Biophysics Sector and Instituto Nazionale Fisica della Materia Unit, International School for Advanced Studies (SISSA), 34014 Trieste, Italy
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ABSTRACT |
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Martina, Marco, Caterina Virginio, and Enrico Cherubini. Functionally distinct chloride-mediated GABA responses in rat cerebellar granule cells cultured in a low-potassium medium. J. Neurophysiol. 77: 507-510, 1997. The patch-clamp technique was used to study whole cell currents evoked by -aminobutyric acid (GABA) in rat cerebellar granule cells cultured in 5 mM potassium, a condition that favors the development of functionalGABAergic synapses. GABA activated both high- and low-sensitivity receptors. The high-sensitivity receptor had an effective concentration producing half the maximum response (EC50) of 13 µM, whereas the low-sensitivity one had an EC50 of 255 µM. TheGABAA receptor agonist isoguvacine activated only the high-sensitivity receptor with an EC50 of 16 µM. When GABA was applied during the desensitized phase of the response elicited by a saturating concentration of isoguvacine, it was still able to induce a small response, whereas when isoguvacine was applied during the desensitizing phase of GABA-evoked current no response was detected. GABA responses were highly heterogeneous regarding their sensitivity to bicuculline. In a small number of cells (3 of 25), bicuculline (10 µM) completely abolished GABA-evoked currents. In the majority of the neurons (22 of 25) the blocking effect of bicuculline (100 µM) was 64 ± 4% (mean ± SE). The bicuculline-resistant component was abolished by picrotoxin (100 µM). In bicuculline, the dose-response curve for GABA was fitted with a sigmoidal curve with an EC50 value of 209 µM. These data indicate that functional new GABA receptor types with unusual pharmacology could be switched on by conditions that maintain cells in their undifferentiated state.
Type A receptors for the transmitter Cell culture
Granule cells were prepared according to the procedure described by Levi et al. (1984) Data acquisition and analysis
The whole cell configuration of the patch-clamp technique was used to characterize the electrophysiological properties of GABA-evoked currents. Borosilicate glass pipettes were fire-polished to a tip resistance of 5-10 M Whole cell patch-clamp recordings were obtained from 38 neurons exhibiting similar morphological features. Cell capacitance measured between 3 and 14 days in culture was 2.28 ± 0.24 (SE) (n = 28). Assuming a membrane specific capacitance of 1 pF per 100 mm2 (Hille 1992
Despite the morphological homogeneity of granule cells cultured in low-potassium medium (see Virginio et al. 1995
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
-aminobutyric acid (GABA) mediate most of the fast inhibitory synaptic transmission in the vertebrate CNS. They inhibit neuronal firing by activating a chloride conductance that hyperpolarizes cells (Sivilotti and Nistri 1991
). At least 14 genes encoding the GABAA receptor subunits have been cloned so far, suggesting an enormous number of possible subunit combinations that can form a receptor-ionophore complex (McKernan and Whiting 1996
). Different combinations of these subunits may account for the functional diversity of native GABAA-receptor-mediated responses (Schönrock and Bormann 1993
). Recently, GABA-gated chloride channels with unusual pharmacology have been detected in vertebrate retina (Feigespan et al. 1993
; Qian and Dowling 1993
) or in hippocampal neurons during a restricted period of postnatal development (Martina et al. 1995
; Strata and Cherubini 1994
). These channels are bicuculline insensitive and therefore do not fit into the conventional scheme for GABAA receptors. They belong to a new class of receptors named GABAC, which are likely to be composed by the recently cloned
1 or
2 receptors. Transcripts for the
1 subunit mRNA are highly expressed in the retina, whereas those for the
2 subunit are expressed also in the CNS, particularly in the hippocampus, cortex, and cerebellum (Bormann and Feigenspan 1995
).
), express both high- and low-sensitivity GABA responses. The receptors with apparent low affinity are less sensitive to bicuculline and in this respect they resemble those present in the retina or in the hippocampus during development, which are supposed to be mediated by GABAC receptor types.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
. Cells were plated with Basal Eagle Medium (Irvine) on petri dishes and were kept in the incubator at 37°C. After 1 day in culture, the K+ concentration of the medium was decreased from 25 to 5 mM. Under these conditions the cells were maintained for ~15 days and were available for experiments starting from the 2nd day in culture.
