Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (I_Ca). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. I_Ca was identified by voltage clamp protocols, ionic substitution and pharmacological tools. Depolarization to –30 mV from a holding voltage of –80 mV induced an inward current consisting of an initial transient and a long-lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 μm nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 μm Bay K 8644 or when the external Ca²⁺ was substituted by equimolar Ba²⁺. Omega-conotoxin (10 μm ω-ctxn GVIA) did not alter either component. We concluded that the transient component is a low-voltage activated T-type I_Ca, while the sustained component is a high-voltage activated L-type I_Ca. T-type I_Ca was recorded in all cells tested, while L-type I_Ca was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L-type I_Ca is generated at the axon terminal and contributes to the transmitter release from the bipolar cell. The present results confirm that in addition to the T-type I_Ca that had been previously described, bipolar cells of the mammalian retina also contain L-type I_Ca similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.     

Ionic channel currents in cultured neurons from human cortex

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na⁺ current, two types of Ca²⁺ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K⁺ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca²⁺-activated K⁺ channels with slope conductances of 146 and 23 pS (K⁺ _iK⁺ _o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca²⁺. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K⁺ and Ca²⁺ channel currents, but Na⁺ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc.     

Vulnerability of Medium Spiny Striatal Neurons to Glutamate: Role of Na+/K+ ATPase

In Huntington's disease neuronal degeneration mainly involves medium-sized spiny neurons. It has been postulated that both excitotoxic mechanisms and energy metabolism failure are implicated in the neuronal degeneration observed in Huntington's disease. In central neurons, >40% of the energy released by respiration is used by Na⁺/K⁺ ATPase to maintain ionic gradients. Considering that impairment of Na⁺/K⁺ ATPase activity might alter postsynaptic responsivity to excitatory amino acids (EAAs), we investigated the effects of the Na⁺/K⁺ ATPase inhibitors, ouabain and strophanthidin, on the responses to different agonists of EAA receptors in identified medium-sized spiny neurons electrophysiologically recorded in the current- and voltage-clamp modes. In most of the cells both ouabain and strophanthidin (1–3 μM) did not cause significant change in the membrane properties of the recorded neurons. Higher doses of either ouabain (30 μM) or strophanthidin (30 μM) induced, per se, an irreversible inward current coupled to an increase in conductance, leading to cell deterioration. Moreover, both ouabain (1–10 μM) and strophanthidin (1–10 μM) dramatically increased the membrane depolarization and the inward current produced by subcritical concentrations of glutamate, AMPA and NMDA. These concentrations of Na⁺/K⁺ ATPase inhibitors also increased the membrane responses induced by repetitive cortical activation. In addition, since it had previously been proposed that dopamine mimics the effects of Na⁺/K⁺ ATPase inhibitors and that dopamine agonists differentially regulate the postsynaptic responses to EAAs, we tested the possible modulation of EAA-induced membrane depolarization and inward current by dopamine agonists. Neither dopamine nor selective dopamine agonists or antagonists affected the postsynaptic responses to EAAs. Our experiments show that impairment of the activity of Na⁺/K⁺ ATPase may render striatal neurons more sensitive to the action of glutamate, lowering the threshold for the excitotoxic events. Our data support neither the role of dopamine as an ouabain-like agent nor the differential modulatory action of dopamine receptors on the EAA-induced responses in the striatum.     

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.     

Patch-Clamp Analysis of the Mechanism of PACAP-Induced Excitation in Rat Supraoptic Neurones

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP)causes an increase in [Ca²⁺]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non-phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca²⁺]i revealed that the membrane depolarization correlated well with increases in [Ca²⁺]i. In the voltage-clamp mode, PACAP induced inward currents at a holding potential of ?70 or ?80 mV in a dose-dependent manner and the time course of the currents was similar to that of the PACAP-induced membrane depolarization. The averaged reversal potential of the PACAP-induced currents obtained from dissociated SON neurones was ?33 mV, which was close to the reversal potential of non-selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation-channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAPreceptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non-selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP-induced increase in [Ca²⁺]i and vasopressin (VP) release observed in SON neurones.     

