Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Extracellular ATP-induced currents in astrocytes: Involvement of a cation channel

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P₂-purinoceptor-mediated depolarization. ATP (100 μM) induced an inward current with a mean amplitude of 130 pA and an EC₅₀ of 17 μM. The response desensitized during a 1 min application. Replacement of extracellular Na⁺ with NMDG or K⁺ abolished the ATP-evoked inward current. Replacement of Na⁺ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na⁺ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K⁺-sensitive microelectrodes showed that 100 μM ATP lowered the intracellular K⁺ concentration. Replacement of extracellular Ca²⁺ or Cl^? did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca²⁺ concentration during ATP application. Removal of extracellular Ca²⁺ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P_2× receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca²⁺ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K⁺ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells. © 1994 Wiley-Liss, Inc.     

Carbachol Potentiates Q Current and Activates a Calcium-dependent Non-specific Conductance in Rat Hippocampus In Vitro

Intracellular recordings were made from CA1 neurons in rat hippocampal slices maintained in vitro. When Na⁺ currents were blocked with tetrodotoxin and K⁺ conductances known to be sensitive to suppression by muscarinic agonists were blocked by 2 mM Ba²⁺, CA1 cells were depolarized by carbachol (3 – 10 μM) with an attendant conductance increase, whereas prior to Ba²⁺ the agonist produced a decrease or no change in conductance. Under voltage clamp at ～–60 mV and in the presence of tetrodotoxin and Ba²⁺, carbachol (3 – 10 μM) induced a variable-latency biphasic inward current of up to 380 pA associated with a conductance increase of ～50%. The first phase was associated with an increase (more than 2-fold) of the Cs⁺-sensitive, hyperpolarization-activated cationic current, I_Q. Carbachol also accelerated the kinetics of I_Q at – 100 mV with an average 24% reduction in its activation time constant. The second phase reflected an additional inward current that was Cs⁺-resistant, displayed little apparent voltage sensitivity and had a mean extrapolated reversal potential, determined in the presence of external Cs⁺ (>5 mM), of ～–20 mV. In a small proportion of cells the second phase of inward current was followed (or overlapped) by an outward current, also associated with a conductance increase, which reversed at ～–70 mV. These carbachol actions were prevented by extracellular 300 μM Cd²⁺ and 2 mM Mn²⁺, by high levels (>5 mM) of extracellular Mg²⁺ or Ca²⁺, and by omission of Ca²⁺ or reduction of extracellular Na⁺ to 25 mM by substitution of NaCl with Tris or N-methyl-d -glucamine. Carbachol action was not mimicked by oxotremorine (≤60 μM), but was irreversibly blocked by this drug. Likewise, atropine (100 nM) irreversibly and gallamine (10 μM) reversibly antagonized carbachol's action. The action of carbachol was blocked shortly after prior exposure of slices to 2 – 5 mM caffeine. Chronic or acute incubation of slices with 2 mM Li⁺ potentiated (between 1- and 2-fold) carbachol responses. The data indicate that muscarinic activation increases cationic flux by a calcium-dependent potentiation of I_Q and activation of a non-selective conductance. The probability that inositol phospholipid metabolism is involved in triggering these events is discussed.     

Voltage-dependent Ca influx into identified leech neurones

We determined the relationships between the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential (E_m) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca²⁺]_i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K⁺ concentration ([K⁺]_o) up to 89 mM caused a persistent increase in [Ca²⁺]_i, which was abolished in Ca²⁺-free solution indicating that it was due to Ca²⁺ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca²⁺]_i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca²⁺]_i and E_m were partly due to the cell-specific generation of action potentials. In Na⁺-free solution, the action potentials were suppressed and the [Ca²⁺]_i/E_m relationships were similar. The K⁺-induced [Ca²⁺]_i increase was inhibited by the polyvalent cations Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, and La³⁺, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K⁺-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca²⁺ channels with similar properties.     

