Possible modulatory role of voltage-activated Ca(2+) currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus.

Voltage-activated Ca(2+) currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6-11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca(2+) current on depolarizations positive to -90 mV (the maximum occurred at about -40 mV). The magnitude of the T-current varied considerably from cell to cell (-56 to -852 pA) while its steady-state inactivation was consistent (E(50)=-88.2+/-1.7 mV, s=-6. 0+/-0.4 mV). The maximum of high-voltage activated (HVA) Ca(2+) currents was observed at about -15 mV. At a membrane potential of -10 mV the L-type Ca(2+) channel blocker nifedipine (10 microM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor omega-Conotoxin GVIA (2 microM) reduced the current by 25% while the P/Q-type channel blocker omega-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca(2+) channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (200 microM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA(B) receptor agonist baclofen (100 microM) reversibly inhibited 25% of the HVA current. Simultaneous application of omega-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca(2+) currents reported in this work are discussed.     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Autoradiographic localization of the binding of calcium channel antagonist, [I]ω-agatoxin IIIA, in rat brain

The calcium channel antagonists ω-agatoxin IIIA (ω-Aga-IIIA) and ω-conotoxin GVIA (ω-CgTx) were radioiodinated and used to locate binding sites in the rat brain by receptor autoradiography. While patterns of regional binding to sagittal sections of rat brain were generally similar for the 2 toxins, notable differences in the cerebellum and hippocampus were observed. Specific [¹²⁵I]ω-Aga-IIIA binding was greatest in the granule cell layers of the cerebellum and of the dentate gyrus. In contrast, binding of [¹²⁵I]ω-CgTx was most intense in the molecular layers of these structures. Less than one-third of [¹²⁵I]ω-Aga-IIIA binding in rat brain slices was inhibited by pre-exposure to 250 nM ω-CgTx, while 40 nM ω-Aga-IIIA virtually eliminated the binding of [¹²⁵I]ω-CgTx under the same conditions. The P-type calcium channel antagonist ω-Aga-IVA blocked only a small fraction of [¹²⁵I]ω-Aga-IIIA and [¹²⁵I]ω-CgTx binding. These autoradiographic data are consistent with membrane binding experiments and indicate that the combined use of agatoxins and conotoxins may be useful in the characterization of separate types of neuronal calcium channels.     

Kinetic and pharmacological properties of Ca currents in postganglionic sympathetic neurones projecting to muscular and cutaneous effectors

Voltage-gated Ca²⁺ channels are expressed in neurones and greatly influence neuronal activity by activating Ca²⁺-dependent K⁺ channels. The whole cell patch-clamp technique was used to compare the kinetic and pharmacological properties of voltage-dependent Ca²⁺ currents in two groups of sympathetic neurones identified by the fluorescent tracer Fast Blue: putative muscular sympathetic neurones (MSN) and putative cutaneous sympathetic neurones (CSN). The tracer was injected into the muscular part of the diaphragm (to mark MSN) and into the skin of the ear (to mark CSN). The capacitance of MSN (23.0 pF) was larger than the capacitance of CSN (12.6 pF). The maximum current in MSN (1.3 nA) was also larger than in CSN (0.93 nA). However, the current density was larger in CSN (77.3 pA/pF) than in MSN (57.7 pA/pF) and the current activation rate was faster in CSN (0.27 nA/ms) than in MSN (0.19 nA/ms). V_1/2 and slope factors of activation and inactivation were not significantly different for MSN and CSN. The majority of Ca²⁺ current was available for activation in both categories of neurones at resting membrane potential. Ca²⁺ currents in MSN and CSN were blocked by nifedipine (7.0 and 3.6%, respectively), ω-Agatoxin-IVA (23.0 and 25.6%, respectively) and ω-conotoxin-GVIA (67.0 and 65.1%, respectively). We found that CSN are twice as small, have higher Ca²⁺ current density and their Ca²⁺ activation rate is faster in comparison to MSN. Such properties may lead to faster rise of Ca²⁺ concentration in the cytoplasm of the CSN comparing to MSN and more effectively dampen their activity due to more effective activation of Ca²⁺-dependent K⁺ current. Both kinds of neurones express high proportion of N and P/Q Ca²⁺ current.     

α-Eudesmol, a P/Q-type Ca channel blocker, inhibits neurogenic vasodilation and extravasation following electrical stimulation of trigeminal ganglion

In this study, we investigated the effect of α-eudesmol, which potently inhibits the presynaptic ω-agatoxin IVA-sensitive (P/Q-type) Ca²⁺ channel, on neurogenic inflammation following electrical stimulation of rat trigeminal ganglion. Treatment with α-eudesmol (0.1–1 mg/kg. i.v.) dose-dependently attenuated neurogenic vasodilation in facial skin monitored by a laser Doppler flowmetry. In addition, α-eudesmol (1 mg/kg. i.v.) significantly decreased dural plasma extravasation in analysis using Evans blue as a plasma marker. On the other hand, α-eudesmol (1 mg/kg, i.v.) did not affect mean arterial blood pressure in rats. The calcitonin gene-related peptide (CGRP) and substance P (SP) released from activated sensory nerves have recently been suggested to be associated with the neurogenic inflammation. In this study, we also showed that α-eudesmol (0.45–45 μM) concentration-dependently inhibits the depolarization-evoked CGRP and SP release from sensory nerve terminals in spinal cord slices. These results indicate that the anti-neurogenic inflammation action of α-eudesmol, which does not affect the cardiovascular system, may be due to its presynaptic inhibition of the neuropeptide release from perivascular trigeminal terminals. We also suggest that the ω-agatoxin IVA-sensitive Ca²⁺ channel blocker, α-eudesmol, may become useful for the treatment of the neurogenic inflammation in the trigemino-vascular system such as migraine.     