Modulation of voltage-activated Ca currents by pain-inducing agents in a dorsal root ganglion neuronal line,F-11

Whole cell currents evoked by pain-inducing agents—bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (I_Na) was 100 μA/cm² and the I_Na was abolished by 10^?6 M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient I_Ca(t) and a high threshold sustained I_Ca(I). In addition, another high threshold transient Ca current, presumably I_Ca(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of ?70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10^?6 M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10^?6 M Bk in the majority of the cells. Similarly, both 10^?6 M Cap and 10^?9 M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (G_i). © 1993 Wiley-Liss, Inc.     

Calcium-activated potassium channels in rat visceral sensory afferents

The purpose of the present study was to describe, at the single-channel level, the activity of a calcium-sensitive potassium channel in rat visceral-sensory neurons which has been suggested to be involved in sensory neuron excitability. Single-channel recordings in the inside-out configuration identified a 220 pS conductance calcium-activated potassium channel (KCa). From a −20 mV holding potential, increasing [Ca²⁺]_i from 0.01 μM to 1.0 μM increased the open probability of this channel 92% (from 0.12 to 0.23). However, from a +20 mV holding potential, increasing [Ca²⁺]_i from 0.01 to 1.0 μM increased the open probability by 326% (from 0.15 to 0.64). In addition, this large conductance KCa channel was blocked by TEA (1.0 μM) and charybdotoxin (40 μM) when applied to the external surface. These results are the first to characterize a large conductance KCa channel in the sensory afferent neurons of the rat nodose ganglia and should further expand the understanding to the ionic currents involved in the regulation of sensory afferent neuronal activity.     

Expression of membrane currents in rat diencephalic neurons in serum-free culture

The whole-cell gigaseal voltage clamp technique has been used to investigate the timing of expression and type of voltage-dependent ionic currents in dissociated primary cultures of fetal rat (E17) diencephalic neurons grown in a serum-free defined medium. The expression of membrane currents varied among cells at any particular time in culture. Despite this variability, certain characteristics of the appearance of ionic currents emerge from this study. These are: (i) the earliest appearing membrane current is a voltage-dependent outward current carried by K+. In some cells, it is the classical delayed rectifier current, whereas in others it is the transient outward current (IA). (ii) The earliest appearing inward current is carried by Na+. In some cells the channels are first expressed in the neurites and then in or near the cell body. The early neuritic Na+ channels are blocked by cobalt or cadmium as well as by tetrodotoxin (TTX). In others, the early Na+ channels appear in or near the cell body and are only blocked by TTX. (iii) With additional time in culture, a majority of cells exhibit a Ca2+ current at the time of Na+ channel appearance in or near the cell body as well as a transient Ca2+-dependent outward current. The Ca2+ current is only a small fraction of the total inward current. These inward currents show the classical pharmacologic profile. The complex pattern of expression of ionic current may reflect multiple populations of neurons with different developmental sequences resulting from differences in cell age and lineage.     

Differences in a K current in schwann cells from normal and neurofibromatosis-infected damselfish

