On the mechanism of GABA-induced currents in cultured rat cortical neurons

 We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by γ-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl^– concentration ([Cl^–]_i). In current-clamp experiments in the presence of 26 mM HCO₃ ^– the application of 50 μM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50–200 μM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO₃ ^–. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO₃ ^–-free saline, indicating a contribution from HCO₃ ^–. The reversal potential of the GABA-induced currents (E _GABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO₃ ^– was –73 mV, while it was shifted to –59 mV in the presence of HCO₃ ^–. Combined patch-clamp and microfluorimetric measurements using the Cl^–-sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl^–]_i in 54% (n=13) of the neurons. We conclude that this increase of [Cl^–]_i in combination with the efflux of HCO₃ ^– results in a shift of E _GABA above the resting membrane potential that gives rise to GABA-mediated depolarizations. Received: 2 June 1998 / Received after revision: 4 August 1998 / Accepted: 10 September 1998     

Two classes of spontaneous GABA-mediated miniature synaptic currents in cultured rat hippocampal neurons.

Amplitude and time course of spontaneous gamma-aminobutyric acid (GABA)-mediated miniature postsynaptic currents (MPSCs), recorded in cultured embryonic hippocampal neurons in presence of either tetrodotoxin (TTX) or increased external [Mg2+/Ca2+] ratio, revealed that they form two classes. The distribution of the most commonly recorded MPSCs was skewed both in terms of peak amplitude and rise-time (skew-MPSCs, mode: 70-120 pS). Another, less frequent class (mode: 1-3 nS) formed bell-shaped (bell-MPSCs) amplitude and rise-time distributions. MPSC initial slope did not correlate with rise time, indicating that smaller MPSCs were not electrotonically attenuated. Bell-MPSCs did not result from the integration of skew-MPSCs and both classes appeared to be composed of subunits.     

Valproate and sodium currents in cultured hippocampal neurons

Cultured rat hippocampal neurons with short processes were investigated using the whole cell voltage clamp under conditions appropriate for isolating Na⁺ currents. After incubation of the neuron culture for a period of 15–30 min in 1 mM sodium valproate, several parameters of the Na⁺ current were changed. The peak Na⁺ conductance g _p, measured using hyperpolarizing prepulses, was reduced by valproate in a voltage-dependent manner. In the membrane voltage range from -30 to +20 mV, this reduction showed a linear dependence on voltage, increasing from about zero to approximately 30% of g _p, the maximum peak Na⁺ conductance of the neuron. At the holding voltage of -70 mV, the inactivation parameter h _t8 decreased from 0.88 in the control to 0.64 in the valproate solution. This reduction originated mainly from a 10 mV shift in the sigmoid relation between h _t8 and membrane voltage along the voltage axis to hyperpolarizing potentials. The decay of the maximum peak Na⁺ current (inactivation) could be fitted by a biexponential function. Time constants of the fast and slow component at -20 mV decreased in valproate by about 50%. Valproate also retarded the recovery from inactivation, as determined at the holding voltage. The sigmoid recovery from inactivation could reasonably be described by an exponential function with time constant _r and delay time t. Both _r and At increased more than 200% in valproate. Our results indicate that valproate affected the Na⁺ current in hippocampal neurons in a way that contributed to a considerable depression of Na⁺ reactivation. This explains the frequency-dependent inhibition of action potentials as observed in mammalian central nervous tissue and may be the principal action of the anticonvulsant.     

Membrane currents induced by L-homocysteic acid in mouse cultured hippocampal neurons.

The concentration-response relationship of membrane currents induced by L-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture (n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3-100 microM L-homocysteic acid which desensitized with a time-constant greater than 1 s in a concentration-dependent manner and were antagonized by 30 microM D-L-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl-D-aspartate receptors. At higher concentrations of L-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM L-homocysteic acid maximum concentration tested) and were not blocked by D-L-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl-D-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to L-homocysteic acid was positively related to concentration and the responses were partially blocked by D-L-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of L-homocysteic acid at concentrations 30 microM up to 300 microM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by D-L-2-amino-5-phosphonovaleric acid (50-100 microM) suggesting that it was produced by activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

