Calcium channels controlling acetylcholine release in the guinea-pig isolated anterior pelvic ganglion: an electropharmacological study

An electropharmacological analysis of the type(s) of calcium channel controlling neurotransmitter release in preganglionic sympathetic nerve terminals in the guinea-pig anterior pelvic ganglion has been carried out. Conventional intracellular recording techniques were used to record excitatory postsynaptic potentials as a measure of neurotransmitter release. Excitatory postsynaptic potentials were abolished by hexamethonium (30-100 microM) and are therefore mediated by acetylcholine acting at nicotinic receptors. In studies of more than 150 cells, the N-type calcium channel blocker omega-conotoxin GVIA (100-300 nM) failed to block the initiation of the nerve impulse by the excitatory postsynaptic potential. In single-cell studies, omega-conotoxin GVIA (1 microM) sometimes altered the configuration of the excitatory postsynaptic potential/cell body nerve action potential complex, but on only one occasion was the excitatory postsynaptic potential reduced below the threshold required to initiate the action potential. Nifedipine (10 microM), omega-agatoxin IVA (100 nM) and omega-conotoxin MVIIC (300 nM), applied alone or in combination with omega-conotoxin GVIA (300 nM), were also ineffective. However, excitatory postsynaptic potentials evoked by trains of stimuli (0.1-0.5 Hz) were markedly reduced or abolished by the non-specific calcium channel blocker omega-grammotoxin SIA (300 nM). When trains of stimuli were delivered at higher frequencies (4 Hz), the block induced by omega-grammotoxin SIA could be overcome, and excitatory postsynaptic potentials were able to initiate action potentials even when omega-conotoxin GVIA, omega-agatoxin IVA and omega-conotoxin MVIIC were also present. The calcium channel(s) controlling acetylcholine release was (were) blocked by low concentrations of cadmium ions (30 microM) at all stimulation frequencies studied (0.1-50 Hz). Thus, the dominant calcium channels controlling acetylcholine release in sympathetic ganglia are not the L, N, P or Q types. At low frequencies of stimulation, omega-grammotoxin SIA-sensitive calcium channels play a dominant role in acetylcholine release, but at higher stimulation frequencies yet another pharmacologically distinct calcium channel (or subtype) supports neurotransmitter release.     

Non-cholinergic synaptic potentials mediated by lumbar colonic nerve in the guinea-pig inferior mesenteric ganglion in vitro

Non-cholinergic slow synaptic potentials mediated by the lumbar colonic nerve have been investigated using an in vitro preparation of the guinea-pig inferior mesenteric ganglion attached to a distal colonic segment. Non-cholinergic potential responses to colonic nerve stimulation, colonic distension and chemical activation of sensory afferents were recorded intracellularly from neurons in the inferior mesenteric ganglion. Electrical stimulation of the lumbar colonic nerve produced either a slow excitatory postsynaptic potential, or a slow inhibitory postsynaptic potential followed by a slow excitatory postsynaptic potential. The extrapolated reversal potential of the slow excitatory postsynaptic potential was in the range of 0 to -20 mV and that of the slow inhibitory postsynaptic potential was -90 to 110 mV. The slow excitatory postsynaptic potential and the slow inhibitory postsynaptic potential were reversibly abolished by perfusion of the ganglion with tetrodotoxin (1 microM), or perfusion with low calcium (200 microM), high magnesium (12 mM) containing solution. Capsaicin (1 microM) evoked a reversible depolarization of inferior mesenteric ganglion cells after which desensitization occurred and the slow excitatory postsynaptic potential was abolished but the slow inhibitory postsynaptic potential was enhanced in amplitude and prolonged in duration. Bath application of substance P (2 microM) evoked a prolonged depolarization of inferior mesenteric ganglion neurons, during which the slow excitatory postsynaptic potential but not the slow inhibitory postsynaptic potential was abolished. Distensions of the colon to pressures in the range of 2-25 cm of water produced a stimulus graded non-cholinergic slow depolarization which was occasionally followed by a late slow hyperpolarization. Both types of response were abolished by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-dependent depolarizing potentials in veratrinized crayfish muscle fibres