Patch clamp techniques were used to study whole cell ionic currents in Schwann cells (SC) from a tropical marine fish, the bicolor damselfish, Pomacentrus partitus. The bicolor damselfish is affected by a disease termed damselfish neurofibromatosis (DNF), being developed as an animal model of neurofibromatosis-type 1 (NF1) in humans. NF1 affects SC, fibroblasts, and perineurial cells. The sole depolarization-activated ionic current present in cultured SC from normal fish peripheral nerve and from neurofibromas of fish with induced or spontaneously occurring DNF was an inactivating K⁺ current (K current), with a strong dependence on the Nernst potential for K⁺. This K current activated at depolarizations to -40 mV and above and inactivated during a maintained test pulse (0.2-1 s), but inactivation was significantly greater in tumored SC. Both currents were inhibited by 4-aminopyridine (K_d ? 1 mM) and by dendrotoxin (15 μM) but were insensitive to extracellular tetraethyammonium (≤ 150 mM), indicating that the whole cell currents were similar pharmacologically. The currents could be distinguished on the basis of their sensitivity to depolarized holding potential, with normal cells less sensitive. Half-inactivation of the current was -32 mV in normal cells and -38 mV in tumored cells. Inactivation curves constructed from the average normalized current for many SC were significantly different in normal and tumored cells. When the depolarized holding potential was maintained between test depolarizations, greater voltage-dependent inactivation in tumored cells was apparent. Normal cells maintained an average of 36% of peak current at a holding voltage of ?40 mV, while in tumored cells this average was 12%, a significant difference. © 1994 Wiley-Liss, Inc.     

Changes in glial K+ currents with decreased extracellular volume in developing rat white matter

Whole cell patch-clamp recordings of K⁺ currents from oligodendrocyte precursors in 10-day-old rats (P10) and, following myelination, in mature oligodendrocytes from 20-day-old rats (P20) were correlated with extracellular space (ECS) diffusion parameters measured by the local diffusion of iontophoretically injected tetramethylammonium ions (TMA⁺). The aim of this study was to find an explanation for the changes in glial currents that occur with myelination. Oligodendrocyte precursors (P10) in slices from corpus callosum were characterized by the presence of A-type K⁺ currents, delayed and inward rectifier currents, and lack of tail currents after the offset of a voltage jump. Mature oligodendrocytes in corpus callosum slices from P20 rats were characterized by passive, decaying currents and large tail currents after the offset of a voltage jump. Measurements of the reversal potential for the tail currents indicate that they result from increases in [K⁺]_e by an average of 32 mM during a 20 msec 100 mV voltage step. Concomitant with the change in oligodendrocyte electrophysiological behavior after myelination there is a decrease in the ECS of the corpus callosum. ECS volume decreases from 36% (P9–10) to 25% (P20–21) of total tissue volume. ECS tortuosity λ = (D/ADC)^0.5, where D is the free diffusion coefficient and ADC is the apparent diffusion coefficient of TMA⁺ in the brain, increases as measured perpendicular to the axons from 1.53 ± 0.02 (n = 6, mean ± SEM) to 1.70 ± 0.02 (n = 6). TMA⁺ non-specific uptake (k′) was significantly larger at P20 (5.2 ± 0.6 × 10⁻³s⁻¹, n = 6) than at P10 (3.5 ± 0.4 × 10⁻³s⁻¹, n = 6). It can be concluded that membrane potential changes in mature oligodendrocytes are accompanied by rapid changes in the K⁺ gradient resulting from K⁺ fluxes across the glial membrane. As a result of the reduced extracellular volume and increased tortuosity, the membrane fluxes produce larger changes in [K⁺]_e in the more mature myelinated corpus callosum than before myelination. These conclusions also account for differences between membrane currents in cells in slices compared to those in tissue culture where the ECS is essentially infinite. The size and geometry of the ECS influence the membrane current patterns of glial cells and may have consequences for the role of glial cells in spatial buffering. J. Neurosci. Res. 49:98–106, 1997. © 1997 Wiley-Liss Inc.     