Possible role of apamin-sensitive K+ channels in myotonic dystrophy

Myotonic muscular dystrophy is a genetic disease characterized mainly by muscle atrophy and myotonia, a repetitive electrical activity of muscle. In the present study, the possible role of apamin-sensitive K⁺ channels in the genesis of myotonia was investigated. Apamin is a peptide from bee venom that specifically blocks small conductance Ca²⁺-activated K⁺ channels. The injection of a small amount of apamin (20–30 μl, 10 μmol/L) into the thenar muscle of myotonic dystrophy patients decreased the basal electrical activity during the electromyogram in the 6 patients studied. Myotonic discharges after muscle percussion were more difficult to trigger and of smaller intensity and duration. In 2 controls and in 2 patients with generalized myotonia, as well as in 1 patient with myotonia congenita (where the defect is in chloride channels), apamin had no effect. These results suggest that apamin-sensitive K⁺ channels participate in the mechanism that generates myotonia in myotonic dystrophy. © 1994 John Wiley & Sons, Inc.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Galanin inhibition of voltage‐dependent Ca2+ influx in rat cultured myenteric neurons is mediated by galanin receptor 1

Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization‐evoked Ca²⁺ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1‐16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ‐57408, and of galanin 2‐11, which has high affinity for GalR2 and GalR3, on Ca²⁺ influx through voltage‐dependent Ca²⁺ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo‐4 and depolarized by high K⁺ concentration to activate voltage‐dependent Ca²⁺ channels. Intracellular Ca²⁺ levels were quantified with confocal microscopy. Galanin 1‐16 (0.01–1 μM) inhibited the depolarization‐evoked Ca²⁺ increase in a dose‐dependent manner with an EC₅₀ of 0.172 μM. The selective GalR1 antagonist, RWJ‐57408 (10 μM), blocked the galanin 1‐16 (1 μM)‐mediated inhibition of voltage‐dependent Ca²⁺ channel. By contrast, the GalR2/GalR3 agonist, galanin 2‐11 did not affect the K⁺‐evoked Ca²⁺ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization‐evoked Ca²⁺ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons. © 2008 Wiley‐Liss, Inc.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Differential inhibition of catecholamine secretion by amitriptyline through blockage of nicotinic receptors,sodium channels,and calcium channels in bovine adrenal chromaffin cells

We investigated the effects of amitriptyline, a tricyclic antidepressant, on [³H]norepinephrine ([³H]NE) secretion and ion flux in bovine adrenal chromaffin cells. Amitriptyline inhibited [³H]NE secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and 70 mM K⁺. The half maximal inhibitory concentration (IC₅₀) was 2 μM and 9 μM, respectively. Amitriptyline also inhibited the elevation of cytosolic calcium ([Ca²⁺]_i) induced by DMPP and 70 mM K⁺ with IC₅₀ values of 1.1 μM and 35 μM, respectively. The rises in cytosolic sodium ([Na⁺]_i) and [Ca²⁺]_i induced by the Na⁺ channel activator veratridine were also inhibited by amitriptyline with IC₅₀ values of 7 μM and 30 μM, respectively. These results suggest that amitriptyline at micromolar concentrations inhibits both voltage-sensitive calcium (VSCCs) and sodium channels (VSSCs). Furthermore, submicromolar concentrations of amitriptyline significantly inhibited DMPP-induced [³H]NE secretion and [Ca²⁺]_i rise, but not veratridine- or 70 mM K⁺-induced responses, suggesting that nicotinic acetylcholine receptors (nAChR) as well as VSCCs and VSSCs can be targeted by amitriptyline. DMPP-induced [Na⁺]_i rise was much more sensitive to amitriptyline than the veratridine-induced rise, suggesting that the influx of Na⁺ and Ca²⁺ through the nAChR itself is blocked by amitriptyline. Receptor binding competition analysis showed that binding of [³H]nicotine to chromaffin cells was significantly affected by amitriptyline at submicromolar concentrations. The data suggest that amitriptyline inhibits catecholamine secretion by blocking nAChR, VSSC, and VSCC. Synapse 29:248–256, 1998. © 1998 Wiley-Liss, Inc.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