ASIC-like, proton-activated currents in rat hippocampal neurons

The expression of mRNA for acid sensing ion channels (ASIC) subunits ASIC1a, ASIC2a and ASIC2b has been reported in hippocampal neurons, but the presence of functional hippocampal ASIC channels was never assessed. We report here the first characterization of ASIC-like currents in rat hippocampal neurons in primary culture. An extracellular pH drop induces a transient Na⁺ current followed by a sustained non-selective cation current. This current is highly sensitive to pH with an activation threshold around pH 6.9 and a pH_0.5 of 6.2. About half of the total peak current is inhibited by the spider toxin PcTX1, which is specific for homomeric ASIC1a channels. The remaining PcTX1-resistant ASIC-like current is increased by 300 μ m Zn²⁺ and, whereas not fully activated at pH 5, it shows a pH_0.5 of 6.0 between pH 7.4 and 5. We have previously shown that Zn²⁺ is a co-activator of ASIC2a-containing channels. Thus, the hippocampal transient ASIC-like current appears to be generated by a mixture of homomeric ASIC1a channels and ASIC2a-containing channels, probably heteromeric ASIC1a+2a channels. The sustained non-selective current suggests the involvement of ASIC2b-containing heteromeric channels. Activation of the hippocampal ASIC-like current by a pH drop to 6.9 or 6.6 induces a transient depolarization which itself triggers an initial action potential (AP) followed by a sustained depolarization and trains of APs. Zn²⁺ increases the acid sensitivity of ASIC channels, and consequently neuronal excitability. It is probably an important co-activator of ASIC channels in the central nervous system.     

Pharmacological characterization of voltage-dependent calcium currents in rat hippocampal neurons.

The kinetics and pharmacology of voltage-dependent calcium (Ca) currents in primary cultures of hippocampal neurons were studied using the whole cell clamp technique. The low voltage-activated (LVA) Ca current was activated at -50 mV and completely inactivated within 100 ms. This current was insensitive to omega-conotoxin (omega-CgTx) and to the calcium agonist Bay K 8644. The high-voltage-activated (HVA) Ca current was activated at -20 mV and inactivated incompletely during pulses of 200 ms duration. The snail toxin omega-CgTx revealed two pharmacological components of the HVA Ca current, one irreversibly blocked and the other insensitive to the toxin. Bay K 8644 had a clear agonistic action mainly on the omega-CgTx insensitive component of the HVA Ca current.     

Non-competitive inhibition of GABA currents by phenothiazines in cultured chick spinal cord and rat hippocampal neurons

The gamma-aminobutyric acid (GABA) inhibiting properties of several classes of antipsychotic medications were studied using gigaseal whole-cell voltage-clamp techniques in cultured chick spinal cord and rat hippocampal neurons. At doses above 1 microM trifluoperazine, chlorpromazine and thioridazine blocked GABA currents in a non-competitive fashion decreasing the maximal transmitter response without altering the half-maximal effective concentration. In contrast, haloperidol was ineffective against GABA at concentrations up to 100 microM. Among the agents studied trifluoperazine was the most potent GABA inhibitor with half maximal effect at 12 microM. Trifluoperazine (100 microM) also inhibited glycine-gated chloride currents in spinal cord neurons to an extent comparable to GABA (85 +/- 6% inhibition) but reduced glutamate currents by less than 35% in either spinal cord or hippocampal neurons.     

Voltage-dependent GABA-induced modulation of calcium currents in chick sensory neurons

Externally applied gamma-aminobutyric acid (GABA) quickly and reversibly reduces by 60% voltage activated Ca2+ currents in chick dorsal root ganglion cells. This action is antagonized by depolarization, with characteristic time and voltage requirements. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) mimicks and blocks the GABA effect, respectively. A 3-state model describing the reactions involved is proposed.     

Two types of kainate response in cultured rat hippocampal neurons.