In the slow abdominal flexor muscle fibers of the crayfish Procambarus clarkii, veratrine or veratridine applied during several minutes produced the persistent transformation of the muscle fibre from a nonspiking into a Ca-dependent spiking one and spikes were followed by a long-duration depolarization. The long-duration potential depends on external Na+ but is not blocked by 30 microM tetrodotoxin (TTX). In solutions containing normal concentrations of Na+ (207 mM) the absence of Ca2+ or the presence of calcium channel blockers abolished both potentials. The results show that alkaloid toxins reveal a Ca2+-dependent, TTX-resistant Na+ conductance in crayfish tonic muscle fibres.     

The firing patterns of rat melanotrophs recorded using the patch clamp technique

Cell-attached and whole-cell recordings were made from adult rat melanotrophs maintained in vitro by standard cell culture techniques. In cell-attached recordings the cells showed small biphasic currents which reflected spontaneous cell firing. Single channel currents often had distinct relaxations and depolarizing currents through single channels could trigger the discharge of an action potential in the cell; both observations are consistent with the high input resistance (1-10 G omega) measured in the whole-cell configuration. The discharge of action potentials occurring either spontaneously or by current injection was eliminated by tetrodotoxin or by removing Na from the external medium. A Na-dependent plateau depolarization which activated near the spike threshold was also seen. In cells exposed to tetrodotoxin and K-channel blocking agents it was possible to evoke a long-lasting (up to 20 s) action potential which was enhanced and reduced, respectively, by Ba and Cd and thus appeared to reflect currents through voltage-activated Ca channels. Small amplitude Ca-dependent depolarizations could also be evoked at membrane potentials as low as -40 mV. In cell-attached and whole-cell recordings 10 mM Ba caused the discharge of tetrodotoxin-insensitive action potentials prior to a maintained depolarization of the membrane. The low threshold for Ca-dependent depolarizations suggest that Ca influx might occur in these cells even at the resting potential. Additionally, both a Ca current and the current underlying the Na-dependent plateau depolarization may influence the rate of cell firing and in doing so further increase Ca influx through voltage-activated channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia-activated Ca2+ currents in pacemaker neurones of rat rostral ventrolateral medulla in vitro.

We examined the effects of brief periods of hypoxia or application of cyanide on the discharge and membrane properties of medullary pacemaker neurones in slices of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata of rats. Stable intracellular recordings were obtained from seventy-nine neurones within the RVL which exhibited spontaneous rhythmic discharge in the absence of excitatory postsynaptic potentials (EPSPs). The membrane potential cycles of these neurones could be reset with an evoked spike without eliciting EPSPs or inhibitory postsynaptic potentials and hence met criteria of RVL pacemaker neurones. Hypoxia, produced by reducing O2 from 95 to 20% for 40 s or exposure to cyanide (30-300 microM for 40 s), reversibly increased neuronal discharge 1.6-fold (20% O2) or 2.6-fold (300 microM cyanide), respectively, in association with membrane depolarization and a significant fall in membrane resistance. The membrane responses to hypoxia and cyanide were observed in the presence of tetrodotoxin (TTX) at a concentration (10 microM) which eliminated spontaneous spikes or spikes evoked by intracellular depolarization. When recorded at a holding potential of -70 mV by single-electrode voltage clamp, hypoxia or cyanide (300 microM) elicited inward currents of 0.44 +/- 0.06 and 0.58 +/- 0.08 nA, respectively, which are attenuated by reducing the concentration of extracellular Ca2+ ions, and abolished by 2 mM CoCl2 and 100 microM NiCl2, but not affected by 50 microM CdCl2, replacement of 83% extracellular Na+, or adenosine deaminase (2U ml-1). We conclude that hypoxia and cyanide directly excite RVL pacemaker neurones in vitro by a common mechanism: activation of Ca2+ channel conductance.     

Reduction of GABAA receptor-mediated inhibition by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione in cultured neurons of rat brain.

