An electrophysiological investigation of the characteristics and function of GABAA receptors on bovine adrenomedullary chromaffin cells

The characteristics and function of -aminobutyric acid_A (GABA_A) receptors expressed on bovine chromaffin cells in culture have been investigated using patch-clamp techniques. In voltage-clamped whole-cells, locally applied GABA (100 M) evoked a transmembrane chloride current which demonstrated outward rectification. The amplitude of such currents was reversibly suppressed by the GABA_A receptor antagonists bicuculline, picrotoxin and RU5135, and enhanced by the general anaesthetic propanidid. Glycine (100 M) and baclofen (100 M) were ineffective as agonists. In support of a physiological role for GABA in the adrenal medulla, the co-existence of GABA_A and nicotinic acetylcholine (ACh) receptors was demonstrated on whole cells and outside-out membrane patches. Ionophoretically applied GABA reduced the amplitude of depolarization and action potential discharge occurring in response to locally applied ACh (100 M), but had no effect upon the underlying ACh-induced current. In addition, an excitatory action of GABA was demonstrated by recording action potential waveforms in cell-attached patches. The results are discussed in the context of a GABA-ergic regulation of catecholamine secretion.     

Alphaxalone activates a Cl- conductance independent of GABAA receptors in cultured embryonic human dorsal root ganglion neurons.

Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 microM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 +/- 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 microM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 microM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors.     

Localisation of the GABA(C) receptors at the axon terminal of the rod bipolar cells of the mouse retina.

In the vertebrate retina, the rod bipolar cells make reciprocal synapses with amacrine cells at the axon terminal. Amacrine cells may perform a fine control of the transmitter release from rod bipolar cells by means of GABAergic synapses acting on different types of GABA receptors. To clarify this possibility GABA-induced currents were recorded by the patch-clamp whole cell method in rod bipolar cells enzymatically dissociated from the mouse retina. All cells tested showed a desensitising chloride-sensitive GABA-induced current. When GABA 30 microM was applied in presence of 100 microM biccuculine, a blocker of the GABA(A) receptors, a slow-desensitising component of the current still remains. This current was blocked when GABA 30 microM was applied in presence of 100 microM 3-aminopropylphosphonic acid, an antagonist of the GABA(C) receptors. The current mediated by GABA(C) receptors showed an EC50 of less that 5 microM; the ionic current through the GABA(A) receptor showed an EC50 of ca. 30 microM. Two pieces of evidence demonstrated that the GABA(C)-mediated current was localised at the axon terminal of rod bipolar cells: (1) cells lacking the axon terminal only showed the biccuculine-sensitive GABA-induced current; and (2) after mechanical section of the axon terminal, bipolar cells lost the slow-desensitising component of the GABA-induced current. We conclude that the rod bipolar cells express two types of ionotropic GABA receptors, and that the high sensitive GABA(C) receptors are mainly localised at the level of the axon terminal and therefore may contribute to the modulation of the transmitter release from the rod bipolar cell.     

Inhibition of type A GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipine's effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.     

Gamma-aminobutyric acid modulates chloride channel activity in cultured primary bovine lactotrophs

The effects of GABA, the analogues muscimol and baclofen, and the antagonist bicuculline were investigated on cultured primary bovine lactotrophs using the patch clamp technique. Under voltage clamp in the whole cell mode using solutions containing chloride as the only permeable ion, GABA application increased the amplitude of mean membrane current and fluctuations of current about this mean. Statistical analysis of current fluctuations induced by GABA showed that the power density spectra in 8 of 12 cells were best fitted to double Lorentzian functions and the variance was smallest around the estimated equilibrium potential of chloride ions. The underlying channel open time estimated from noise analysis was only weakly voltage-dependent. The variance of current noise increased with GABA concentration within the range of 1-30 microM, although a slight decrease of variance in one cell could be observed at 30 microM, suggesting that desensitization to GABA might occur. Muscimol mimicked the effect of GABA but baclofen was without effect under these conditions. Bicuculline reduced the GABA-activated membrane current fluctuations. GABA- or muscimol-activated channels recorded in isolated outside-out patches had a slope conductance of about 20 pS. Mean open times of the channel were characterized by two exponential decay functions. We conclude that bovine lactotrophs have GABA-activated chloride channels, which appear to be voltage-independent. In addition, the action of GABA appears to be mediated through the GABAA receptor subtype.     