1. Two different types of kainate response were recorded in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. There was an outward rectification in the current-voltage (I-V) plot of the kainate-induced current (type I response) in relatively large neurons bearing a morphological resemblance to young pyramidal cells. In smaller neurons with elliptical somata and fine neurites, the kainate response was characterized by a remarkable inward rectification in the I-V plot of the kainate-induced current and a significant permeability to Ca2+ (type II response). 3. Both type I and type II responses were negligible below 2 microM and almost saturated at 500 microM kainate. The concentrations producing half-maximal responses and the Hill coefficients were 68 microM and 1.76 and 56 microM and 1.21 for type I and type II responses, respectively. Both responses were suppressed similarly by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 4. The mean single-channel conductance (gamma) of the type II kainate response was estimated, from the relation between the whole-cell mean currents and current variances, to be 8.7 pS. The power spectrum for the current noise was fitted with the sum of two Lorentzians with cutoff frequencies (fc) of 61.1 +/- 1.4 and 327.8 +/- 10.5 Hz (n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual-component miniature excitatory synaptic currents in rat hippocampal CA3 pyramidal neurons.

1. Spontaneous miniature synaptic events were studied with tight-seal whole-cell recordings from CA3 neurons maintained in the hippocampal slice from immature rats (3-15 days). CA3 neurons suffer a constant, high-frequency barrage of inhibitory synaptic input. When inhibitory postsynaptic currents were suppressed by bicuculline, a smaller contribution from excitatory synapses was revealed. 2. Addition of tetrodotoxin (TTX) removed a persistent inward current and substantially reduced the baseline noise facilitating the detection of "miniature" excitatory currents. Addition of hyperosmotic media increased the frequency of spontaneous excitatory postsynaptic currents (EPSCs). 3. Under both physiological and elevated potassium conditions, individual spontaneous miniature EPSCs (10-30 pA amplitude) were composed of components mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors as determined by their voltage dependence, time course, and sensitivity to selective antagonists. 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX) or D-2-amino-5-phosphonovaleric acid (D-APV) shifted the amplitude distribution of miniature EPSCs to a smaller mode at both +40 mV and -40 mV. Similar to EPSCs recorded in CA1 neurons, the rise and decay times of the NMDA receptor component were slower than those of the non-NMDA component. The time course of the non-NMDA component was voltage independent. 4. In 13 of 21 neurons, no correlation existed between individual EPSC rise times and their corresponding halfwidth, peak amplitude, or decay time constant. This suggests that the large range of EPSC kinetics observed in each individual neuron was not due solely to cable attenuation of EPSCs widely distributed over the dendritic tree. Plots of the mean EPSC rise time against mean halfwidth for each cell, however, revealed a striking correlation, suggesting that in neonates, active synapses may be grouped in a restricted region of the dendritic tree and as such are subject to similar amounts of dendritic filtering. 5. The electrotonic length of CA3 neurons (L = 0.52) predicted that at this maturity the electrotonic compactness of the neuron facilitated voltage control over all but the most distal synapses. The reversal potential of the fast component of spontaneous events was close to 0 mV, whereas the reversal potential of exogenously applied kainate and NMDA was more positive. This discrepancy likely reflects a compromise of the voltage clamp by the activation of conductances distributed over the entire cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

Chloride permeable intermediate conductance single channel events activated on patch excision were found in outside-out patches from cultured postnatal hippocampal neurons. A majority of the channels had a conductance of 83 ± 2.1 pS when recorded in a symmetrical TEAC1 solution. Two other populations of channels with conductance values of 62 ± 2.1 pS and 145 ± 1.9 pS were also observed. The reversal potentials for these intermediate conductance Cl⁻ channels coincided with that of the GABA activated channels. The channels characteristically appeared 5–15 min after patch excision, suggesting that these channels may be blocked by some diffusible factors under physiological conditions. Based on the measurements of channel burst durations while the channel was partially blocked, and the channel open times after complete relief from the block, the mechanism of blockade does not appear to be a simple open channel blockade. The high prevalence and its potential regulation by cytosolic factors suggest an important physiological role for these Cl⁻ channels coupling neuronal excitability with cellular metabolism.     

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.     

