Physiological Properties of GABAA Receptors From Acutely Dissociated Rat Dentate Granule Cells

Jaideep Kapur,1 Kevin F. Haas,1 and Robert L. Macdonald1,2

 1Department of Neurology; and  2Department of Physiology, University of Michigan Medical Center, Ann Arbor, Michigan 48104-1687


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kapur, Jaideep, Kevin F. Haas, and Robert L. Macdonald. 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 ~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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA mediates inhibitory neurotransmission in the forebrain by activation of GABAA and GABAB receptors. Study of fast, GABAA receptor-mediated, inhibitory postsynaptic currents (IPSCs) in the dentate granule cells of the hippocampus has produced a model of central inhibitory synaptic function that postulated quantal transmission and a small number of postsynaptic receptors activated by a saturating concentration of GABA (Edwards et al. 1990; Mody et al. 1994). This model predicted that properties of GABAA receptors were likely to influence the amplitude and time course of the IPSCs. IPSC amplitude is regulated by GABAA receptor single channel conductance, ion selectivity of the channel, desensitization, and rectification, whereas IPSC time course is determined by the processes of activation, desensitization, and deactivation (Jones and Westbrook 1995, 1996). In addition to these factors, the state of phosphorylation of GABAA receptors played a role in maintenance of their function. Progressive loss or "run-down" of GABAA receptor currents occurred during whole cell recording from many types of neurons, including acutely isolated CA1 pyramidal neurons (Stelzer et al. 1988), cultured chick spinal cord neurons (Gyenes et al. 1994), and recombinant receptors expressed in fibroblasts (Lin et al. 1994). This ATP dependence was likely due to ATP-dependent phosphorylation of GABAA receptors or of a closely associated protein by a protein kinase (Chen et al. 1990; Gyenes et al. 1994).

The identification of multiple subunits and subtypes of GABAA receptors and the heterogeneous distribution of their mRNAs suggested the existence of substantial diversity of native GABAA receptors (Macdonald and Olsen 1994). Expression of specific GABAA receptor isoforms in different neurons may result in distinct forms of GABAergic inhibition because GABAA receptors composed of different subunit subtype combinations expressed in heterologous expression systems have been shown to have different pharmacological and biophysical properties (Burgard et al. 1996; Gingrich et al. 1995; Saxena and Macdonald 1994). In situ hybridization studies of GABAA receptor subtype mRNAs suggested that many subtypes with a restricted distribution were expressed in hippocampal dentate granule cells (Wisden et al. 1992).

GABAA receptor subunit composition has been shown to influence the pharmacological properties of GABAA receptors (Macdonald and Olsen 1994) and activation, deactivation, desensitization, and voltage dependence (Burgard et al. 1996) of the chloride channel. GABAA receptors have been shown to be regulated by phosphorylation of different sites on different subunit subtypes. We have reported previously that GABAA receptors present on dentate granule cells acutely isolated from 28- to 35-day-old rats have distinct pharmacological properties (Kapur and Macdonald 1996). Here we describe physiological properties, including activation, deactivation, desensitization, rectification, and ATP dependence of GABAA receptor currents recorded from dentate granule cells acutely isolated from 18- to 35-day-old rats.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of dentate granule cells

All experiments were performed on dentate granule cells isolated according to the method described originally by Kay and Wong (1986). Male or female Sprague-Dawley rats (18-35 days old) were euthanized and decapitated. The brain was dissected free, and the region containing the hippocampus was blocked and chilled in an oxygenated PIPES-buffered medium (4°C) for 1 min. The PIPES buffer solution contained (in mM) 120 NaCl, 2.5 KCL, 1.5 CaCl2, 1 MgCl2, 25 D-glucose, and 20 PIPES (pH 7.0). After blot drying, the brain was mounted on a vibratome stage, and 500-µm coronal sections containing the hippocampus were cut. The sections were allowed to recover in oxygenated (95% O2-5% CO2) PIPES buffer for 30-60 min. Hippocampal sections were then incubated in oxygenated SIGMA type XXIII (Sigma Chemical; St. Louis, MO) protease enzyme in the buffer at 32°C for 30-45 min. The dentate gyrus was dissected out and cut into 0.5-mm cubes that were triturated in a cold (4°C) PIPES-buffered medium in fire-polished glass pipettes to isolate neurons. The isolated neurons were plated on poly-L-lysine-coated 35-mm polystyrene Petri dishes (Corning Glass Works; Corning, NY), and the recordings were made within 1 h of isolation.