The action of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on gamma-aminobutyric acid-A (GABAA) receptor-mediated currents was studied in dissociated rat midbrain and hypothalamic cultures using whole-cell recording. Spontaneous synaptic activity consisted of excitatory (EPSCs) and inhibitory postsynaptic currents (IPSCs). Bicuculline (20 microM) blocked IPSCs and increased the frequency of EPSCs. CNQX (1 microM) reduced both EPSCs and IPSCs. In the presence of 0.3 microM tetrodotoxin (TTX), CNQX (1-20 microM) blocked miniature EPSCs and reduced IPSCs. In TTX, increasing K+ (20 mM) evoked EPSCs and IPSCs in a Ca-dependent manner. CNQX (10 microM) blocked evoked EPSCs and diminished evoked IPSCs similarly as miniature IPSCs. Muscimol-(0.2-5 microM) induced currents were dose-dependently reduced by CNQX (10-50 microM). It is concluded that CNQX reduces GABAA receptor-mediated inhibition primarily by reducing the excitatory drive in the evolving network, but, in addition, has a significant blocking effect on the GABAA receptor-channel complex.     

Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission.     

Calcium action potential and prolonged afterhyperpolarization in developing myotubes of a mouse clonal myogenic cell line

Under a high-Ca condition (greater than 5 mM), myotubes of a mouse myogenic cell line MC3T3-A1/M13 generated a long-lasting Ca action potential and a prolonged afterhyperpolarization (a.h.p.) during their in vitro development. The action potential was sensitive to Co or verapamil. Under a voltage-clamp condition, membrane depolarization more positive than -20 mV evoked a Ca-dependent inward current, which was apparently prolonged and responsible for the generation of the long-lasting action potential. The appearance of the Ca action potential preceded that of a Na spike by about 24 h, and it developed so that the maximum rate of rise became 26 +/- 4 V/s by day 7. Then this Ca-dependent potential faded as the myotubes matured, until the action potentials became solely Na-dependent. The a.h.p. was evoked accompanying the Ca action potential and was inhibited by quinine or quinidine, showing that it is operated by Ca-activated K channels. This channel developed together with the Ca channel and continued to exist during the myotube maturation process. These results indicate that the MC3T3-A1/M13 myotube at the initial stage of development has a highly developed Ca spike system that is due mostly to a high-threshold-type Ca channel.     

Inhibition by neosurugatoxin and omega-conotoxin of acetylcholine release and muscle and neuronal nicotinic receptors in mouse neuromuscular junction.

Neosurugatoxin and omega-conotoxin, known to be specific ligands for the neuronal nicotinic receptor and Ca2+ channel, respectively, were previously claimed to exert no depressant action on the mouse neuromuscular junction. It was found that in preparations partially blocked with tubocurarine or with low Ca(2+)-high Mg2+ Tyrode's, both toxins, at 3-10 microM, depressed indirect twitches and either produced wanings (neosurugatoxin) or waxings (omega-conotoxin) of indirectly elicited tetanic contractions whilst in normal Tyrode's the contractile forces were not changed. In normal Tyrode's, neosurugatoxin decreased the amplitudes of spontaneous and evoked endplate potentials and enhanced the run-down of endplate potentials as did tubocurarine though with lesser potency. By contrast, omega-conotoxin (10 microM) decreased the amplitude of the evoked but not of the spontaneous endplate potential in low Ca(2+)-high Mg2+ Tyrode's, and produced facilitation of endplate potentials, instead of run-down, on repetitive stimulations. Higher concentrations of omega-conotoxin appeared to depress quantal release in normal Tyrode's. The effects were all reversible. The prolonged endplate depolarization found in preparations treated with neostigmine or 3,4-diaminopyridine, was partially depressed by both toxins. The results suggest that neosurugatoxin blocks the neuron and muscle nicotinic receptors in the neuromuscular junction with comparable potency. The pharmacology of the nicotinic receptor on motor nerve terminal seems more similar to the muscle nicotinic receptor than to that on autonomic ganglia or brain. On the other hand, omega-conotoxin seems to block a small fraction of Ca2+ channels on the motor nerve and decreases the quantal release of evoked endplate potentials.     