(in working solutions). The external (bath) solution contained (in mM) 137 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES)-NaOH, and 10 D-glucose. The pipette solution contained (in mM) 137 CsCl, 4 MgCl2, 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid, 1 CaCl2, 2 ATP-Na2, and 10 HEPES-tetraethylammonium-OH. The pH of both solutions was adjusted to 7.3. Granule cells were kept at room temperature (22-24°C) and continuously superfused with extracellular (control) solution applied by gravity (at 2 ml/min). GABA agonists and antagonists were applied by a fast superfusion system (Akaike et al. 1991
). With this method a complete exchange of the external solution surrounding the neuron was achieved within 100 ms. GABA-evoked currents were recorded with a patch-clamp amplifier (EPC-7, List Medical Instruments, Darmstadt, Germany). The current signal, filtered at 10 kHz, was recorded on a video tape. Data were transferred to a microcomputer (ATARI 1040ST) after digitization with an analog-digital converter (ITC-16, Instrutech). Whole cell GABA currents were sampled at 100 Hz and filtered at 1 kHz. Drugs used were GABA, isoguvacine, bicuculline methiodide, and picrotoxin, all purchased from Sigma. GABA responses were normalized to peak currents induced by GABA (1 mM). Data points in the dose-response curves were fitted with the logistic Hill equation Inorm = 1/1 + (k/c)n, where Inorm is equal to I/Imax, k is the agonist concentration activating one half of the receptors (EC50), c is the agonist concentration, and n is the Hill coefficient. When two components were present, data points in the dose-response curves were fitted with the use of the sum of two Hill functions: Inorm = a/[1 + (k/c)n] + (1
a)/[1 + (k
/c)n
], where 0
a
1; Inorm, k, c, and n have the usual meanings; and k
and n
represent the half-maximum concentration and the Hill coefficient of the second component, respectively.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
), the surface area of individual cells was 228 ± 24 µm2. This value was similar to that obtained in the same cells cultured in a high-potassium medium (Kili
et al. 1993
). Plotting the peak amplitude of GABA currents versus agonist concentrations revealed three distinct populations of responses. In the first population (n = 8) the dose dependence of the normalized current was described by a sigmoidal curve (Fig. 1A). The threshold for a detectable current was in the range of 0.5-1 µM and apparently saturating responses were obtained with GABA concentrations of ~100 µM. The estimated EC50 value, obtained by fitting data points with the empirical Hill equation, was 7.2 ± 3 µM (n = 8). The Hill coefficient was equal to 1. In the second population (n = 11), detectable currents were still obtained with low concentrations of GABA (<10 µM). However, the dose-response curve, after reaching a first plateau, increased again with higher GABA concentrations (Fig. 1B). Normalized peak current amplitudes were fitted by two sigmoidal curves with different apparent affinities for GABA. The EC50 value for the first component was 22 ± 18 µM (n = 11). This value was not significantly (P > 0.5) different from that found in the first population of GABA responses. The third group of cells(n = 3) exhibited only low-affinity responses. The dose-response curve was fitted by a sigmoidal curve. The threshold for a detectable current was in the range of 10 µM and a saturating response was obtained with a GABA concentration of 1 mM.
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FIG. 1.
Representative dose-response relationships from 2 cells expressing only high-affinity (A) or both high- and low-affinity -aminobutyric acid (GABA)-mediated responses (B). The effective concentration producing half the maximum response (EC50) values were 10.5 µM in A and 20 or 305 µM for the 1st or 2nd component of the dose-response curve in B, respectively. Above the curves are original traces illustrating some of the data points. Holding potential:
50 mV. C: dose-response relationship for isoguvacine-evoked currents. Each point is from 6 experiments. Data points were fitted by a single sigmoidal curve. Estimated EC50 value was 16 µM. D: dose-response curve for GABA-evoked currents. Each point is from 22 experiments. Data points were fitted with the use of the sum of 2 Hill functions (see METHODS). Note the presence of a high- and a low-affinity component. Estimated EC50 values were 13 and 255 µM for the 1st and 2nd component, respectively.
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FIG. 2.
Cross-desensitization between isoguvacine and GABA-evoked currents. When a submaximal concentration of GABA was applied during the desensitizing phase of the response induced by a saturating concentration of isoguvacine, this aminoacid was still able to induce a small current (top trace). On the contrary, no response was detected when isoguvacine was applied during the desensitizing phase of GABA response (bottom trace). Holding potential: 50 mV.