Recording and analysis of GABAA receptor currents from outside-out macropatches excised from acutely isolated dentate granule cells

Patch-clamp recordings were performed on outside-out membrane patches (Hamill et al. 1981) pulled from acutely isolated dentate granule cells bathed in an external solution consisting of (in mM) 142 NaCl, 8 CsCl, 6 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose (pH 7.4, 330 mosmol) at room temperature. Intrapipette CsCl was used to block potassium currents. Glass microelectrodes were formed from thick-walled borosilicate glass (World Precision Instruments) with a Flaming Brown electrode puller, fire polished to tip resistances of 10-20 MOmega , and then coated with Q-dope. Patch electrodes were filled with an internal solution consisting of (in mM) 153 CsCl, 1 MgCl2, 2 MgATP, 10 HEPES, and 5 EGTA (pH 7.3, 300 mosmol). This combination of internal and external solutions produced a chloride equilibrium potential of 0 mV. Outside-out membrane patches were voltage clamped at -50 mV with an EPC-7 amplifier (List; Darmstadt, Germany).

GABA was applied to outside-out membrane patches with a rapid application system (Franke et al. 1987) consisting of a double-barreled theta tube (FHC, Brunswick, ME) connected to a piezoelectric translator (Burleigh Instruments; Fishers, NY). One barrel was perfused with the external recording solution, and the other was perfused with a GABA-containing external solution. Activation of the translator drove the solution interface rapidly across the patch surface. The solution exchange time was routinely monitored at the end of each recording by blowing out the patch and stepping a dilute (90%) external solution across the open electrode tip to measure a liquid junction current; 10-90% rise times for solution exchange were consistently <400 µs. The recording chamber was continuously perfused with external solution to prevent accumulation of GABA in the bath. All experiments were performed at room temperature (22-23°C).

Outside-out patch data were low-pass filtered at 3 kHz, digitized at 10 kHz, and analyzed with the pClamp6 software suite (Axon Instruments; Foster City, CA) and Origin 4.1 (Microcal; Northhampton, MA). Multiple GABA-elicited responses (5-20) were acquired for each patch at 30-s intervals and then were averaged to form ensemble currents for analysis. Activation of ensemble currents was measured as a 10-90% rise time to the peak current. The desensitization or deactivation time courses of ensemble GABAA receptor currents were fit with the Levenberg-Marquardt least-squares method with one or two component exponential functions. The number of exponential components was determined by statistically comparing the sum of squared residuals for one and two component fits (F test, P < 0.001). The extent of desensitization was measured as (peak current - fitted steady state current)/(peak current). Numerical data were expressed as means ± SE. Statistical significance was determined with paired two-tailed t-tests (P < 0.05).

Recording and analysis of whole cell GABAA receptor currents from acutely isolated dentate granule cells

Whole cell GABAA receptor currents were recorded from hippocampal dentate granule cells acutely isolated from 18- to 35-day-old rats with the technique described by Hamill et al. (1981). The extracellular recording solution consisted of (in mM) 142 NaCl, 1.0 CaCl2, 8 KCl, 6 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 and osmolarity of 310-320 mosmol (all reagents from Sigma). For ATP-dependence experiments, glass recording patch pipettes were filled with a solution consisting of the following (in mM): 115 Trizma phosphate (dibasic), 30 Trizma base, 11 EGTA, 2 MgCl2, and 0.5 CaCl2, pH 7.35. Recording pipettes contained ATP as specified in the RESULTS. For voltage-dependence experiments the pipette solution was altered to obtain symmetrical intracellular and extracellular chloride concentration. The pipette solution consisted of (in mM) 153 CsCl, 1 MgCl2, 5 EGTA, 10 HEPES, and 2 ATP, pH 7.4. GABA dissolved in extracellular solution was applied to neurons with a modified U-tube "multipuffer" rapid application system (Bormann 1992; Greenfield and Macdonald 1996) with the tip of application pipette placed 100-200 µm from the cell.

