Differences in the Cs block of baclofen and 4-aminopyridine induced potassium currents of guinea pig CA3 neurons in vitro

Single-electrode current- and voltage-clamp techniques were employed to study responses elicited by (?)baclofen or γ-aminobutyric acid (GABA) and 4-aminopyridine (4-AP) induced inhibitory postsynaptic potentials in CA3 pyramidal neurons in guinea pig hippocampal slices. All drugs were applied by the bath to submerged slices in which fast synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM), bicuculline (50 μM), and picrotoxin (50 μM). (?)Baclofen (0.5 μM) and GABA (1 μM) induced equivalent-sized hyperpolarizations and input resistance decreases. The agonist induced hyperpolarization or current and 4-AP induced hyperpolarizations or currents (4-AP induced K-IPSPs or IPSCs) reversed in sign near the K-equilibrium potential (E_K). The GABA_B receptor antagonists, OH-saclofen (500 μM) and CGP 35348 (100 μM), reduced (?)baclofen responses, and 4-AP induced K-IPSPs, suggesting that they were mediated by GABA_B receptors. Intracellular tetraethylammonium-, and extracellular barium-ions (1 μM) diminished the (?)baclofen induced current and 4-AP induced K-IPSCs. Intracellular Cs-ions blocked the (?)baclofen induced outward current at resting membrane potential but did not grossly affect the inward current recorded at membrane potentials negative to E_K. 4-AP induced inwardly or outwardly directed KIPSCs were not blocked by intracellular Cs-ions. Extracellular Cs-ions (5 μM) blocked the (?)baclofen induced inward K-current, but did not block 4-AP induced inwardly directed K-IPSCs. In conclusion, we found differences in the Cs block of K-channels activated by (?)baclofen or the endogenous transmitter GABA. One reason could be that (?)baclofen predominantly activated extra synaptic GABA_B receptors provided that extrasynaptic and subsynaptic receptors couple to different potassium channels. © 1994 Wiley-Liss, Inc.     

Pharmacological characterization of GABAB-mediated responses in the CA1 region of the rat hippocampal slice.

It is generally accepted that the bicuculline-resistant responses to GABA are mediated through the activation of GABAB receptors that mediate a slow IPSP. However, a number of reported observations are difficult to reconcile with this model. Specifically, GABAB antagonists only partially block bicuculline-resistant GABA responses, and both 4-aminopyridine (4-AP) and carbachol have been reported to block responses to the selective GABAB agonist baclofen, but not GABA itself. Thus, it has been argued that baclofen and GABA increase potassium conductance through separate receptor mechanisms. This suggestion is not easily reconcilable with the postulated physiological role of GABAB receptors in mediating the slow IPSP. We have addressed these discrepancies by using the new GABAB antagonists 2-hydroxy-saclofen (2-OH-SAC) and CGP 35348 in the presence of the GABA uptake inhibitor SKF 89976A. The weak antagonism of 2-OH-SAC against the bicuculline-resistant GABA response was improved when the GABA uptake was inhibited with SKF 89976A, allowing for the application of lower GABA concentrations. Under these circumstances, 2-OH-SAC and CGP 35348 strongly antagonized GABA and baclofen responses, but did not have any effect on outward currents evoked by 5-HT. The slow IPSP evoked in the presence of glutamate antagonists was reversibly inhibited by CGP 35348 (IC50 = 14 microM), without affecting the fast IPSP. Carbachol (0.3-20 microM) had no effect on outward currents evoked by either baclofen or GABA. 4-AP (5 microM to 1 mM), despite causing a large increase in cell excitability, did not change baclofen responses. Higher concentrations of 4-AP (5 mM) induced inward current, and reduced both baclofen and GABA outward currents to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro studies of the role of gamma-aminobutyric acid in inhibition in the lateral septum of the rat