GABA-Induced Cl- current in cultured embryonic human dorsal root ganglion neurons.

gamma-Aminobutyric acid (GABA)-activated channels in embryonic (5-8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 microM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 microM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 microM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl- equilibrium potential, shifting with changes in intracellular Cl- concentration in a manner expected for Cl--selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl- concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl- channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 microM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.     

Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells.

Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells. Study of fast, GABAA receptor-mediated, inhibitory postsynaptic currents (IPSCs) in hippocampal dentate granule cells has suggested that properties of GABAA receptors influence the amplitude and time course of the IPSCs. This study describes the physiological properties of GABAA receptors present on hippocampal dentate granule cells acutely isolated from 18- to 35-day-old rats. Rapid application of 1 mM GABA to outside-out macropatches excised from granule cells produced GABAA receptor currents with rapid rise time and biexponential decay of current after removal of GABA. After activation, granule cell GABAA receptor currents desensitized incompletely. During a 400-ms application of 1 mM GABA, peak current only desensitized approximately 40%. In symmetrical chloride solutions there was no outward rectification of whole cell current. Activation rates and peak currents elicited by rapid application of GABA to macropatches were also similar at positive and negative holding potentials. However, deactivation of GABAA receptor currents was slower at positive holding potentials. When whole cell currents were recorded without ATP in the pipette, current run-down was not apparent for 30 min in 50% of neurons, but run-down appeared to start soon after access was established in the remaining neurons. When 2 mM ATP was included in the recording pipette no run-down was apparent in 30 min of recording. The efficacy and potency of GABA were lower in cells recorded with no ATP in the pipette and during run-down compared with those recorded with 2 mM ATP and no run-down.     

Actions of steroids and bemegride on the GABAA receptor of mouse spinal neurones in culture

The effects of a synthetic and an endogenous steroid were studied on the GABAA receptors of isolated mouse spinal neurones, maintained in culture. Low doses of alphaxalone reversibly increased GABA-evoked whole-cell currents. Alphaxalone at higher doses (10-50 microM), when pressure ejected onto spinal neurones, also directly evoked a membrane chloride current. Such currents were reversibly suppressed by bicuculline (a GABAA antagonist) and enhanced by phenobarbitone. 5 beta-Pregnan-3 alpha-ol-20-one, a progesterone metabolite, dose-dependently potentiated the amplitude of GABA-evoked whole-cell currents. The mechanism of potentiation was examined at the single-channel level using outside-out patches from spinal neurones. The main action of the steroid on the GABAA receptor appears to be similar to that found for barbiturates, in that they prolonged GABA-activated bursts of channel openings. Bemegride had an antagonistic action on the GABAA receptor, suppressing both GABA- and pentobarbitone-evoked whole-cell currents to similar extents.     

Differential activation of GABAA and GABAB receptors by spontaneously released transmitter.

1. Whole-cell patch-clamp techniques were used to record from dentate gyrus granule cells in adult rat brain slices when N-methyl-D-aspartate (NMDA) and non-NMDA type glutamate receptors were blocked by D-2-amino-5-phosphonovaleric acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. Spontaneous inhibitory postsynaptic currents (sIPSCs), each presumably due to vesicular release of gamma-aminobutyric acid (GABA), selectively activated GABAA-type receptors. None of the individual sIPSCs showed a slow-onset potassium current characteristic of GABAB receptor activation. 2. In contrast, stimulation in the molecular layer with a bipolar stimulating electrode or bath application of the convulsant drug 4-aminopyridine (4-AP, 10-30 microM) elicited fast GABAA IPSCs followed by slower outward currents that were sensitive to the selective GABAB antagonist CGP 35348 (0.1-1 mM) and that reversed polarity near the potassium equilibrium potential. 3. CGP 35348 (0.5-1 mM) or the GABAB agonist (-)baclofen (1 microM) had no significant effect on the frequency or average amplitude of sIPSCs. However, either bath application of (-)baclofen (1 microM) or a preceding conditioning stimulus caused large reductions in the amplitude of stimulus-evoked IPSCs, suggesting a strong GABAB-mediated presynaptic inhibition of stimulus-evoked GABA release. 4. We conclude that under normal conditions spontaneous transmitter release does not activate GABAB receptors in dentate gyrus slices. These findings are consistent with either of two general possibilities. Separate groups of interneurons with different basal firing rates may selectively form GABAA and GABAB synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effect of the GABA uptake blockers, tiagabine and nipecotic acid, on inhibitory synaptic efficacy in hippocampal CA1 neurones.