Membrane properties underlying patterns of GABA-dependent action potentials in developing mouse hypothalamic neurons

Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons (n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2-9 (P2-9) mice. With conventional patch pipettes (containing 29 mM Cl-), action potentials were also elicited by GABA from neurons of 2-13 days in vitro (2-13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 microM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 microM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd(2+) (200 microM) and Ni(2+) (300 microM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl-] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl-]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current (E(GABA)) was positive to the resting membrane potential, suggesting a high intracellular [Cl-] in developing mouse neurons. Varying the holding potential from -80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca(2+) from the extracellular solution did not block spikes, indicating GABA-evoked Na+ -based spikes. Although E(GABA) was more positive within 2-5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the E(GABA) is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl-, not bicarbonate, was primarily responsible for generating multiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.     

Intrinsic membrane properties of neostriatal neurons can account for their low level of spontaneous activity

Electroresponsiveness of neostriatal neurons was studied by intracellular recording in a rat brain slice preparation maintained in standard solution or in solution containing K-channel blockers. In standard solution, the neurons fired repetitively at increasing frequencies with increasing amplitude of direct depolarization. The firing pattern was independent of the membrane potential from which firing was induced. In the presence of tetraethylammonium (20 mM), long-lasting (300-500 ms) plateau potentials could be elicited by the injection of short (5-10 ms) current pulses. Plateau potentials persisted in Na-free solution, in the presence of tetrodotoxin (1-3 microM) and if Ca in the perifusate was replaced by Ba. The plateau was blocked by Cd (500 microM). The plateaux were followed by depolarizing after-potentials. When the plateau potential failed due to fatigue, a small slow depolarization of short duration (10-30 ms) was elicited in Na-free or tetrodotoxin-containing solution, which increased in amplitude with membrane hyperpolarization. This slow depolarization was blocked by Cd, indicating that it was also mediated by Ca. By intrastriatal stimulation in the presence of 4-aminopyridine a long-lasting, voltage-dependent depolarization was triggered from the enhanced postsynaptic potential. In contrast, in the presence of tetraethylammonium, postsynaptic potentials were only slightly increased if they were compared at sizes subthreshold for the plateau potentials. It is concluded that neostriatal neurons, although being characterized as "silent" and "non-bursting", possess slow conductances for inward currents which they share with other mammalian central neurons. However, in contrast, to other central neurons, their Ca-spikes are suppressed by their K-conductances and, in contrast to oscillating neurons, low-threshold Ca-potentials are not prominent.     

Effects of Ca antagonists on the action potential and their relationship to the muscarinic ACh actions in isolated sympathetic neurons of rabbits

Muscarinically induced depressions of the shoulder in the falling phase as well as the after-spike-hyperpolarization and -depolarization of the action potential in the isolated sympathetic neurons of rabbits were mimicked by a novel peptide Ca channel blocker, omega-conotoxin (synthetic; 0.1-0.5 microM). Cobalt ions (0.1-2 mM) showed bidirectional effects on the shoulder, an early depression followed by a later prolongation, while they consistently induced depressions of other components. Organic Ca channel blockers, verapamil and D-600 (1-50 microM) and nifedipine (0.1-1 microM) appeared to have other effects as they rather caused a prolongation of the falling phase that was shortened by further application of acetylcholine.     

Different ion channel mechanisms between low concentrations of capsaicin and high concentrations of capsaicin and nicotine regarding peptide release from pulmonary afferents.