Suppression of presynaptic calcium influx by metabotropic glutamate receptor agonists in neonatal rat hippocampus

Mechanisms of presynaptic inhibition by metabotropic glutamate receptor (mGluR) agonists were investigated in neonatal rat hippocampal CA1 region using the optical recording technique recently developed. Following selective loading of presynaptic terminals with a fluorescent Ca²⁺ indicator dye rhod-2 AM, changes in Ca²⁺ signals and the corresponding field excitatory postsynaptic potentials (EPSPs) induced by single electrical stimuli to the Schaffer collateral-commissural (SCC) pathway were recorded simultaneously. Application of a mGluR agonist, 1 S,3 R-1-aminocyclopentane-1,3-dicar?ylic acid (1 S,3 R-ACPD; 100 μM) or (±)-1-aminocyclopentane-trans-1,3-dicar?ylic acid (trans-ACPD; 100 μM), reversibly reduced both the field EPSP and the presynaptic Ca²⁺ transient, and the quantitative relationship between them was quite similar to that observed during application of Cd²⁺, a non-selective Ca²⁺ channel blocker, or in a Ca²⁺-free solution. Application of 4-aminopyridine (4-AP; 1 mM), a blocker of certain subtypes of voltage-dependent K⁺ channels, significantly inhibited the 1 S,3 R-ACPD effect. Application of DCG-IV, a novel mGluR2/mGluR3-selective agonist, suppressed field EPSPs only slightly even at a high dose (3 μM). These results suggest that activation of presynaptic mGluR different from mGluR2/mGluR3 suppresses the action potential-triggered Ca²⁺ influx, probably via 4-AP-sensitive mechanisms, and thereby reduces glutamate release in neonatal rat hippocampal CA1 region.     

Membrane depolarization in LA-N-1 cells

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K⁺, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K⁺ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na⁺ and Ca²⁺. Ouabain (5 mM), an inhibitor of the Na⁺, K⁺-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 μM), a Na⁺ channel activator, only in the presence of 10 μg of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca²⁺ ions. On the other hand, Na⁺ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na⁺ channels and the removal of Na⁺ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca²⁺- and Na⁺-dependent, although the latter only partially, and that this effect is dependent on Ca²⁺ influx into the cells likely through a voltage-insensitive calcium-entry system.     

Effects of Na gradient on the intracellular Ca oscillation in the sympathetic ganglion cell: Na---Ca exchange in the neurone cell soma?

(+)-Tubocurarine ((+)-Tc:10–100 μM) reduced the duration of the afterhyperpolarization, which was induced by the activation of Ca²⁺-dependent K⁺-conductance (G_K,Ca) following an action potential in the bullfrog sympathetic ganglion cell, but did not affect the maximum rates of rise and fall of Na⁺- and Ca²⁺-dependent action potentials. The amplitudes of slow rhythmic membrane hyperpolarizations produced by rhythmic rises in the G_K,Ca were also decreased by (+)-Tc without a change in their intervals. Thus, (+)-Tc appears to block the Ca²⁺-dependent K⁺-channel of the bullfrog sympathetic ganglion cell.     

Differential effect of a dihydropyridine derivative to Ca entry pathways in neuronal preparations

Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca²⁺ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca²⁺-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheochromocytoma cells, while the high K^+- stimulated Ca²⁺ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K⁺-stimulated Ca²⁺ influx and transmitter releases. It was then suggested that the voltage-dependent Ca²⁺ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility.     

Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K⁺]_i/140 mM [K⁺]_o and 140 mM [K⁺]_i/3 mM [K⁺]_o respectively. From the reversal potential the channel was highly selective for K⁺, the P_K+/P_na+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca²⁺]_i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca²⁺]_i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K⁺ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.     