The action of the novel gamma-aminobutyric acid (GABA) uptake blocker, tiagabine, has been studied on isolated GABAergic fast inhibitory postsynaptic potentials (IPSP) and currents (IPSC) in rat hippocampal CA1 pyramidal cells in the slice preparation. Tiagabine (20-50 microM) had little effect on the peak amplitude of the IPSC, but caused a robust increase in the half-width (by 109 +/- 15%). These results contrasted with those obtained using the established uptake blocker, nipecotic acid (100 microM to 1 mM), which reduced the amplitude of the IPSC by 35 +/- 6% and caused only a modest prolongation of the recovery phase. These effects, which were poorly reversible, are probably explained by the fact that nipecotic acid is a substrate for the GABA-uptake carrier and can act as a false transmitter. Tiagabine is not transported by the GABA carrier and results with this substance demonstrate the role of uptake in determining the kinetics of activation of GABAA receptors. Tiagabine is proposed as the blocker of choice for the GABA uptake system.     

Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study.

The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However, the density of the GABAA receptors was significantly increased in the THIP-treated cultures as compared to the control cultures and this effect of THIP was particularly pronounced in the processes. GABAA receptors were occasionally observed to form 'hot spots' in process-like structures and again the frequency of these areas with an extremely high density of GABAA receptors was greatly increased in the THIP-treated cultures compared to the controls. It has thus been demonstrated that the ability of GABA agonists to induce formation of low-affinity GABA receptors can be directly visualized and quantified at the EM level using the preembedding immunogold technique. It is likely that low-affinity GABAA receptors are preferentially located in the cell processes and to a considerable extent in the form of 'hot spots'. However, these 'hot spots' also contain high-affinity receptors.     

Opposing roles for GABAA and GABAC receptors in short-term memory formation in young chicks

The inhibitory neurotransmitter GABA has both inhibitory and enhancing effects on short-term memory for a bead discrimination task in the young chick. Low doses of GABA (1-3 pmol/hemisphere) injected into the multimodal association area of the chick forebrain, inhibit strongly reinforced memory, whereas higher doses (30-100 pmol/hemisphere) enhance weakly reinforced memory. The effect of both high and low doses of GABA is clearly on short-term memory in terms of both the time of injection and in the time that the memory loss occurs. We argue on the basis of relative sensitivities to GABA and to selective GABA receptor antagonists that low doses of GABA act at GABAC receptors (EC50 approximately 1 microM) and the higher doses of GABA act via GABAA receptors (EC50 approximately 10 microM). The selective GABAA receptor antagonist bicuculline inhibited strongly reinforced memory in a dose and time dependent manner, whereas the selective GABAC receptor antagonists TPMPA and P4MPA enhanced weakly reinforced in a dose and time dependent manner. Confirmation that different levels of GABA affect different receptor subtypes was demonstrated by the shift in the GABA dose-response curves to the selective antagonists. It is clear that GABA is involved in the control of short-term memory formation and its action, enhancing or inhibiting, depends on the level of GABA released at the time of learning.     