Focal stimulation, stimulation of the fimbria, and stimulation of the medial septal area result in an inhibitory postsynaptic potential (IPSP) in lateral septal neurons. Increased stimulus intensity results in the appearance of a late hyperpolarizing potential (LHP). Treatment of the slice with bicuculline methiodide or picrotoxin results in blockade of the IPSP. When present, LHPs are enhanced in the presence of bicuculline or picrotoxin. Spontaneous and evoked IPSPs reverse near -70 mV, and LHPs reverse near -90 mV. Iontophoretic application of gamma-amino-butyric acid (GABA) results in hyperpolarizing, depolarizing, or biphasic potentials. Treatment with bicuculline or picrotoxin results in depression of biphasic GABA responses that appears selective for the depolarizing portion of the potential. At high concentrations of bicuculline, a portion of the hyperpolarizing GABA potential persists. The reversal potential of the depolarizing GABA potential is near -30 mV, and the reversal potential of monophasic hyperpolarizing GABA potential is near -70 mV. The bicuculline-resistant hyperpolarizing GABA response has a reversal potential near -90 mV. GABA activates three separate conductances on septal neurons, which are similar to those reported on hippocampal neurons. The resistance of the hyperpolarizing GABA potential to bicuculline appears to be due to the presence of a GABA-activated potassium conductance, which is similar to that activated by baclofen.     

Shunting and hyperpolarizing GABAergic inhibition in the high-potassium model of ictogenesis in the developing rat hippocampus

Ontogenesis of GABAergic signaling may play an important role in developmental changes in seizure susceptibility in the high-potassium model of ictogenesis in vitro. The age-dependent effects of [K(+)](o) on the reversal potential of the GABA(A)-mediated responses and membrane potential in hippocampal slices in vitro were compared with the effect of GABA(A)-receptors antagonists and GABA(A) modulators on high-potassium induced seizures in the CA3 pyramidal layer of rat hippocampus in vivo. GABA(A) responses were depolarizing at P8-12 and hyperpolarizing at P17-21. In P8-12 rats, GABA(A) responses switch their polarity from depolarizing to hyperpolarizing upon elevation of extracellular potassium. At approximately 10 mM [K(+)](o), activation of GABA(A) receptors produced an isoelectric, purely shunting response characterized by no changes in the membrane potential but an increase in the membrane conductance. In P17-21 rats, the hyperpolarizing GABA(A) driving force progressively increased with elevation of [K(+)](o). In P8-12 rats in vivo, GABA(A)-receptor antagonists did not affect the occurrence of ictal discharges induced by intrahippocampal injection of 10 mM [K(+)](o), but significantly increased seizure duration. Diazepam and isoguvacine completely prevented seizures induced by 10 mM [K(+)](o). In P17-21 rats, GABA(A)-receptor antagonists strongly increased the occurrence of ictal activity induced both by 10 mM [K(+)](o). Taken together, these results suggest that anticonvulsive effects of GABA are because of the combination of shunting and hyperpolarizing actions of GABA. Although shunting GABA is already efficient in the young age group, a developmental increase in the hyperpolarizing GABA(A) driving force likely contributes to the increase in the GABAergic control of seizures upon maturation.     

Excitatory effect of Cd2+ on cat adrenal chromaffin cells.

To understand the mechanism(s) underlying the Cd2+- and Co2+-induced increases in the cytosolic free Ca2+ concentration ([Ca]i) in cat adrenal chromaffin cells, we used nystatin-perforated patch recording method and fura-2 microfluorometry. Under the current-clamp conditions, the external application of 5x10(-7) M Cd2+ slowly depolarized the cells resulting in the bursting of action potentials. Under the voltage-clamp conditions, Cd2+ evoked a slow inward current accompanied by a decrease of K+ conductance at a holding potential of -40 mV, and Co2+ mimicked Cd2+ action. In some cells (16%), Cd2+ evoked an additional rapid transient outward current associated with an increased K+ conductance and a successive slow inward current. The Cd2+-induced inward current was activated in a concentration-dependent manner with a half-maximum concentration of 9.3x10(-8) M. The Cd2+- and Co2+-induced [Ca]i increases measured with fura-2 microfluorometry were maximal at 10(-6) and 10(-5) M, respectively, and the higher concentrations of both cations caused the smaller responses. Additional transient increase in [Ca]i was often evoked upon the removal of relatively higher concentrations of these metals. It was concluded that the Cd2+-induced membrane depolarization due to the decrease in K+ conductances evoked the bursting firings resulting in the increase in [Ca]i, and consequently might stimulate the catecholamine secretion.     