Vagal nerve stimulation (1 Hz for 1 min), capsaicin (10(-8) M and 10(-6) M), resiniferatoxin (3 x 10(-10) M) and nicotine (10(-4) M) evoked a non-cholinergic bronchoconstriction in the isolated perfused guinea-pig lung preparation. Simultaneously there was an increase in the perfusate levels of calcitonin gene-related peptide-like immunoreactivity, suggesting release from sensory nerves. Both the bronchoconstriction and peptide release evoked by a low concentration of capsaicin (10(-8) M) and that evoked by nerve stimulation were depressed by tetrodotoxin, suggesting involvement of Na+ channel dependent depolarization. Since the effects of capsaicin (10(-8) M) and vagal nerve stimulation were inhibited by omega-conotoxin but not influenced by nifedipine, the Ca(2+)-channel dependent is probably of N-type. Furthermore, the capsaicin analogue resiniferatoxin also evoked omega-conotoxin sensitive peptide release and bronchoconstriction. At the higher capsaicin concentration (10(-6) M), the functional response was only slightly inhibited by omega-conotoxin or tetrodotoxin indicating that capsaicin at this concentration evoked peptide release and functional effects through other mechanisms, probably involving Ca2+ fluxes in the non-selective cation channel associated with the proposed capsaicin receptor. The nicotine (10(-4) M) evoked peptide release and bronchoconstriction were only marginally influenced by omega-conotoxin or tetrodotoxin. It is concluded that the ion-channel mechanisms underlying the peptide releasing properties of antidromic nerve stimulation and low concentrations of capsaicin are similar and depend on action potential propagation, whereas capsaicin in high, toxic concentration and nicotine mainly act via receptor operated channels.     

Persistent Na+ current and Ca2+ current boost graded depolarization of rat retinal amacrine cells in culture

Retinal amacrine cells are depolarized by the excitatory synaptic input from bipolar cells. When a graded depolarization exceeds the threshold level, trains of action potentials are generated. There have been several reports that both spikes and graded depolarization are sensitive to tetrodotoxin (TTX). In the present study, we investigated the contribution of voltage-gated currents to membrane depolarization by using rat GABAergic amacrine cells in culture recorded by the patch-clamp method. Injection of a negative current induced membrane hyperpolarization, the waveform of which can be well fitted by a single exponential function. Injection of positive current depolarized the cell, and the depolarization exceeded the amplitude expected from the passive properties of the membrane. The boosted depolarization sustained after the current was turned off. Either 1 microM TTX or 2 mM Co2+ suppressed the boosted depolarization, and co-application of TTX and Co2+ blocked it completely. Under the voltage clamp, we identified a transient Na+ current (fast I(Na)), a TTX-sensitive persistent current that reversed the polarity near the equilibrium potential of Na+ (I(NaP)), and three types of Ca2+ currents (I(Ca)), L, N, and the pharmacological agent-resistant type (R type). These findings suggest that the I(NaP) and I(Ca) of amacrine cells boost depolarization evoked by the excitatory synaptic input, and they may aid the spread of electrical signals among dendritic arbors of amacrine cells.     

Serotonergic stretch receptors induce plateau properties in a crustacean motor neuron by a dual-conductance mechanism.