GABA increases refractoriness of adult rat dorsal column axons

We applied randomized double pulse stimulation for assessing the effects of GABA and a GABAA antagonist on compound action potentials in dorsal column axons isolated from adult rat. We stimulated the axons with double pulses at 0.2 Hz and randomly varied interpulse intervals between 3, 4, 5, 8, 10, 20, 30, 50 and 80 ms. Action potentials were measured using glass micropipettes. The first pulse was used to condition the response activated by the second test pulse. Concentrations of GABA of 1 mM, 100 microM and 10 microM did not affect action potential amplitudes or latencies activated by conditioning pulses. In the control studies, before drug administration, test pulses induced response amplitudes that were significantly decreased at 3-, 4- and 5-ms interpulse intervals. The test action potential amplitudes were 84.6 +/- 2.5%, 89.0 +/- 3.9% and 93.3 +/- 3.6% (mean +/- S.E.M.) of conditioning pulse levels, respectively. At 3-ms interpulse intervals, test response latencies were prolonged to 104.3 +/- 1.0%, but were unchanged at the other interpulse intervals. The 10 microM, 100 microM and 1 mM concentrations of GABA affected test response amplitudes. Application of 100 microM GABA reduced the amplitudes of test responses at 3-, 4-, 5- and 8-ms interpulse intervals, to 59.2 +/- 3.0%, 70.0 +/- 3.0%, 80.2 +/- 1.1% and 88.6 +/- 3.6% of the conditioning pulse amplitudes, respectively. At both 100 microM and 1 mM concentrations, GABA significantly prolonged the latencies of test responses. Treatment with 100 microM GABA prolonged the latencies of test responses at 3-, 4- and 5-ms interpulse intervals, to 119.3 +/- 3.1%, 107.3 +/- 2.8% and 105.5 +/- 2.5% of conditioning pulse latencies, respectively. The addition of 100 microM bicuculline methochloride, a GABAA antagonist, eliminated the effects of 100 microM GABA. The combined application of GABA and bicuculline (both 100 microM) did not affect amplitudes or latencies of test responses. These results suggest that GABA(A) receptor subtypes are present on the spinal dorsal column axons of adult rat, and that they modulate the excitability of the axons. The randomized double pulse methods reveal that GABA increases refractoriness of adult rat dorsal column axons.     

Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells

The modulation of a transient T-type calcium current by the five muscarinic receptor subtypes, stably expressed in NIH 3T3 cells, was studied with the whole-cell patch-clamp technique. Voltage-step depolarizations applied to the NIH 3T3 cells revealed a low-voltage-activated (LVA) T-type calcium current that was inhibited by Ni2+ and unaffected by omega-conotoxin GVIA. In cells transfected with the m3 and m5 muscarinic receptors, application of acetylcholine (ACh) resulted in a pertussis-toxin-insensitive increase in peak T-type calcium current amplitude. The m3-induced atropine-sensitive increase in current amplitude was accompanied by a shift in the voltage dependence of activation to more hyperpolarized potentials. The increase in peak T-type calcium current amplitude and the shift in voltage dependence was mimicked by incubation with 500 microM 8-bromo-cAMP. Conversely, T-type calcium current amplitudes were reduced by incubation with 10 microM RpcAMPS, an inhibitor of cAMP-dependent protein kinase (PKA). Preincubation with 500 microM 8-bromo-cAMP or with 10 microM RpcAMPS abolished the increase in T-type calcium current amplitude previously noted on stimulation of the m3 muscarinic receptor by ACh. Application of ACh to NIH 3T3 cells stably transformed with the m1 muscarinic receptor resulted in no discernable change in T-type calcium current amplitude. However, on pre-incubation of the cells with calphostin C, an inhibitor of protein kinase C (PKC), application of ACh to the cells now resulted in a robust increase in T-type calcium current amplitude. Application of 500 nM PDBu, an activator of PKC, reduced the T-type calcium current amplitude. No significant changes in T-type calcium currents were observed on application of ACh to cells stably transfected with the m2 or m4 muscarinic receptors. However, after pre-incubation with forskolin, the m2 muscarinic receptor induced a decrease in T-type calcium current amplitude. Stimulation of the ml, m3 and m5 muscarinic receptors in the NIH 3T3 cell resulted in dose-dependent increases in the concentration of intracellular cAMP in comparison to control as determined by cAMP immunoassay. Conversely, stimulation of the m2 and m4 muscarinic receptors by carbachol resulted in a dose-dependent reduction in intracellular concentrations of cAMP, as compared with control basal levels. It is concluded that the m3 and m5 muscarinic receptors enhance T-type calcium channel activity. At least in the case of the m3 muscarinic receptor, the increased T-type channel activity appeared to be mediated via increased cAMP levels and subsequent activation of PKA. The lack of effect of the ml muscarinic receptor on the T-type calcium channel was probably due to the opposing actions of concomitant activation of both PKC and PKA. The physiological significance of these findings is discussed.     