Differential effects of zinc on hyperpolarizing and depolarizing GABAA synaptic potentials in hippocampal slice cultures

We have examined the changes in GABA_A-mediated synaptic potentials recorded from CA3 pyramidal neurons in hippocampal slice cultures following application of zinc (Zn²⁺). Unlike 4-AP, Zn²⁺ did not enhance fast hyperpolarizing potentials but primarily enhanced depolarizing GABA_A potentials. Zn²⁺ did not alter the postsynaptic response of pyramidal neurons to pressure applied GABA, consistent with previous reports that Zn²⁺ enhances the release of GABA from presynaptic terminals. To examine the role of local circuitry in the production of Zn²⁺ responses, we recorded from cultures maintained for 7–10 days following removal of the dentate and hilus to allow complete degeneration of the mossy fibers (DGX cultures). Zn²⁺ produced giant depolarizing potentials (GDPs) in DGX cultures that were identical to those in intact cultures. In contrast, the 4-AP response was dramatically altered in DGX cultures. In DGX cultures, Zn²⁺ co-applied with 4-AP appeared to inhibit the production of fast hyperpolarizing GABA_A synaptic potentials produced by 4-AP alone. This inhibition of fast hyperpolarizing potentials suggests that Zn²⁺ may reduce the release of GABA onto pyramidal cell somata. These observations suggest that Zn²⁺ enhances GABA release from local circuit neurons that synapse onto pyramidal cell dendrites, and inhibits GABA release onto pyramidal cell somata.     

Effects of GABA and baclofen on pyramidal cells in the developing rabbit hippocampus: an 'in vitro' study

Using the in vitro hippocampal slice preparation, we have investigated the effects of gamma-aminobutyric acid (GABA) and its analogue beta-(p-chlorophenyl)-GABA (baclofen) on CA1 and CA3 pyramidal cells in the developing rabbit hippocampus. Somatic applications: both GABA and baclofen, when applied to CA1 pyramidal cells from immature tissue, led to cell depolarization from resting membrane potential; this baclofen depolarization may be indirectly mediated. In contrast, CA3 pyramidal cells at the same age were primarily hyperpolarized by both drugs. In mature tissue, both GABA and baclofen applied at the soma induce cell hyperpolarizations. Dendritic applications: immature CA1 cells responded to dendritic GABA and baclofen application with depolarizations associated with increased cell excitability; here, too, the baclofen depolarization may be due to indirect 'disinhibition'. Both depolarizing and hyperpolarizing responses were recorded in immature tissue when GABA was applied to CA3 pyramidal cell dendrites: baclofen produced only hyperpolarizations. In mature CA1 cells, dendritic GABA application produced membrane depolarization, but dendritic baclofen application produced hyperpolarizations. In mature CA3 cells, dendritic GABA and baclofen application produced predominant hyperpolarizations. Mature CA1 pyramidal cells appear to retain some of the GABA-induced depolarizations characteristic of immature tissue. In contrast, mature CA3 neurons show only hyperpolarizing responses to GABA and baclofen application. In all cases, responses to GABA and baclofen are associated with a decrease in cell input resistance. We conclude that the GABAergic receptor/channel complexes mature differently in the CA1 and CA3 regions of the hippocampus.     

Temperature dependence of intrinsic membrane properties and synaptic potentials in hippocampal CA1 neurons in vitro

The temperature dependence of intrinsic membrane conductances and synaptic potentials in guinea pig hippocampal CA1 pyramidal neurons were examined in vitro as they were cooled from 37 degrees C to between 33 and 27 degrees C. Cooling reversibly increased resting input resistance in a voltage-independent manner (Q10 = 0.58 to 0.75). The amplitude and duration of orthodromically evoked action potentials were increased by cooling (Q10 = 0.87 and 0.52 to 0.53, respectively), whereas the maximum rates of rise and fall were reduced (Q10 = 1.27 to 1.49 and 2.19 to 2.44, respectively). The amplitude and duration of the afterhyperpolarization which follows a directly evoked train of action potentials were substantially increased at low temperatures. It is possible to attribute this increase to an augmentation of Ca2+ influx during the train and also to a slowing of Ca2+ removal from the cytoplasm. Spike frequency adaptation during prolonged depolarizing pulses was enhanced at low temperatures. In addition, there was a decrement in spike amplitude during the train of action potentials. These observations all suggest an increase in Ca2+-activated K+ conductance at low temperature. A late, slow, hyperpolarizing synaptic potential in response to orthodromic stimulation became apparent at low temperature. This potential had an apparent reversal potential more negative than the early inhibitory postsynaptic potential, suggesting that it was mediated by a K+ conductance, possibly activated by Ca2+ influx. We conclude that reductions in temperature of as little as 5 to 10 degrees C from normal can significantly alter the intrinsic and synaptic physiology of hippocampal neurons and should, therefore, be considered an important variable in in vitro brain slice experiments.     