Effects of funnel web spider toxin on Ca currents in neurohypophysial terminals

Funnel web spider toxin (FTX) is reportedly a specific blocker of P-type Ca²⁺ channels. The effects of FTX on the Ca²⁺ currents of isolated neurohypophysial nerve terminals of the rat were investigated using the ‘whole-cell’ patch-clamp technique. Both the transient and long-lasting Ca²⁺ current components were maximally elicited by depolarization from a holding potential equal to the normal terminal resting potential (−90 mV). Externally applied FTX inhibited the high-voltage-threshold, transient component of the Ca²⁺ current in a concentration-dependent manner, with a half-maximal inhibition at a dilution of approximately 1: 10000. FTX also shifted the peak current of the I–V relationship by + 10 mV. The long-lasting Ca²⁺ current component, which is sensitive to L-type Ca²⁺ channel blockers, was insensitive to FTX. The transient current, which is sensitive to ω-conotoxin GVIA, was completely blocked by FTX. These results suggest that there could be a novel, inactivating Ca²⁺ channel in the rat neurohypophysial terminals which is affected by both N-type and P-type Ca²⁺ channel blockers.     

Effects of ω-conotoxin MVIIC on veratridine-induced cytotoxicity and cytosolic Ca oscillations

External Ca²⁺ entry through various Ca t+-channel subtypes is responsible for the large oscillations of the cytosolic Ca²⁺ concentrations, [Ca²⁺]_i, and cell death induced by veratridine in primary cultures of bovine chromaffin cells. Blockade by ω-conotoxin GVIA (GVIA) of N-type Ca²⁺ channels, by ω-agatoxin GIVA (IVA) of P-type Ca²⁺ channels, or by furnidipine of L-type Ca²⁺ channels did not afford cytoprotection. However, ω-conotoxin MVIIC (MVIIC), a wide-spectrum blocker of N-, P- and Q-type Ca²⁺ channels greatly protected the cells against the cytotoxic effects of veratridine. Furnidipine further enhanced the cytoprotecting effects of MVIIC. MVIIC but not fumidipine, markedly reduced the oscillations of [Ca²⁺]_i induced by veratridine in single fura-2-loaded chromaffin cells. The results suggest that Ca²⁺ entry through any of the different Ca²⁺ channel subtypes present in bovine chromaffin cells might be cytotoxic. They also support two ideas: (i) that wide-spectrum neuronal Ca²⁺ channel blockers (i.e. MVIIC) might be better cytoprotecting agents than more specific neuronal Ca²⁺ channel blockers (i.e., GVIA, IVA, furnidipine); and (ii) that combined Ca²⁺ channel blockers may provide greater cytoprotection than single compounds.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

Activation of ATP receptor increases the cytosolic Ca concentration in nucleus accumbens neurons of rat brain

ATP increased the cytosolic Ca²⁺ concentration ([Ca]_i) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca²⁺ and Na⁺, and was blocked by voltage-dependent Ca²⁺ channel blockers. The results suggest that the ATP-induced depolarization increases Ca²⁺ influx resulting in the increase in [Ca]_i.     

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.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

A new type of Ca channel blocker, NC-1100, inhibits the low- and high-threshold Ca currents in the rat CNS neurons

The effect of a new type of organic Ca²⁺ channel blocker, NC-1100 [(±)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)ethanol dihydrochloride], on both low- and high-threshold Ca²⁺ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca²⁺ current (HVAI_Ca) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC₅₀) were 1.3 × 10⁻⁵ and 9.1 × 10⁻⁶M in external solution containing 10 and 2.5 mM Ca²⁺, respectively. The NC-1100 also decreased the low-threshold Ca²⁺ current (LVAI_Ca) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and / or decreasing the extracellular Ca²⁺ concentration to a physiological range (2.5 mM). The IC₅₀ value decreased to 7.7 × 10⁻⁷M in external solution containing 2.5 mM Ca²⁺ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca²⁺ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca²⁺ channel but also the inactivated one.     

Norepinephrine efflux evoked by potassium chloride in cat sympathetic nerves: dual mechanism of action

Using a dialysis technique, prominent efflux of norepinephrine (NE) from cardiac sympathetic nerve endings was observed under local administration of potassium chloride (KCl, 100 mM). KCl induced NE efflux was suppressed by ω-conotoxin GVIA or desipramine but residual efflux of NE was still detectable. In the presence of ω-conotoxin GVIA, KCl induced efflux of NE was augmented by pretreatment with reserpine, indicating that this efflux of NE was derived from axoplasma with neurotransporter. These data suggest that a KCl induced brisk increase in dialysate NE levels might occur as a consequence of exocytotic NE release and carrier mediated outward NE transport from nerve endings.     