Blockade of GABAA receptors facilitates induction of NMDA receptor-independent long-term potentiation.

An N-methyl-D-aspartate (NMDA)-independent form of long-term potentiation (LTP), which depends on postsynaptic, voltage-dependent calcium channels (VDCCs), has been demonstrated in area CA1 of hippocampus. GABA acting at GABAA receptors limits postsynaptic depolarization during LTP induction. Blockade of GABAA receptors should therefore enhance activation of postsynaptic VDCCs and facilitate the induction of this NMDA receptor-independent, VDCC-dependent LTP. In agreement with this hypothesis, pharmacological blockade of GABAA receptors in the in vitro rat hippocampal slice increased the magnitude of LTP resulting from a normally effective, high-frequency (200 Hz) tetanic stimulation protocol. In addition, GABAA receptor blockade allowed a lower frequency (25 Hz) and normally ineffective tetanic stimulation protocol to induce this form of LTP. Intracellular recordings from CA1 pyramidal cells revealed that blocking GABAA receptors during tetanic stimulation allowed greater postsynaptic depolarization, increased the number of postsynaptic action potentials fired during the tetanization, and also increased the duration of synaptically evoked action potentials. To mimic the increased action potential firing observed when GABAA receptors were blocked, we paired 25-Hz antidromic stimulation with 25-Hz orthodromic stimulation. Paired antidromic + orthodromic 25-Hz stimulation induced NMDA receptor-independent LTP, whereas neither antidromic nor orthodromic stimulation alone induced LTP. Increased action potential firing can therefore at least partially account for the facilitation of NMDA receptor-independent LTP caused by blockade of GABAA receptors. This conclusion is consistent with prior studies demonstrating that action potentials are particularly effective stimuli for the gating of VDCCs in CA1 pyramidal cell dendrites.     

Membrane voltage differently affects mIPSCs and current responses recorded from somatic excised patches in rat hippocampal cultures

Recent analysis of current responses to exogenous GABA applications recorded from excised patches indicated that membrane voltage affected the GABAA receptor gating mainly by altering desensitization and binding [M. Pytel, K. Mercik, J.W. Mozrzymas, Membrane voltage modulates the GABAA receptor gating in cultured rat hippocampal neurons, Neuropharmacology, in press]. In order investigate the impact of such voltage effect on GABAA receptors in conditions of synaptic transmission, mIPSCs and current responses to rapid GABA applications were recorded from the same culture of rat hippocampal neurons. We found that I-V relationship for mIPSCs amplitudes showed a clear outward rectification while for current responses an inward rectification was seen, except for very low GABA concentrations. A clear shift in amplitude cumulative distributions indicated that outward rectification resulted from the voltage effect on the majority of mIPSCs. Moreover, the decaying phase of mIPSCs was clearly slowed down at positive voltages and this effect was represented by a shift in cumulative distributions of weighted decaying time constants. In contrast, deactivation of current responses was only slightly affected by membrane depolarization. These data indicate that the mechanisms whereby the membrane voltage modulates synaptic and extrasynaptic receptors are qualitatively different but the mechanism underlying this difference is not clear.     

Depolarizing actions of GABA and glycine on amphibian retinal horizontal cells.

1. The effects of inhibitory amino acid transmitters on horizontal cells in the superfused amphibian retina were studied by the use of conventional intracellular recording techniques. 2. Gamma-aminobutyric acid (GABA) caused a calcium-independent depolarization of horizontal cells in mud puppy and tiger salamander. This action was mimicked by muscimol but not baclofen (BAC) and blocked by bicuculline and picrotoxin (PTX), matching the GABAa receptor profile. 3. The purported GABA uptake inhibitors nipecotate (NPA) and guvacine (GUV) acted as GABAa agonists, having pharmacological properties very similar to GABA itself. These agents also activated receptors of amacrine and ganglion cells, causing membrane polarizations similar to GABA. Concentrations of these analogues that did not activate the GABAa receptor (submillimolar) did not lower the effective dose of GABA, even after prolonged application. 4. Glycine (GLY) also depolarized horizontal cells, but only in approximately 25% of the horizontal cells was the amplitude of the depolarization as great as GABA. The glycine response was blocked by both strychnine (STR, 10 microM) and PTX (100 microM). In contrast, the action of GABA was unaffected by STR. 5. Ion substitution and channel-blocking agents indicated that the effects of applied GABA and GLY were independent of both external sodium and calcium. 6. The results suggest that GABA receptors on horizontal cells may act 1) as a positive feedback system to modulate the light response and 2) as a mechanism for chemical coupling between horizontal cells.     

