as a function of prepulse voltage (i.e., inactivation curves) and accelerated the recovery time from the inactivation evoked by depolarizing prepulses (>35%). The effects outlasted GABA application up to 1 h. The observed changes in inactivation properties may be of functional importance because they indicate that previous depolarization relieves the GABA-induced inhibition of I(Ca), implying that this long-lasting inhibition is under the regulation of the prepulse potential and the subsequent Ca2+ entry.

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.

Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells.

Neurotrophins, acting through tyrosine kinase family genes, are essential for neuronal differentiation. The expression of tyrosine kinase family genes is prognostic in neuroblastoma, and neurotrophins reduce proliferation and induce differentiation, indicating that neuroblastomas are regulated by neurotrophins. We tested the effects of nerve growth factor and brain-derived neurotrophic factor on Na(+) and Ca(2+) currents, using the whole-cell patch-clamp technique, in human neuroblastoma NB69 cells. Control cells exhibited a slow tetrodotoxin-resistant (IC(50)=98 nM) Na(+) current and a high-voltage-activated Ca(2+) current. Exposure to nerve growth factor (50 ng/ml) and/or brain-derived neurotrophic factor (5 ng/ml) produced the expression of a fast tetrodotoxin-sensitive (IC(50)=10 nM) Na(+) current after day 3, and suppressed the slow tetrodotoxin-resistant variety. The same type of high-voltage-activated Ca(2+) current was expressed in control and treated cells. The treatment increased the surface density of both Na(+) and Ca(2+) currents with time after plating, from 17 pA/pF at days 3-5 and 1-5 to 34 and 30 pA/pF after days 6-10, respectively. Therefore, both nerve growth factor and brain-derived neurotrophic factor, acting through different receptors of the tyrosine kinase family and also possibly the tumor necrosis factor receptor-II, were able to regulate differentiation and the expression of Na(+) and Ca(2+) channels, partially reproducing the modifications induced by diffusible astroglial factors. We show that neurotrophins induced differentiation to a neuronal phenotype and modified the expression of Na(+) and Ca(2+) currents, partially reproducing the effects of diffusible astroglial factors.

Fast BK-type channel mediates the Ca(2+)-activated K(+) current in crayfish muscle.

The role of the Ca(2+)-activated K(+) current (I(K(Ca))) in crayfish opener muscle fibers is functionally important because it regulates the graded electrical activity that is characteristic of these fibers. Using the cell-attached and inside-out configurations of the patch-clamp technique, we found three different classes of channels with properties that matched those expected of the three different ionic channels mediating the depolarization-activated macroscopic currents previously described (Ca(2+), K(+), and Ca(2+)-dependent K(+) currents). We investigated the properties of the ionic channels mediating the extremely fast activating and persistent I(K(Ca)). These voltage- and Ca(2+)-activated channels had a mean single-channel conductance of approximately 70 pS and showed a very fast activation. Both the single-channel open probability and the speed of activation increased with depolarization. Both parameters also increased in inside-out patches, i.e., in high Ca(2+) concentration. Intracellular loading with the Ca(2+) chelator bis(2-aminophenoxy) ethane-N, N,N',N'-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (< or =560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic I(K(Ca)). However, the intrinsic properties of these single channels do not account for the partial apparent inactivation of the macroscopic I(K(Ca)), which probably reflects temporal Ca(2+) variations in the whole muscle fiber. We conclude that the channels mediating I(K(Ca)) in crayfish muscle are voltage- and Ca(2+)-gated BK channels with relatively small conductance. The intrinsic properties of these channels allow them to act as precise Ca(2+) sensors that supply the exact feedback current needed to control the graded electrical activity and therefore the contraction of opener muscle fibers.

In vitro electrophysiological properties of rat dorsal column nuclei neurons.