Effects of the DAG analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents through α-bungarotoxin-insensitive nicotinic acetylcholine receptors expressed on cockroach neurosecretory cells

We previously demonstrated that the cockroach α-bungarotoxin-sensitive nicotinic acetylcholine receptors, nAChR1 and nAChR2 subtypes, are differently sensitive to intracellular calcium pathways. Here, using whole cell patch-clamp recordings, we studied the effects of the diacylglycerol (DAG) analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents under an α-bungarotoxin treatment. Our results demonstrated that DiC8 reduced nicotine and clothianidin evoked currents. 10 μM DiC8 suppressed the increase in nicotine-induced currents which was brought about by application of 5 mM caffeine or 9 mM Ca²⁺, whereas DiC8 did not affect the decrease in nicotine-induced currents induced by BAPTA. Similarly, bath application of caffeine or 9 mM Ca²⁺ did not change the clothianidin effects, and the amplitude of clothianidin-induced currents was not affected. However, co-application of both 10 μM DiC8 with 9 mM Ca²⁺, caffeine or BAPTA reduced clothianidin current amplitudes. We conclude that nicotine and clothianidin differently modulate nAChR1 and nAChR2 subtypes under DiC8 treatment, and that nicotine activates nAChR1, whereas clothianidin activates both nAChR1 and nAChR2 subtypes.     

Serotonin- and proton-induced and modified ionic currents in frog sensory neurons

Using the whole-cell patch-clamp technique, the effects of serotonin (5-HT) and increased acidity to produce membrane currents and to modify high threshold voltage-dependent calcium currents were studied in isolated dorsal root ganglion (DRG) cells of the frog maintained in short-term culture. DRG cells were classified by morphology into two types: (1) cells with a large number of dark rusty brown granules, and (2) cells devoid of these granules or with few scattered pale granules. Fast application of 5-HT (10–30 μM) induced a rapidly desensitizing inward current with a reversal potential at about 0 mV in 38 of 50 granule-containing neurons (76%) which was never observed (0/35) in “clear” neurons. This current was blocked by 10 nM (+)-tubocurarine. In addition, a small noninactivating outward current was also observed in most DRG neurons during 5-HT superfusion. A sudden decrease of pH from 7.4 to 6 or 5.8 induced a fast inactivating inward current of 100–300 pA in 74% of the “clear” neurons and only 24% of the granule-containing neurons. Small noninactivating membrane currents induced by lowering pH were observed in all neurons. Both 5-HT and increased extracellular H⁺ reduced the magnitude of high threshold calcium currents in all DRG neurons. It is suggested that the 5-HT receptors are expressed on a morphologically distinct population of neurons while the cells with channels responsible for the fast inactivating proton-induced current cannot be related to any distinct morphological cell type. © 1995 Wiley-Liss, Inc.     

The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier.

Sodium and potassium currents in the node of Ranvier decrease exponentially with time during long lasting voltage clamp experiments. This decline is strongly dependent on temperature (Q10 approximately 3). Thiamine and, particularly, its diand triphosphoric acid esters are shown to prevent this exponential decline of the ionic currents. Thiamine acts from the outside and from the inside of the nodal membrane, but more potently from the inside. Thiamine diphosphate prevents the exponential decline of the ionic currents only when applied internally. Thiamine triphosphate, the most effective thiamine derivative was tested form the inside only. Bacterial thiaminases applied externally were not effective, presumably because they do not permeate the nodal membrane. Tetrodotoxin, that has been shown by other investigators to induce a release of thiamine from nerve membranes, does not alter the action of thiamine on the exponential decline of current and vice versa. It is concluded that: (1) thiamine diphosphate or thiamine triphosphate are the active thiamine compounds in nerve membranes; (2) the site of action is located at the internal suface of the membrane; (3) the reduction of the thiamine concentration in the membrane or in the axoplasm could cause the exponetial decline of currents; (4) the release of thiamine from nerve membranes induced by tetrodotoxin is interpreted as a side effect not even related to the mechanism by which tetrodotoxin blocks the sodium channels; (5) thiamine polyphosphates appear to stabilise the intrinsic electric field strength of the nodal membrane in the resing state. Threfore, as a working hypothesis, it is suggested that the thiamine derivatives control the number of functioning ionic channels by stabilising the density of negative surface charges at the inner side of the nerve membrane.     