1. The mechanisms for induction of bistable plateau potential properties by a set of serotonergic/cholinergic peripheral stretch receptor cells [gastropyloric receptor (GPR) cells] were examined in the crab stomatogastric ganglion (STG) with the use of intracellular recording techniques. 2. GPR cell stimulation evoked nicotinic excitatory postsynaptic potentials (EPSPs) and induced plateau potential capability in the dorsal gastric (DG) motor neuron. The plateau potential could be triggered during a GPR train either by the summating nicotinic EPSPs or by brief intracellular current injection. After pharmacological blockade of nicotinic and muscarinic receptors, a slow depolarization in response to GPR stimulation was revealed. Prolonged plateau potentials could still be evoked after this treatment. Local application of serotonin (5-HT; 10 microM to 1 mM) mimicked the noncholinergic plateau inducing effects of GPR stimulation in the DG motor neuron. 3. The synergistic action of acetylcholine (ACh) and 5-HT was examined by stimulating the GPR cells at different frequencies (1-20 Hz). The plateau induction was present down to 2 Hz. The time to onset for triggering a plateau during a GPR train was determined by the co-released ACh. 4. The 5-HT-evoked slow depolarization persisted in tetrodotoxin (TTX; 0.1-1 microM), and the DG motor neuron could still produce a plateau potential on brief depolarization in the absence of the spike-generating mechanism. 5. In normal TTX-containing saline, the 5-HT-evoked depolarization was accompanied by a weak and variable decrease in apparent input conductance. After substituting one-half of the extracellular sodium with either Trisma-HCl or choline, the decrease in apparent input conductance became more pronounced. This decrease was converted to an increase in apparent input conductance when extracellular Ca2+ was replaced with Mg2+. 6. Under voltage-clamp conditions, local application of 5-HT caused a slow inward current of prolonged duration in DG. The current versus voltage relationship had an inverted U-shape with no apparent reversal potential in the entire voltage range investigated (-90 to -5 mV). The 5-HT-induced changes in input conductance showed a complex voltage dependence, with a conductance decrease from moderately depolarized voltages. 7. Extracellular Cs+ (2-4 mM) caused the DG to hyperpolarize 2-4 mV from rest, whereas lowering extracellular Ca2+ caused it to depolarize 7-15 mV. The combined action of low extracellular Ca2+ and 2-4 mM Cs+ caused an almost complete block of the slow 5-HT-evoked depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitatory postsynaptic potentials in rat neocortical neurons in vitro. III. Effects of a quinoxalinedione non-NMDA receptor antagonist

1. Intracellular microelectrodes were used to obtain recordings from neurons in layer II/III of rat frontal cortex. A bipolar electrode positioned in layer IV of the neocortex was used to evoke postsynaptic potentials. Graded series of stimulation were employed to selectively activate different classes of postsynaptic responses. The sensitivity of postsynaptic potentials and iontophoretically applied neurotransmitters to the non-N-methyl-D-asparate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was examined. 2. As reported previously, low-intensity electrical stimulation of cortical layer IV evoked short-latency early excitatory postsynaptic potentials (eEPSPs) in layer II/III neurons. CNQX reversibly antagonized eEPSPs in a dose-dependent manner. Stimulation at intensities just subthreshold for activation of inhibitory postsynaptic potentials (IPSPs) produced long-latency (10 to 40-ms) EPSPs (late EPSPs or 1EPSPs). CNQX was effective in blocking 1EPSPs. 3. With the use of stimulus intensities at or just below threshold for evoking an action potential, complex synaptic potentials consisting of EPSP-IPSP sequences were observed. Both early, Cl(-)-dependent and late, K(+)-dependent IPSPs were reduced by CNQX. This effect was reversible on washing. This disinhibition could lead to enhanced excitability in the presence of CNQX. 4. Iontophoretic application of quisqualate produced a membrane depolarization with superimposed action potentials, whereas NMDA depolarized the membrane potential and evoked bursts of action potentials. At concentrations up to 5 microM, CNQX selectively antagonized quisqualate responses. NMDA responses were reduced by 10 microM CNQX. D-Serine (0.5-2 mM), an agonist at the glycine regulatory site on the NMDA receptor, reversed the CNQX depression of NMDA responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methyllycaconitine-sensitive neuronal nicotinic receptor-operated slow Ca2+ signal by local application or perfusion of ACh at the mouse neuromuscular junction

Local application of acetylcholine (ACh; 0.3 mM, 20 microl) elicited bi-phasic elevation of intracellular Ca2+ concentrations (contractile fast and non-contractile slow Ca2- signal measured as aequorin luminescence) in diaphragm muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (MLA; 0.01-1 microM), which did not affect the fast Ca2+ transients and twitch tension, concentration-dependently depressed only the slow Ca2+ component. Ca2+ channel blockers, Cd2+ (200 microM), nitrendipine (1 microM), verapamil (1 microM) and diltiazem (1 microM), or a Na+ channel blocker tetrodotoxin (TTX; 0.1 microM) failed to prevent the generation of slow Ca2+ response. Perfusion of ACh (1 microM) to isolated single skeletal (flexor digitorum brevis) muscle cells pretreated with TTX (0.1 microM) also elicited a slow Ca2+ signal measured as confocal imaging with a fluorescent dye, fluo-3, at the endplate region. MLA (1 microM) antagonized against the ACh perfusion-elicited slow Ca2+ signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also elicited the MLA-sensitive slow Ca2+ signal. These results strongly suggest that the ACh-induced slow Ca2+ signal reflects Ca2+ entry through a postsynaptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromuscular junction.     