Speeding of miniature excitatory post-synaptic currents in Ts65Dn cultured hippocampal neurons

Down syndrome (DS) is the leading non-heritable cause of mental retardation and is due to the effects of an extra chromosome 21. Mouse models of DS have been developed which parallel many of the cognitive and behavioral deficits of DS individuals. Of these, Ts65Dn mice show abnormal hippocampal properties including learning and memory deficits, altered synaptic plasticity and irregular dendritic spines. We assessed synaptic function of cultured postnatal Ts65Dn hippocampal neurons through examination of spontaneous miniature excitatory post-synaptic currents (mEPSCs) and compared them to those from diploid neurons. Averaged amplitudes and frequency of mEPSC events were similar to diploid suggesting presynaptic function is not overtly disrupted in Ts65Dn hippocampal neurons. However, both averaged decay and rise times (10-90% of peak) were significantly faster (approximately 20% for both rise and decay) in Ts65Dn neurons compared to diploid. The distribution of both decay and rise times, indicates global scaling of all percentile groups and is independent of amplitude suggesting normal electrotonic filtering in spite of abnormal expression of GIRK2 channel in Ts65Dn mouse. Western blot analysis suggests overexpression of GluR4 subunit of AMPA receptors which may contribute to faster mEPSC in Ts65Dn neurons. Intrinsic synaptic properties influenced by genetics or epigenetics factors in Ts65Dn postnatal cultured neurons are therefore disrupted and may contribute to the cognitive deficits associated with DS.     

Two types of acid-sensing ion channel currents in rat hippocampal neurons

Two types of acid-sensing ion channel (ASIC)-like currents in cultured rat hippocampal neurons were recorded and their characteristics were studied by using a whole-cell recording technique. The results revealed that the ASIC-like currents, induced by a quick drop of the extracellular pH, decayed with different time constants (τ) of 229 ± 16 (Type I) and 1209 ± 56 ms (Type II). The ASIC-like currents displayed different sensitivities to extracellular proton (pH_0.5 was 6.17 ± 0.04 for Type I and 5.70 ± 0.07 for Type II) and amiloride, a specific ASIC channel blocker (IC₅₀ was 1.19 ± 0.37 μM for Type I and 0.14 ± 0.02 μM for Type II). Among all the 360 recorded neurons, ASIC-like currents were induced in 314 neurons (87.2%). In the neurons expressing ASICs, Type I currents were evoked from 269 neurons (85.7%) and Type II currents were induced only from 45 neurons (14.3%). As these ASIC-like currents presented various electrophysiological and pharmacological properties, further experiments should be conducted to decipher the complex subunit composition of ASICs in the hippocampus.     

Molecular correlates of the M-current in cultured rat hippocampal neurons

M-type K⁺ currents ( I _K(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K⁺ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I _K(M) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I _K(M) with an IC₅₀ of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed I _K(M) with an IC₅₀ of 0.7 ± 0.1 m m . In the remaining cells, TEA maximally reduced I _K(M) by only 59.7 ± 5.2 % with an IC₅₀ of 1.4 ± 0.3 m m ; residual I _K(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I _K(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.     

Agonist-dependent postsynaptic effects of opioids on miniature excitatory postsynaptic currents in cultured hippocampal neurons

Although chronic treatment with morphine is known to alter the function and morphology of excitatory synapses, the effects of other opioids on these synapses are not clear. Here we report distinct effects of several opioids (morphine, [d-ala(2),me-phe(4),gly(5)-ol]enkephalin (DAMGO), and etorphine) on miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons: 1) chronic treatment with morphine for >3 days decreased the amplitude, frequency, rise time and decay time of mEPSCs. In contrast, "internalizing" opioids such as etorphine and DAMGO increased the frequency of mEPSCs and had no significant effect on the amplitude and kinetics of mEPSCs. These results demonstrate that different opioids can have distinct effects on the function of excitatory synapses. 2) mu opioid receptor fused with green fluorescence protein (MOR-GFP) is clustered in dendritic spines in most hippocampal neurons but is concentrated in axon-like processes in striatal and corticostriatal nonspiny neurons. It suggests that MORs might mediate pre- or postsynaptic effects depending on cell types. 3) Neurons were cultured from MOR knock-out mice and were exogenously transfected with MOR-GFP. Chronic treatment with morphine suppressed mEPSCs only in neurons that contained postsynaptic MOR-GFP, indicating that opioids can modulate excitatory synaptic transmission postsynaptically. 4) Morphine acutely decreased mEPSC amplitude in neurons expressing exogenous MOR-GFP but had no effect on neurons expressing GFP. It indicates that the low level of endogenous MORs could only allow slow opioid-induced plasticity of excitatory synapses under normal conditions. 5) A theoretical model suggests that morphine might affect the function of spines by decreasing the electrotonic distance from synaptic inputs to the soma.     