Robust tonic GABA currents can inhibit cell firing in mouse newborn neocortical pyramidal cells

Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl^? reversal potential (E_Cl). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA_A receptors (GABA_ARs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2–4] then decreases dramatically by the second postnatal week (P7–10 and P30–40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA_AR α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 μM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E_Cl recorded from P2–5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability.     

Intracellular calcium stores modulate miniature GABA‐mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature γ-aminobutyric acid_A (GABA_A) receptor-mediated currents (in tetrodotoxin, 1 μm and kynurenic acid 1 mm ) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6–9) old rats. Switching from a Ca²⁺-containing to a nominally Ca²⁺-free medium (in which Ca²⁺ was substituted with Mg²⁺, in the presence or in the absence of 100 μm EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 μm over control) was 1.8 ± 0.5, (n = 9). In nominally Ca²⁺-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mm ), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mm ) reversibly reduced the amplitude of the current induced by GABA (100 μm ) indicating a clear postsynaptic effect on GABA_A receptor. Superfusion of ryanodine (30 μm ), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine–ryanodine-sensitive stores facilitates GABA release.     

A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus

The pyramidal neurons in the CA1 area of hippocampal slices from 17- to 19-day-old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μm 2-amino-5-phosphonovaleric acid (D-APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 ± 11% of the total EPSC when measured at a holding potential of ?50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) 10 μm and bath incubation with non-hydrolysable ATP analogues, ATP-γ-S and α,β-methylene-ATP at 50 and 20 μm , respectively. The rEPSC was dramatically potentiated by external Zn²⁺ (10 μm ). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one-fifth to one-quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.     

Postnatal development of GABAB receptor-mediated modulation of voltage-activated Ca2+ currents in mouse brain-stem neurons.

GABA_B receptors modulate respiratory rhythm generation in adult mammals. However, little is currently known of their functional significance during postnatal development. In the present investigation, the effects of GABA_B receptor activation on voltage-activated Ca²⁺ currents were examined in rhythmically active neurons of the pre-Bötzinger complex (PBC). Both low- (LVA) and high-voltage-activated (HVA) Ca²⁺ currents were present from the first postnatal day (P1). The density of LVA Ca²⁺ currents increased during the first week, whilst the density of HVA Ca²⁺ currents increased after the first week. In the second postnatal week, the HVA Ca²⁺ currents were composed of L- (47 ± 10%) and N-type (21 ± 8%) currents plus a ‘residual’ current, whilst there were no N-type currents detectable in the first few days. The GABA_B receptor agonist baclofen (30 μm ) increased LVA Ca²⁺ currents (30 ± 11%) at P1–P3, but it decreased the currents (35 ± 11%) at P7–P15 without changing its time course. At all ages, baclofen (30 μm ) decreased the HVA Ca²⁺ currents by ≈ 54%. Threshold of baclofen effects on both LVA and HVA Ca²⁺ currents was 5 μm at P1–P3 and lower than 1 μm at P7–P15. The effect of baclofen was abolished in the presence of the GABA_B receptor antagonist CGP 55845A (50 n m ). We conclude that both LVA and HVA Ca²⁺ currents increased postnatally. The GABA_B receptor-mediated modulation of these currents undergo marked developmental changes during the first two postnatal weeks, which may contribute essentially to modulation of respiratory rhythm generation.     

Neonatal Rat Cerebellar Granule and Purkinje Neurons in Culture Express Different GABAA Receptors

We have established a culture system for microexplants of rat cerebellar cortical tissue in which cells develop morphologically, express type-A receptors for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and form GABAergic synaptic connections. Criteria of cell size and shape allow reliable identification of granule and Purkinje neurons, criteria confirmed by studies of the binding of antibodies to calbindin D28K and GABA. Both granule and Purkinje neurons express GABA_A receptors, but granule neurons fall into two classes in terms of their sensitivity. Granule neurons which do not show spontaneous synaptic currents are relatively insensitive to GABA, while granule neurons with synaptic currents are much more sensitive. The responses of Purkinje neurons to applications of 1 μM GABA are relatively insensitive to Zn²⁺ ions (10 μM), and are potentiated by chlordiazepoxide (100 μM) and La³⁺ ions (100 μM). Responses of innervated granule neurons, on the other hand, are blocked more strongly by Zn²⁺ ions, are less affected by chlordiazepoxide and are equally potentiated by La³⁺ ions. Hence these cultures provide a source of identifiable, functionally innervated cells which express distinct types of GABA_A receptors.     