Calcium transport in and out of brain nerve endings in vitro—the role of synaptosomal plasma membrane Ca-ATPase in Ca-extrusion

The effects of cellular cations and ATP on calcium transport in and out of the nerve endings (synaptosomes) of mice brain were studied. The synaptosomes accumulated⁴⁵Ca time-dependently in the absence of ATP or other additions for at least 10 min. When ATP was present, the overall⁴⁵Ca accumulation was decreased and was maximal at about 4 min, after which it started to decline. Studies on the effects of cations with or without ATP at 4 min revealed selective activities for different cations. Mg²⁺ inhibited⁴⁵Ca accumulation in the absence of ATP but increased⁴⁵Ca accumulation when ATP was present. Similarly, ATP increased⁴⁵Ca accumulation only when Mg²⁺ was present. Na⁺, on the other hand, inhibited⁴⁵Ca accumulation both in the presence and absence of ATP and/or Mg²⁺. K⁺ increased⁴⁵Ca accumulation in the presence of ATP with or without Mg²⁺; however, K⁺-stimulation was not noted in the presence of 100 mM Na⁺, and in fact, K⁺ became inhibitory. The ATP-stimulated⁴⁵Ca accumulation in the presence of Mg²⁺ peaked within 4–6 min and then declined, suggesting release of⁴⁵Ca. Compatible with this notion, in⁴⁵Ca-loaded synaptosomes, ATP evoked⁴⁵Ca release which was accompanied by the appearance of P_i in the medium. Although ATP-activated⁴⁵Ca-release can occur in the presence of Mg²⁺, Mg²⁺ is not required and, infact, is inhibitory. Rapid release of⁴⁵Ca was also noted when⁴⁵Ca-loaded synaptosomes were incubated in the presence of Na⁺ without ATP. It is concluded that Mg²⁺, Na⁺,⁺K and ATP each has a specific role in regulating Ca²⁺ permeability of the plasma membrane, calcium binding and calcium extrusion.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Short-term synaptic plasticity in layer II/III of the rat anterior cingulate cortex

Recent in vivo electrophysiological studies in our laboratory demonstrated medial thalamus (MT) induced short-term facilitation in the middle layers of the anterior cingulate cortex (ACC). The aim of the present study was to investigate different forms of short-term plasticity (STP) in layer II/III of the ACC in an in vitro slice preparation. Extracellular field potentials in layer II/III consisting of an early component (fAP) and a late component (fPSP) were activated by electrical stimulation of the deep layers. The fPSP and intracellularly recorded excitatory post-synaptic potential (EPSP) could be facilitated by paired-pulse stimulation at a low frequency (0.033Hz, pulse interval 20-400ms). An initial facilitation and subsequent depression were obtained when high frequency (12.5, 25 and 50Hz) tetanus stimulations were applied to the ACC slice. A post-tetanic augmentation 30s in duration was also observed. The effects of tetanic stimulation were altered in the presence of an increased or a decreased calcium concentration. Application of omega-conotoxin GVIA (CTX) in normal calcium concentration conditions decreased overall responses during tetanic stimulation similar to reducing calcium exposure. However CTX application did not increase paired-pulse facilitation (PPF) as is seen under low calcium conditions. These results indicate that calcium is involved in the formation of certain features of STP in layer II/III of the ACC and that N-type calcium channels contribute to some, but not all, components of these plastic changes. Two-site electrical stimulation testing showed that two separate presynaptic inputs can produce short-term facilitation. Our findings implicate a post-synaptic mechanism in STP in layer II/III of the ACC.     

Ca2+ depletion-induced disconnection of tight junctions in isolated rat brain microvessels

Summary Cerebral microvessels were isolated from rat brains. One part of the microvessel pellets was incubated for 25 or 90 min in Krebs-Henseleit bicarbonate buffer (KHB) at pH 7.5 (control group). The other part of the pellets was treated for the same periods of time with Ca²⁺-free KHB, containing 2.2 mM EGTA and 2 mM glucose (experimental group). Morphological changes of endothelial tight junctions were evaluated in 100 randomly selected interendothelial clefts from isolated cerebral microvessels of each groups by electron microscopy. Following 25 min of incubation time, either with Ca²⁺-containing or with Ca²⁺-free KHB, no significant changes of tight junctions were observed. After 90 min of incubation in Ca²⁺-free medium, 58% of tight junctions were altered (in 42% partial, and in 16% complete disconnection of tight junctions were found). This contrasted the control group, where only 14% of tight junctions were disconnected (12% partially and 2% completely). Our results are consistent with a role for intercellular Ca²⁺ in maintaining structural integrity of cerebral tight junctions.Supported by the Medical Research Council of Canada, grant MT-5958 and the Hungarian Ministry of Health, grant 06/1-44/313     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.