Membrane currents of cultured rat sympathetic neurons under voltage clamp

Sympathetic neurons, dissociated from neonatal rat superior cervical ganglia, were voltage clamped with two microelectrodes. Depolarization from resting potential activated a rapid transient inward current carried by sodium and a slow inward current blocked by cobalt. Depolarization from resting potential also activated up to three kinetically distinct outward currents, which were further studied by tail current analysis. Following long depolarizing steps, outward current decayed biphasically. The fast phase (delayed rectifier) decayed over 10-20 ms. The slow phase (calcium dependent) required as much as 1-2 s to decay to base line. A small component of the total outward current was a persistent current activated between -70 and -30 mV (M-current), which decayed over 200-300 ms. This current was studied in isolation following hyperpolarizing steps from potentials negative to the threshold for activation of the other delayed outward currents. Tetraethylammonium (TEA) blocked the fast tail current, partially inhibited the slow tail current, and reduced M-currents. Cobalt selectively decreased the slow tail current. Muscarine blocked M-current but not other outward currents. A transient outward current was activated by depolarization from only holding potentials negative to -60 mV. This current peaked in 10-20 ms and decayed over about 50 ms. A persistent ("anomalous") inward current was evoked by hyperpolarizing steps from only holding potentials negative to -50 to -60 mV. These seven membrane currents may be separately characterized on the basis of their voltage- and time-dependent properties. Further identification is aided by the use of channel-blocking chemicals, although the latter may lack specificity, especially when used to study potassium channels.     

Differential oxidative modulation of voltage-dependent K+ currents in rat hippocampal neurons

Oxidative stress is enhanced by [Ca2+]i-dependent stimulation of phospholipases and mitochondria and has been implicated in immune defense, ischemia, and excitotoxicity. Using whole cell recording from hippocampal neurons, we show that arachidonic acid (AA) and hydrogen peroxide (H2O2) both reduce the transient K+ current I(A) by -54 and -68%, respectively, and shift steady-state inactivation by -10 and -15 mV, respectively. While AA was effective at an extracellular concentration of 1 microM and an intracellular concentration of 1 pM, extracellular H2O2 was equally effective only at a concentration >800 microM (0.0027%). In contrast to AA, H2O2 decreased the slope of activation and increased the slope of inactivation of I(A) and reduced the sustained delayed rectifier current I(K(V)) by 22% and shifted its activation by -9 mV. Intracellular application of the antioxidant glutathione (GSH, 2-5 mM) blocked all effects of AA and the reduction of I(A) by H2O2. In contrast, intracellular GSH enhanced reduction of I(K(V)) by H2O2. Decrease of the slope of activation and increase of the slope of inactivation of I(A) by hydrogen peroxide was blocked and reversed to a decrease, respectively, by intracellular application of GSH. Intracellular GSH did not prevent H2O2 to shift inactivation and activation of I(A) and activation of I(K(V)) to more negative potentials. We conclude, that AA and H2O2 modulate voltage-activated K currents differentially by oxidation of GSH accessible intracellular and GSH inaccessible extracellular K+-channel domains, thereby presumably affecting neuronal information processing and oxidative damage.     

GABA activated chloride currents in cultured rat hippocampal and septal region neurons can be inhibited by curare and atropine

In order to study the influence of curare and atropine on the gamma-aminobutyric acid (GABA)-evoked chloride current, we have investigated cultures of hippocampal and septal region neurons from embryonic rats (E18). The neurons were cultured under the trophic influence of spatially separated astrocytes in serum-free medium. By means of the patch clamp technique, the excitable cells displayed GABA induced chloride currents within 1-15 days in vitro. In both cultures picrotoxin or bicuculline as well as curare or atropine reversibly inhibited the chloride current in a dose dependent manner. We conclude that curare and atropine are not specifically anti-cholinergic for cultured central neurons. Our results provide evidence for common ligand binding properties shared by GABAA and acetylcholine (ACh) receptors supporting the recent concept of a receptor superfamily.