Voltage-dependent membrane currents of cultured human neurofibromatosis type 2 Schwann cells

Previous experimental observations indicate that inhibition of voltage-dependent K⁺ currents suppresses proliferation of normal Schwann cells. In the present study we tested the opposite relationship, i.e., whether Schwann cells from tumors with abnormally high rates of proliferation would have an increase in membrane K⁺ currents. Whole-cell membrane currents were studied in cultured cells from schwannomas of two neurofibromatosis type 2 (NF2) patients (n = 53), one patient with a sporadic schwannoma (n = 22), and two control subjects (n = 41). Five different types of voltage-dependent membrane currents were found in all of the Schwann cells tested. Membrane depolarization activated outward K⁺ and Cl⁻ currents; quinidine was found to block the K⁺ current (IC₅₀ ≈ 1 μM), and NPPB reduced the Cl⁻ current. Ba²⁺-sensitive inward rectifier K⁺ currents, fast Na⁺ currents, and a transient, inactivating K⁺ current were less frequently observed. On average, NF2 cells were found to have statistically significant higher membrane potential and larger non-inactivating K⁺ outward current as compared to controls. Electrophysiological parameters of Schwann cells from a sporadic schwannoma showed a tendency for larger outward currents; however, the difference did not reach statistical significance. Together the data support the suggestion of a possible link between K⁺ outward current and proliferation of Schwann cells. GLIA 24:313–322, 1998. © 1998 Wiley-Liss, Inc.     

Enantioselective modulation of GABAergic synaptic transmission by steroids and benz[e]indenes in hippocampal microcultures

The effects of enantiomers of the neurosteroid analogues, 3α-hydroxy-5α-pregnan-20-one (DHP) and 3α-hydroxy-5α-androstane-17β-carbonitrile (ACN), and the benz[e]indene, BI-1, on synaptic currents were examined in microcultures of rat hippocampal neurons. Over the range of 0.1–10 μM, the (+)-enantiomers were more potent and effective than their (−)-enantiomeric counterparts in enhancing γ-aminobutyric acid (GABA)_A receptor-mediated evoked synaptic currents. The (+)-enantiomers had small effects on peak currents, but slowed the decay of inhibitory synaptic currents, resulting in 2–3-fold increases in charge transfer during inhibitory synaptic events at 10 μM. Similar prolongations of spontaneous miniature inhibitory postsynaptic currents (IPSCs) and responses to brief GABA pulses to outside-out patches suggest that the prolongations of evoked synaptic currents result primarily from postsynaptic effects. In contrast, the (−)-enantiomers had little effect on evoked IPSCs at concentrations ≤1 μM, but enhanced inhibitory transmission at 10 μM. At concentrations ≤1 μM, neither the (+)- nor (−)-enantiomers altered glutamate-mediated excitatory synaptic currents. At 10 μM, (+)-DHP and (+)-ACN depressed excitatory responses in a bicuculline-sensitive fashion, suggesting that direct chloride channel gating by the steroids contributed to the depression. These data indicate that certain steroids and benz[e]indenes augment inhibitory synaptic transmission enantioselectively and provide strong support for the hypothesis that steroids act at specific sites on synaptic GABA_A receptors rather than via alteration of membrane lipids. Synapse 29:162–171, 1998. © 1998 Wiley-Liss, Inc.