Complex effects of baclofen on synaptic transmission of the frog optic tectum in vitro

In the frog optic tectum in vitro, low concentrations of the GABAB agonist (-)-baclofen reversibly depressed field potentials evoked by optic nerve stimulation. This effect, which was dose-dependent and stereo-specific, differed from the potent facilitatory action of bath-applied GABA. A transient enhancement in field potential amplitude often preceded the more consistently observed depression. Both phases of the baclofen response were insensitive to the GABAA antagonist picrotoxin. Since the effects of baclofen and GABA were different, it is suggested that GABAB receptors were unlikely to mediate the action of GABA on synaptic transmission in the optic tectum.     

GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways

GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca2+ concentration of guinea-pig myenteric neurons (at 35 degrees C) using Fura-2-AM. Neurons were identified by 75 mM K+ depolarization (5 s), which evoked a transient intracellular Ca2+ concentration increase. GABA (10 s) induced a dose dependent (5 nM-1 microM) transient intracellular Ca2+ concentration rise in the majority of neurons (500 nM GABA: 251+/-17 nM, n=232/289). Interestingly, the response to 5 microM GABA (n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly (P<0.001) reduced by GABAA and GABAB receptor antagonists (10 microM) bicuculline and phaclofen. The GABAA agonist isoguvacine (10 microM) and GABAB agonist baclofen (10 microM) induced similar responses as 50 nM GABA, while the GABAC agonist cis-4-aminocrotonic acid (CACA) (10 microM) only elicited small responses in a minority of neurons. Removal of extracellular Ca2+ abolished all responses while depletion of intracellular Ca2+ stores by thapsigargin (5 microM) did not alter the responses to 500 nM GABA (n=13), but reduction of Ca2+ influx through voltage-dependent Ca2+ channels did. The nicotinic antagonist hexamethonium (100 microM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and the 5-HT3 receptor blocker ondansetron only had minor effects. Conclusion: GABA elicits transient intracellular Ca2+ concentration responses in the majority of myenteric neurons through activation of GABAA and GABAB receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5 microM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.     

Electrophysiological properties of mouse horizontal cell GABAA receptors

GABA-induced currents have been characterized in isolated horizontal cells from lower vertebrates but not in mammalian horizontal cells. Therefore horizontal cells were isolated after enzymatical and mechanical dissociation of the adult mouse retina and visually identified. We recorded from horizontal cell bodies using the whole cell and outside-out configuration of the patch-clamp technique. Extracellular application of GABA induced inward currents carried by chloride ions. GABA-evoked currents were completely and reversibly blocked by the competitive GABAA receptor antagonist bicuculline (IC50 = 1.7 microM), indicating expression of GABAA but not GABAC receptors. Their affinity for GABA was moderate (EC50 = 30 microM), and the Hill coefficient was 1.3, corresponding to two GABA binding sites. GABA responses were partially reduced by picrotoxin with differential effects on peak and steady-state current values. Zinc blocked the GABA response with an IC50 value of 7.3 microM in a noncompetitive manner. Furthermore, GABA receptors of horizontal cells were modulated by extracellular application of diazepam, zolpidem, methyl 6,7-dimethoxy-4-ethyl-beta-carboxylate, pentobarbital, and alphaxalone, thus showing typical pharmacological properties of CNS GABAA receptors. GABA-evoked single-channel currents were characterized by a main conductance state of 29.8 pS and two subconductance states (20.2 and 10.8 pS, respectively). Kinetic analysis of single-channel events within bursts revealed similar mean open and closed times for the main conductance and the 20.2-pS subconductance state, resulting in open probabilities of 44.6 and 42.7%, respectively. The ratio of open to closed times, however, was significantly different for the 10.8-pS subconductance state with an open probability of 57.2%.