Whole cell currents were recorded with an Axopatch 1-D amplifier and low-pass filtered at 2 kHz with a eight-pole Bessel filter before digitization, storage, and display. Currents were displayed on a Gould 2400S chart recorder, and peak whole cell currents were measured manually from the chart paper. Currents were also recorded on a hard disk (Acer-286 turbo personal computer) with the Axotape program (Axon Instruments) (digitized at 208 Hz) and on a videotape recorder (Sony SL-HF360) via a digital audio processor (Sony PCM-501 ES, 14-bit, 44 kHz). Peak GABAA receptor currents at various GABA concentrations were fitted to a sigmoidal function with a four-parameter logistic equation (sigmoid concentration response) with a variable slope. The equation used to fit the concentration-response relationship was
<IT>I</IT><IT>=</IT><FR><NU><IT>I</IT><SUB><IT>max</IT></SUB></NU><DE><IT>1+10</IT><SUP>(<IT>Log<SUB>EC50</SUB>−Log</IT><SUB><IT>drug</IT></SUB>)<IT>·Hill slope</IT></SUP></DE></FR>
where I was the GABAA receptor current at a given GABA concentration and Imax was the maximal GABAA receptor current. Maximal current and concentration-response curves were obtained after pooling data from all neurons. Concentration-response curves were also obtained from all individual neurons to derive EC50 for GABA. The curve-fitting algorithm minimized the sum of the squares of the actual distance of points from the curve. Convergence was reached when two consecutive iterations changed the sum of squares by <0.01%. The curve fit was performed on an IBM PC-compatible personal computer with Graphpad prism program. All values are reported as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation and deactivation of dentate granule cell GABAA receptor outside-out patch currents

In the synaptic cleft of central GABAergic synapses, the GABA concentration has been estimated to be high (0.5-1 mM) after release of GABA from presynaptic terminals (Edwards et al. 1990; Jones and Westbrook 1995; Maconochie et al. 1994). To mimic the time course of GABA in the synaptic cleft, 1 mM GABA was applied briefly to outside-out membrane patches excised from acutely isolated dentate granule cells with a piezoelectric translator to make rapid switches between solutions. GABA was applied to individual patches every 20-30 s during each experiment. Open-tip currents were routinely checked at the end of experiments and indicated a rapid solution exchange time under these experimental conditions (10-90% rise time 360 ± 120 µs, n = 6).

Transient (2 ms) application of 1 mM GABA to outside-out membrane patches voltage clamped to -50 mV produced rapidly activating and deactivating currents (Fig. 1). In the example shown, the current activated with a 10-90% rise time of 0.90 ms (Fig. 1A) and deactivated with a biexponential time course with time constants of 9.7 (58.1%) and 100 ms (41.9%) (Fig. 1B). The average rise time of the outside-out patch GABAA receptor currents, although rapid (10-90% rise time, 0.90 ± 0.09 ms, n = 6), was two to three times slower than the solution exchange time. This rise time was similar to reported dentate granule cell IPSC rise times (Edwards et al. 1990) but was clearly slower than that reported by De Koninck and Mody (1994).



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Fig. 1. Biphasic deactivation of an outside-out membrane patch current after a 2-ms application of 1 mM GABA. An outside-out membrane patch pulled from an acutely isolated dentate granule cell was voltage clamped at -50 mV and rapidly exposed to 1 mM GABA for 2 ms. A: current activated rapidly with a 10-90% rise time of 0.90 ms. B: current deactivated with a biphasic time course (tau f = 9.7 ms, tau s = 100 ms) with 58.1% of the fitted decay in the fast component.

Deactivation of dentate granule cell GABAA receptor currents was studied after brief (2 ms), rapid application of 1 mM GABA. The current decay time course was fitted best by the sum of two exponential functions (tau fast = 9.9 ± 1.8 ms and tau slow = 95.8 ± 10.3 ms) (Table 1). The fast component contributed 63.1% and the slow component contributed 36.9% of the peak current decay. The biphasic deactivation time course of outside-out patch currents after transient application of GABA was similar to the biexponential IPSC decay in the dentate granule cells reported by Edwards et al. (1990) but differed from the monoexponential decay reported for dentate granule cell mIPSCs (DeKonnick and Mody 1994).