Effects of ethanol and other drugs on excitatory and inhibitory neurotransmission in the crayfish.

1. Crayfish exposed to 434 mM ethanol (EtOH) showed signs of hyperactivity within 0.5-2 h, at which times crayfish hemolymph EtOH concentration had reached 60-90 mM. 2. A 10-min exposure to 60-90 mM EtOH reduced presynaptic inhibition of excitatory postsynaptic currents (EPSCs) at the crayfish opener neuromuscular junction (NMJ) in vitro but did not significantly alter excitatory neurotransmission. The same concentrations of EtOH did not alter other potentials or currents associated with inhibition at this synapse, such as presynaptic inhibitory potentials (PIPs), inhibitory postsynaptic potentials (IPSPs), and inhibitory postsynaptic currents (IPSCs). 3. Intermediate EtOH concentrations (120-180 mM) applied for 10 min in vitro reduced the amplitude of excitatory postsynaptic potentials (EPSPs) by decreasing the membrane resistance of opener muscle fibers and by reducing the amplitude of EPSCs. 4. High EtOH concentrations (434 mM) applied for 10 min in vitro had yet greater depressive effects on measures of postsynaptic properties described above. The time course of EPSCs was also significantly reduced. In addition, presynaptic properties such as action-potential (AP) amplitude and frequency of spontaneous release of neurotransmitter were reduced by 434 mM EtOH. 5. Presynaptic inhibition, gamma-aminobutyric acid (GABA; 250-500 microM), muscimol (50 microM), and baclofen (75 microM) all reduced the depolarizing afterpotential of APs in the excitor axon and reduced EPSPs in opener muscle fibers. GABA (500 microM) and baclofen (75 microM) significantly reduced presynaptic AP amplitudes, whereas presynaptic inhibition, GABA (250 microM), and muscimol (50 microM) had no effect on AP amplitude. Bicuculline (250-500 microM), a GABAA antagonist, did not entirely eliminate presynaptic inhibition, whereas picrotoxin (50 microM), another GABAA antagonist, completely removed presynaptic inhibition. Thus presynaptic inhibitory mechanisms may involve both GABAA and GABAB receptors on the opener excitor axon. 6. Our data suggest that the behavioral hyperactivity seen at hemolymph EtOH concentrations of 60-90 mM is not accompanied by a change in excitatory synaptic transmission observed at the opener NMJ. Rather, crayfish hyperactivity may be due to depressive effects of EtOH on inhibitory synapses in the CNS similar to the disinhibition evoked by EtOH at the opener NMJ.     

Effects of tetrodotoxin on changes in extracellular free calcium induced by repetitive electrical stimulation and iontophoretic application of excitatory amino acids in the sensorimotor cortex of cats

Extracellular free calcium ([Ca²⁺]_o) was measured with double barreled ion-sensitive reference electrodes in the sensorimotor cortex of cats before and after application of tetrodotoxin (TTX). Electrical stimulation of the cortical surface or of the thalamic ventrobasal complex resulted in reductions of [Ca²⁺]_o (ΔCa) by up to 0.45 mM (baseline 1.2–1.3 mM). Iontophoretic applications of the excitatory amino acids glutamate, aspartate and dl-homocysteate evoked ΔCa by up to 1.2 mM. ΔCa were largest at a depth of 100–300 μm below cortical surface. After application of 10^?5 M TTX to the cortical surface, the ΔCa evoked by electrical stimulation disappeared and the accompanying slow negative potentials were reduced in amplitude. In contrast, ΔCa evoked by excitatory amino acids were only slightly affected. It is suggested that excitatory amino acids activate voltage-dependent postsynaptic Ca²⁺ conductances in neocortical neurones.