The dorsal column nuclei include the gracile and cuneate nuclei, which receive somatosensory information from the periphery and project to the ventroposterior nucleus of the contralateral thalamus. The aim of this study was to determine the electrophysiological and morphological characteristics of the neurons of the dorsal column nuclei and to identify synaptic events evoked by electrical stimulation of the dorsal column, using an in vitro slice preparation. The results show two types of neurons, termed type I and II. A repolarizing sag distinguished type I cells during hyperpolarizing current injection, suggesting the activation of a Q-current. Moreover, type I cells, but not type II cells, were capable of maintaining spontaneous rhythmic activity at 9-15 Hz. Both types of cells displayed a delay in their return to the resting membrane potential following hyperpolarizing current pulses, indicating the existence of an A-current. Electrical stimuli applied to the dorsal column elicited brief EPSPs and IPSPs in both cell types. EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that they were mediated through non-NMDA receptors. IPSPs were blocked by picrotoxin, implying the activation of GABAA receptors. Intracellular staining with carboxyfluoresceine revealed that type I neurons had elongated somas and primary dendrites that extended radially. Type II cells were smaller and had round somas with few primary dendrites, most of them emerging from one pole of the soma. The axon of many type I neurons was stained and could be followed running ventrally and in rostral direction.

The activity-dependent potentiation of the slow Ca2+-activated K+ current regulates synaptic efficacy in rat CA1 pyramidal neurons.

Activity-dependent modifications of neuronal excitability are of key functional importance because they accomplish general postsynaptic control of the flow of synaptic signals. We tested the modifications of synaptic efficacy evoked in rat CA1 hippocampal pyramidal neurons during the short-term activity-dependent reduction in excitability termed "response depression". The in vitro slice technique and recordings with sharp electrodes in the current- and voltage-clamp modes were used. Depression was induced by repeatedly stimulating the Schaffer collateral and stratum oriens. Repeated synaptic stimuli also depressed subsequent responses evoked by transmembrane current pulse injection and vice versa. Depression was characterised by a marked decrease in synaptic efficacy that outlasted stimuli for several minutes and was generalized to all pyramidal cells. The action potential frequency adaptation, the slow after-hyperpolarization and the underlying slow Ca2+-dependent K+ current (IAHP) were potentiated during depression. The potentiated IAHP caused depression by acting as a cumulative negative feedback that reduced synaptic efficacy by increasing the membrane conductance and hyperpolarizing the neurone. This depression may act as a homeostatic negative feedback mechanism to limit the rise in intracellular Ca2+ concentration and stabilize the membrane potential following intense synaptic activation.

BAPTA-AM blocks both voltage-gated and Ca2+-activated K+ currents in cultured bovine chromaffin cells.

The effects of the membrane permeant Ca2+ chelator BAPTA-AM on voltage-gated Na+, Ca2+, K+ (I(Na), I(Ca) I(K), respectively) and Ca2+-activated K+ (I(KCa)) currents in cultured bovine chromaffin cells were investigated using the whole-cell patch-clamp technique. Superfusion with BAPTA-AM (50 microM) induced a rapid (< 60 s) and reversible block of both I(KCa) and I(K) (approximately 50%), without affecting either I(Ca) or I(Na). Preincubation with BAPTA-AM (50 microM, 30 min) or cell loading with the nonpermeable active form of BAPTA (10 mM in the pipette solution) permanently blocked I(KCa). BAPTA-AM superfusion (50 microM) also blocked I(K) (approximately 53%) after BAPTA-loading or BAPTA-AM preincubation. In conclusion, we show a fast and reversible block of I(KCa) and I(K) by BAPTA-AM, acting directly on K+ channels before it operates as a Ca2+ chelator, in cultured bovine chromaffin cells.

Neurotransmitter actions on oral pontine tegmental neurons of the rat: an in vitro study.

The actions of neurotransmitters involved in the sleep-wakefulness cycle on neurons located in the ventral part of the oral pontine tegmentum were studied in a rat brain-slice preparation. Results show that glutamate and histamine evoke depolarizations and spike firing while serotonin and gamma-aminobutyric acid evoke hyperpolarizations. The excitatory and inhibitory actions of these neurotransmitters increase pontine neuron activity during the conditions occurring during paradoxical sleep.

Voltage-gated and Ca2+-activated conductances mediating and controlling graded electrical activity in crayfish muscle.