Distinct types of ionic modulation of GABA actions in pyramidal cells and interneurons during electrical induction of hippocampal seizure-like network activity

It has recently been shown that electrical stimulation in normal extracellular fluid induces seizure-like afterdischarge activity that is always preceded by GABA-dependent slow depolarization. These afterdischarge responses are synchronous among mature hippocampal neurons and driven by excitatory GABAergic input. However, the differences in the mechanisms whereby the GABAergic signals in pyramidal cells and interneurons are transiently converted from hyperpolarizing to depolarizing (and even excitatory) have remained unclear. To clarify the network mechanisms underlying this rapid GABA conversion that induces afterdischarges, we examined the temporal changes in GABAergic responses in pyramidal cells and/or interneurons of the rat hippocampal CA1 area in vitro. The extents of slow depolarization and GABA conversion were much larger in the pyramidal cell group than in any group of interneurons. Besides GABA(A) receptor activation, neuronal excitation by ionotropic glutamate receptors enhanced GABA conversion in the pyramidal cells and consequent induction of afterdischarge. The slow depolarization was confirmed to consist of two distinct phases; an early phase that depended primarily on GABA(A)-mediated postsynaptic Cl- accumulation, and a late phase that depended on extracellular K+ accumulation, both of which were enhanced by glutamatergic neuron excitation. Moreover, extracellular K+ accumulation augmented each oscillatory response of the afterdischarge, probably by further Cl- accumulation through K+-coupled Cl- transporters. Our findings suggest that the GABA reversal potential may be elevated above their spike threshold predominantly in the pyramidal cells by biphasic Cl- intrusion during the slow depolarization in GABA- and glutamate-dependent fashion, leading to the initiation of seizure-like epileptiform activity.     

Evidence for the involvement of G-proteins in the generation of the slow poststimulus afterdepolarisation (sADP) induced by muscarinic receptor activation in rat olfactory cortical neurones in vitro

The involvement of G-proteins in generating the slow poststimulus afterdepolarising potential (sADP) induced by muscarinic receptor activation in immature (P10-20) rat olfactory cortical brain slice neurones was investigated under whole-cell patch clamp, using GTP-gamma-S (G-protein activator) or GDP-beta-S (G-protein blocker)-filled electrodes. In control experiments using K methylsulphate electrodes, cell resting potential (V(m)) and spike firing properties were unaffected over 10-15 min recording, although input resistance (R(N)) was slightly increased ( approximately 14%). Oxotremorine-M (OXO-M; 10 microM) produced a reversible slow depolarisation, an increase in R(N) ( approximately 90%) and induction of a slow poststimulus inward tail current (I(ADP)) (measured under voltage clamp at -60 mV) that was sustained during drug exposure (up to 15 min); the amplitude of slow inward rectifier (I(h)) currents activated from -50 mV were also apparently increased. By contrast, in GTP-gamma-S-loaded cells, R(N) was consistently decreased ( approximately 22%) and spike firing threshold (V(th)) was raised ( approximately 5 mV) after 10 min recording. In approximately 60% of loaded cells, a persistent muscarinic slow inward current and I(ADP) were induced by OXO-M; I(h) relaxation amplitude was also significantly decreased. The effects of GTP-gamma-S on R(N), V(th) and I(h) were partly counteracted by adding Ba(2+) (100 microM) to the bathing medium or mimicked by adding baclofen (GABA(B) receptor agonist; 100 microM) to normally-recorded cells. Intracellular GDP-beta-S (up to 30 min) had no effect on cell membrane properties or I(h), but irreversibly blocked the muscarinic slow inward current and I(ADP) induced by OXO-M. We conclude that both muscarinic responses require G-protein-linked transduction mechanisms for their generation.     

gamma-Aminobutyric acid (GABA) causes consistent depolarization of neurons in the guinea pig supraoptic nucleus due to an absence of GABAB recognition sites