0.26 and
0.52 for 10 and 100 µM of GABA, respectively, giving support to the hypothesis that at least two different GABA receptors having different sensitivities for the antagonist were activated by different concentrations of GABA. In bicuculline (100 µM), the dose-response curve for GABA was fitted by a sigmoidal curve with an EC50 value of 209 µM (Fig. 3C). This value is similar to that calculated for the second component of the dose-response curve obtained in the absence of bicuculline. The Hill coefficient was 1.7. The discrepancy between this value and that for the second component of the plot obtained in the absence of bicuculline from the entire population of cells (Fig. 1D) could be due to the fact that in that plot are included cells showing different degrees of sensitivity to bicuculline. Moreover, the threshold for activation of GABA currents recorded in the presence of bicuculline varied consistently from cell to cell (between 100 and 300 µM), leading to a shallower slope of the dose-response curve. The bicuculline-resistant GABA currents, like the bicuculline-sensitive ones, were chloride mediated, because they reversed at a potential very close to that predicted by the Nernst equation for chloride permeant channels (3.2 ± 2 mV, n = 5, not shown).
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FIG. 3.
Different sensitivity to bicuculline of GABA-evoked currents. A: original traces from 2 representative neurons showing bicuculline-sensitive (top) or -insensitive (bottom) GABA-mediated responses (100 µM). Bicuculline was coapplied with GABA. Horizontal bars: period of drug application. Holding potential: 50 mV. B: bicuculline inhibition of responses evoked by GABA at 10 µM (
, n = 16) or 100 µM (
, n = 9), supposed to activate only the high-affinity or both the high- and low-affinity receptors, respectively. Slope values of the regression lines were
0.26(r = 0.99) and
0.52 (r = 0.99) for 10 or 100 µM GABA, respectively. C: dose-response relationship for normalized currents evoked by GABA in the presence of bicuculline. Data points (from 6 experiments) could be fitted with a single sigmoidal curve. Estimated EC50 value was 209 µM.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
), we found that when data obtained from the entire cell population were pooled together the dose-response curve for GABA had a bimodal behavior. This suggests that in our culture conditions, cerebellar granule cells express at least two binding sites for GABA, having high- or low-sensitivity for the ligand. The EC50 value of the first component was very close to that found in cells grown in a high-potassium medium (Kili
et al. 1993
), a condition in which only high-affinity binding sites for GABA are expressed (Meier and Schousboe 1982
). Evidence in favor of two different receptor populations is also given by the occlusion experiments in which responses to a submaximal concentration of GABA could still be obtained during receptor desensitization caused by a prolonged application of a saturating concentration of isoguvacine, which is a selective ligand for GABAA receptors (Sivilotti and Nistri 1991
). Responses with lower affinity for GABA were also found by Zhu et al. (1995)
in granule cells maintained in 12.5 µM extracellular potassium. However, these authors did not test the sensitivity of GABA-evoked currents to bicuculline.
and by their sensitivity to picrotoxin. In this respect, the low-sensitivity GABA responses resemble those described in the optic tectum (Sivilotti and Nistri 1989
), in the retina (Feigespan et al. 1993
; Qian and Dowling 1993
), or in the developing hippocampus (Martina et al. 1995
; Strata and Cherubini 1994
) and supposed to be mediated by a new receptor type, named GABAC. In the retina, when bicuculline-sensitive GABA receptors are coexpressed with the bicuculline-insensitive ones, the latterexhibit higher affinity for the agonist (Bormann and Feigenspan 1995
). Similarly to the present experiments, however, in the developing hippocampus, the bicuculline-insensitive responses had a lower affinity for GABA in comparison with the bicuculline-sensitive ones, indicating the involvement of different receptor subtypes. In conclusion, we have demonstrated that lowering the extracellular potassium concentration, a condition that favors the development of functional GABAergic synapses, switches on the expression of low-sensitivity GABA receptors. Interestingly, in previous work in which binding techniques were used, it was shown that additional low-affinity GABA receptors could be expressed by exposing cerebellar granule cells to GABA or GABAA agonists (Meier et al. 1984
).
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ACKNOWLEDGEMENTS |
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The authors thank A. Nistri for helpful discussion and suggestions during the course of this work.
This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CNR 95.01664.CT04) and Human Capital and Mobility program network from the European Union.
Present addresses: M. Martina: Physiologisches Institut der Universität, Hermann-Herder Str. 7, 79104 Freiburg, Germany; C. Virginio: Glaxo Institute for Molecular Biology, Geneva, Switzerland.
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FOOTNOTES |
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Address for reprint requests: E. Cherubini, Biophysics Sector, International School for Advanced Studies (SISSA), Via Beirut 2-4, 34014 Trieste, Italy.
Received 19 June 1996; accepted in final form 30 September 1996.
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REFERENCES |
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