Crayfish opener muscle fibers provide a unique preparation to quantitatively evaluate the relationships between the voltage-gated Ca2+ (ICa) and Ca2+-activated K+ (IK(Ca)) currents underlying the graded action potentials (GAPs) that typify these fibers. ICa, IK(Ca), and the voltage-gated K+ current (IK) were studied using two-electrode voltage-clamp applying voltage commands that simulated the GAPs evoked in current-clamp conditions by 60-ms current pulses. This methodology, unlike traditional voltage-clamp step commands, provides a description of the dynamic aspects of the interaction between different conductances participating in the generation of the natural GAP. The initial depolarizing phase of the GAP was due to activation of the ICa on depolarization above approximately -40 mV. The resulting Ca2+ inflow induced the activation of the fast IK(Ca) (<3 ms), which rapidly repolarized the fiber (<6 ms). Because of its relatively slow activation, the contribution of IK to the GAP repolarization was delayed. During the final steady GAP depolarization ICa and IK(Ca) were simultaneously activated with similar magnitudes, whereas IK aided in the control of the delayed sustained response. The larger GAPs evoked by higher intensity stimulations were due to the increase in ICa. The resulting larger Ca2+ inflow increased IK(Ca), which acted as a negative feedback that precisely controlled the fiber's depolarization. Hence IK(Ca) regulated the Ca2+-inflow needed for the contraction and controlled the depolarization that this Ca2+ inflow would otherwise elicit.

Sustained GABA-induced regulation of the L-type Ca2+ conductance in crustacean muscle fibers.

The sustained effects of gamma-aminobutyric acid (GABA) on voltage-gated conductances, and excitatory and inhibitory postsynaptic currents (EPSC and IPSC, respectively) in crayfish opener muscle fibers were analyzed using the two-electrode voltage-clamp technique. GABA (1.0 mM) was applied for 1-2 min and measurements were performed 30 min after restoring control Ringer solution. The L-type Ca2+ current (ICa) was reduced by > 33%. The ICa conductance (gCa) was reduced and the activation and inactivation were slowed down by GABA. The ICa regulation outlasted GABA superfusion (150 min). A small decrease (< 19%) of the Ca2+-activated K+ current (IKCa), due to the ICa reduction, was also recorded. The leak (IL), the delayed-rectifier (IK) and the hyperpolarization-activated (IAB) currents were not affected. Picrotoxin (0.5 mM) and bicuculline (0.2 mM) blocked the ICa reduction. Neither the GABAB antagonist saclofen (1.0 mM) nor the agonist baclofen (1.0 mM) had any effect. Therefore, the ICa regulation was probably mediated through GABAA receptors. EPSCs, but not IPSCs, were reduced (30%) for prolonged periods (> 100 min.) after GABA application. We describe a new, potentially functional, role for GABA receptors in the mediation of a sustained reduction of presynaptic and postsynaptic excitability in crustacean muscle.

Differential expression of voltage-gated Ca2+ conductances in human neuroblastoma NB69 cells cultured in defined serum-free and astrocyte-conditioned media.

Voltage-gated Ca2+ conductances were investigated with the whole-cell patch-clamp technique-either using Ca2+ or Ba2+ as charge carriers-in NB69 human neuroblastoma cells plated in "defined" serum-free (DM) and in "astroglial-conditioned" media (CM). Cells expressed the microtubule associated protein 1A when plated in both media, indicating neuronlike differentiation. Cells of similar sizes and shapes were selected for recordings. Different sets of voltage-gated Ca2+ current types were usually expressed in DM- and CM-plated cells. DM-plated cells exhibited a high-voltage-activated current (HVAC) in isolation, whereas 43% of the CM-plated cells also displayed the low-voltage-activated current (LVAC). The membrane surface density of the HVAC was about twofold higher in CM than in DM-plated cells and increased with plating time from 10 and 16pA/pF (days 1-4) to 24 and 37 pA/pF (days 5-10) in DM- and CM-plated cells, respectively. However, the amplitude of the LVAC did not change significantly with culture age. In conclusion, NB69 cells expressed HVAC in isolation when plated in DM, whereas both HVAC and LVAC were present in many CM-plated cells, suggesting that the CM contained diffusible factors secreted by astroglial cells which: (1) could induce the appearance of the LVAC and (2) increased HVAC current expression.