The action of gamma-aminobutyric acid (GABA) in the supraoptic nucleus was investigated using guinea pig brain slices. GABA produced a membrane depolarization accompanied by a decrease in the input resistance. The action of GABA was concentration-dependent throughout a wide range of concentrations (10(-7)-10(-3) M). In none of the cells examined, a membrane hyperpolarization was observed. The reversal potential for the depolarization induced by GABA was about 25 mV positive to the resting membrane potential. The amplitude of the GABA-induced depolarization was increased to 1.5 X the control by reducing the external Cl- from 134.2 mM to 10.2 mM. The action of GABA was readily antagonized by relatively low concentrations of bicuculline (10(-5) M). The action of GABA in the hippocampus or in the anterior hypothalamus was markedly different from that in the supraoptic nucleus, i.e. GABA produced both depolarizing and hyperpolarizing responses in the hippocampus and consistently a hyperpolarization in the anterior hypothalamus. The depolarizing but not the hyperpolarizing response in the hippocampus was selectively blocked by picrotoxin (2 X 10(-5) M) or by bicuculline (10(-5) M). The depolarizing component was dependent on the external Cl- concentration and had a reversal potential similar to that of the depolarization induced by GABA in the supraoptic nucleus. The hyperpolarizing component was resistant to bicuculline and had a reversal potential about 30 mV negative to the resting membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of neurokinin receptors modulates K and Cl channel activity in cultured astrocytes from rat cortex

Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP-dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch-clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential.     

Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area

We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.     

In vitro electrophysiology of rat subicular bursting neurons

Intracellular recordings were used to study the electrophysiological properties of rat subicular neurons in a brain slice preparation in vitro. Cells were classified as bursting neurons (n = 102) based on the firing pattern induced by depolarizing current pulses. The bursting response recorded at resting membrane potential (−66.1 ± 6.2 mV, mean ± SD n = 94) was made up of a cluster of fast action potentials riding on a slow depolarization and was followed by an afterhyperpolarization. Tonic firing occurred at a membrane potential of approximately −55 mV. A burst also occurred upon termination of a hyperpolarizing current pulse. Tetrodotoxin (TTX, 1 μM) blocked the burst and decreased or abolished the underlying slow depolarization. These effects were not induced by the concomitant application of the Ca²⁺ channel blockers Co²⁺ (2 mM) and Cd²⁺ (1 mM). Subicular bursting neurons displayed voltage- and time-dependent inward rectifications of the membrane during depolarizing and hyperpolarizing current pulses. The inward rectification in the depolarizing direction was abolished by TTX, while that in the hyperpolarizing direction was blocked by extracellular Cs⁺ (3 mM), but not modified by Ba²⁺ (0.5–1 mM), TTX, or Co²⁺ and Cd²⁺. Tetraethylammonium (10 mM)-sensitive, outward rectification became apparent in the presence of TTX. These results suggest that neurons in the rat subiculum can display voltage-dependent bursts of action potentials as well as membrane rectification in the depolarizing and hyperpolarizing directions. These results also indicate that activation of a voltage-gated Na⁺ conductance may be instrumental in the initiation of bursting activity. Hippocampus 7:48–57, 1997. © 1997 Wiley-Liss, Inc.     

Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation

The electrical membrane properties of rat substantia nigra pars compacta (SNC) neurons were studied in an in vitro slice preparation. Some of the recorded neurons were intracellularly labeled with HRP and were found to have morphological characteristics resembling the presumed SNC dopaminergic neurons, as reported by others. The input resistance of SNC neurons at resting membrane potential ranged between 70 and 250 M omega. The membrane resistance showed strong anomalous rectification when the membrane was hyperpolarized by current injection. The anomalous rectification was decreased by the addition of tetraethylammonium bromide (TEA) to the bathing Ringer solution. Injection of depolarizing current or termination of hyperpolarizing current induced slow depolarizing potentials. Their amplitude was dependent on the membrane potential and the current intensity. In neurons treated with tetrodotoxin (TTX) and TEA, slow action potentials were triggered from the slow depolarizing potentials. Both the slow depolarizing potential and slow action potential were TTX resistant and abolished by superfusion of Ca2+-free medium. Long duration hyperpolarizations were observed following the injection of depolarizing current pulses. The hyperpolarization was abolished by the superfusion of Ca2+-free medium or decreased by addition of TEA to the Ringer solution indicating an involvement of a Ca2+-dependent K+-conductance in generation of the hyperpolarization. The long duration hyperpolarization was also observed following action potentials. The spike after hyperpolarization consisted of an initial short duration fast component and a long lasting component. The amplitude of both components seems to be reduced but not abolished by TEA (up to 10 mM). When hyperpolarizing current pulses were applied to neurons that were held either continuously depolarized or were superfused with Ca2+-free medium, the pattern of the membrane potential after the offset of current pulses consisted of an initial fast and a later slow ramp-shaped phase. The latter was associated with a membrane conductance increase and interpreted to be due to an early K+ current. This early K+ current was relatively resistant to TEA. Injections of strong depolarizing currents triggered action potentials with multiple inflections on their rising phase. The amplitudes of action potentials changed abruptly during current application. These data indicate that SNC neurons have multiple generation sites for action potential.     

Effects of acute and chronic ethanol treatment on pre- and postsynaptic responses to baclofen in rat hippocampus

Interactions between the GABA_B receptor and acute or chronic ethanol treatment were studied using extracellular and intracellular electrophysiological recording techniques. Bath application of the GABA_B receptor agonist, (−)-baclofen (0.1–100 μM) induced concentration-dependent inhibition of extracellularly recorded dendritic excitatory postsynaptic potentials (EPSPs) in the CA1 region of hippocampal slices. Responses to baclofen were unchanged relative to control either by simultaneous application of ethanol (10–60 mM) or by previous chronic ethanol exposure. The membrane potential of CA1 pyramidal neurons was reversibly hyperpolarized an average of 5 mV by pressure ejection of baclofen (1 mM). Bath application of ethanol (30 mM) alone occasionally caused a small depolarization of resting membrane potentials in CA1 neurons but failed to increase hyperpolarizing responses to pressure-ejected baclofen. However, in slices from chronic ethanol-treated animals hyperpolarizing responses to bath-applied baclofen (10 μM) were reduced by approximately 30% relative to controls. These results suggest that GABA_B-mediated responses in CA1 hippocampal pyramidal neurons are relatively resistant to the acute effects of ethanol, but that continuous exposure to ethanol sufficient to induce physical dependence may evoke an adaptive reduction in some GABA_B receptor mediated responses.     

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons

The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage- and current-clamp whole-cell techniques. The resting potential of SCG cells varied from -47 to -80 mV (-58.3 +/- 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 +/- 0.7 mV, n = 22) and a hyperpolarisation (7.2 +/- 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 +/- 0.2 mV, n = 8). Inhibition of voltage-gated currents unmasked a voltage-independent resting conductance reversing at -50 mV. The reversal potential of the voltage-independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage-independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage-independent conductance, with voltage-activated M and h currents playing a prominent stabilising role.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

Ionic mechanism of 4-aminopyridine action on leech neuropile glial cells

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K+ channels, but also induced Na+ and Ca2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was -5 mV. Application of 5 mM Ni2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K+ and Cl- channels and 4-AP-induced Ca2+ influx could activate Ca2+-sensitive K+ and Cl- channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K+ conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.     

Activation of a non-specific cation conductance by intracellular injection of inositol 1,3,4,5-tetrakisphosphate into identified neurons of Aplysia

The ionic mechanism of the effect of intracellularly injected inositol 1,3,4,5-tetrakisphosphate (IP4) on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure injection, and ion-substitution techniques. Intracellular injection of IP4 into a neuron voltage-clamped at -45 mV reproducibly induced a slow inward current (20-60 s in duration, 3-5 nA in amplitude) associated with a conductance increase. The current was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential was -21 mV. The IP4-induced inward current was sensitive to changes in the external Na+, Ca2+ and K+ concentration but not to changes in Cl- concentration, and was resistant to tetrodotoxin (50 microM). When the cell was perfused with tetraethylammonium (5 mM) but not with 4-aminopyridine (5 mM), the IP4-induced inward current recorded at -45 mV slightly increased. The IP4-induced inward current was partially reduced by calcium channel blockers (Co2+ and Mn2+). These results suggest that intracellularly injected IP4 can activate a non-specific cation conductance.