Apamin, a blocker of the calcium-activated potassium channel, induces neurodegeneration of Purkinje cells exclusively

Following acute intracerebroventricular injections of 1 ng of apamin and chronic apamin infusion (0.4 ng/μl, 0.5 μl/h, 14 days), the rat brains exhibited bilateral damage only in the cerebellum. The argyrophilic cells were Purkinje cells in copula pyramis, flocculus, paraflocculus, and paramedian lobules. These data demonstrate that the inactivation of small conductance Ca²⁺-activated K⁺ channels by apamin induces a non-limbic neurodegeneration.     

Light-increased cGMP and K conductance in the hyperpolarizing receptor potential of Onchidium extra-ocular photoreceptors

The phototransduction mechanism of the extra-ocular photoreceptor cells Ip-2 and Ip-1 in the mollusc Onchidium ganglion was examined. Previous work showed that the depolarizing receptor potential of another extra-ocular photoreceptor cell, A-P-1 is produced by a decrease of the light-sensitive K⁺ conductance activated by a second messenger, cGMP and is inactivated by the hydrolysis of cGMP. Here, a hyperpolarizing receptor potential of Ip-2 or Ip-1 was associated with an increase in membrane conductance. When Ip-2 or Ip-1 was voltage-clamped near the resting membrane potential, light induced an outward photocurrent corresponding to the above hyperpolarization. The spectral sensitivity had a peak at 510 nm. The shift of reversal potentials of the photocurrent depended on the Nernst equation of K⁺-selective conductance. The photocurrent was blocked by 4-AP and l-DIL, which are effective blockers of the A-P-1 light-sensitive K⁺ conductance. These results suggested that the hyperpolarization is mediated by increasing a similar light-sensitive K⁺ conductance to that of A-P-1. The injection of cGMP or Ca²⁺ into a cell produced a K⁺ current that mimicked the photocurrent. 4-AP and l-DIL both abolished the cGMP-activated K⁺ current, while TEA suppressed only the Ca²⁺-activated K⁺ current. These results indicated that cGMP is also a second messenger that regulates the light-sensitive K⁺ conductance. The photocurrent was blocked by LY-83583, a guanylate cyclase (GC) inhibitor, but was unaltered by zaprinast, a phosphodiesterase (PDE) inhibitor. Together, the present results suggest that increasing the internal cGMP in Ip-2 or Ip-1 cells light-activates GC rather than inhibits PDE, thereby leading to an increase of the light-sensitive K⁺ conductance and the hyperpolarization.     

Multiple Types of Ca2+ Channels in Mouse Motor Nerve Terminals

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca²⁺-channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω-Aga-IVA (0.3 μM), a blocker of P-type Ca²⁺channels] or ω-conotoxin MVIIC [ω-CTx-MVIIC (2 μM), a P-and Q-type Ca²⁺-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L-type Ca²⁺ channels) plus ω-conotoxin GVIA [ω-CTx-GVIA (2 μM), a blocker of N-type Ca²⁺ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca²⁺ signals, a small decrease in presynaptic Ca²⁺ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4-diaminopyridine (3, 4-DAP, a k⁺-channel blocker) or high Ca²⁺(10 mM) to accelerate and amplify the endplate potentials and Ca²⁺ currents. The endplate potentials amplified by 3, 4-DAP or by high Ca²⁺ correspondingly proved to be quite resistant to both ω-Aga-IVA and ω-CTx-MVIIC; ωAga-IVA exerted only a partial inhibitory effect on endplate potentials, and the ω-Aga-IVA-resistant component was further inhibited by ω-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca²⁺ channel blocker Cd²⁺ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na⁺ and K⁺ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca²⁺ currents in the triangularis sterni also revealed partial inhibition by ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA. Cd²⁺ (300 μM) abolished the toxin-resistant component of the Ca²⁺ current. In contrast, a combination of nifedipine (50 μM) with ω-CTx-GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca²⁺channels, i.e. ω-Aga-IVA-sensitive, ω-CTx-MVIIC-sensitive and toxin-resistant Ca²⁺ channels, coexist in mouse motor nerve terminals.