                              
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Table 1. Rapid kinetic properties of dentate granule cell GABAA receptors

Desensitization of dentate granule cell GABAA receptor outside-out patch currents

Desensitization of GABAA receptor currents was studied after rapid application of 1 mM GABA for 400 ms to outside-out patches excised from acutely isolated dentate granule cells. In the example shown, the current desensitized with two exponential components with time constants (and relative proportions) of 12.4 ms (26.0%) and 218 ms (74.0%) (Fig. 2). During the 400-ms period of application, average GABAA receptor currents desensitized with a biexponential time course to 60 ± 4.9% of the peak response (n = 7). The fast phase of desensitization (tau fast 16.4 ± 2.7 ms) contributed to 76% of the total desensitization, whereas the slow phase (tau slow 211 ± 31.6 ms) contributed the remainder of the desensitization (Table 1).



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Fig. 2. Biphasic desensitization of an outside-out membrane patch current after 400-ms application of 1 mM GABA. An outside-out membrane patch pulled from an acutely isolated dentate granule cell was voltage clamped at -50 mV and rapidly exposed to 1 mM GABA for 400 ms. The current desensitized with a biphasic time course (tau f = 12.4 ms, tau s = 218 ms) with 26.0% of the fitted desensitization in the fast component. The extent of desensitization after 400 ms was 56.7%.

Voltage dependence of whole cell dentate granule cell GABAA receptor currents

The voltage dependence of dentate granule cell GABAA receptor currents was studied with symmetrical transmembrane chloride concentration. Under these conditions, reversal of the currents occurred at 0 mV, and identical amplitude outward and inward currents were recorded in response to 10 µM GABA applied to cells voltage clamped to +50 mV and -50 mV (Fig. 3A). The current-voltage relationship of dentate granule cell GABAA receptor currents elicited by 10 µM GABA with symmetrical transmembrane chloride concentrations was linear (n = 5 neurons) (Fig. 3B).



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Fig. 3. No rectification of whole cell GABAA receptor current in a dentate granule cell when the transmembrane chloride concentration was symmetrical. A: acutely isolated dentate granule cell was voltage clamped to -60 and +60 mV. The horizontal bar indicated the duration of application of 10 µM GABA for each trace. B: plot of the current elicited by 10 µM GABA when the cell in A was voltage clamped to various potentials ranging from -70 to +70 mV was linear, suggesting no rectification dentate granule cell GABAA receptor currents

Voltage dependence of dentate granule cell GABAA receptor outside-out patch currents

The effects of transmembrane potential on rectification, activation, and deactivation of GABAA receptor currents in outside-out patches were determined. GABA (1 mM) was rapidly applied for 2 ms to outside-out patches excised from acutely isolated dentate granule cells when the same membrane patch was voltage clamped to -50 and +50 mV (Fig. 4). The +50/-50-mV peak current ratio of 1.05 ± 0.11:1 (n = 5) demonstrated no difference in the peak currents at +50 and -50 mV.



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Fig. 4. Outside-out membrane patch currents deactivated more slowly at positive voltages without any rectification of peak currents. A: outside-out membrane patch pulled from an acutely isolated dentate granule cell was rapidly exposed to a 2-ms application of 1 mM GABA while voltage clamped at -50 and +50 mV. At +50 mV (top trace), the deactivation time course was biphasic (tau f = 11.3 ms, tau s = 85.7 ms) with 51.2% of the fitted decay in the fast component. At -50 mV, deactivation was biphasic (tau f = 12.4 ms, tau s = 103.4 ms) with 72.9% of the fitted decay in the fast component. The +50/-50-mV peak current ratio was not significantly different at 1.05 ± 0.11:1 (n = 5). B: weighted sum of the fitted deactivation time constants (see METHODS) was used to compare the deactivation time courses at -50 and +50 mV. Deactivation was significantly slower at positive holding potentials at 42.8 ± 6.8 ms than at negative potentials 35.9 ± 6.8 ms (*Student's paired t-test, P < 0.05) caused by greater contribution of the slow deactivation phase.

Although peak current amplitudes were identical at positive and negative holding potentials, the currents deactivated at different rates when patches were voltage clamped to +50 mV or to -50 mV. In the example shown, the outside-out patch currents deactivated with two similar time constants at both positive (tau f = 11.3 and tau s = 85.7 ms) and negative (tau f = 12.4 ms and tau s = 103.4 ms) holding potentials (Fig. 4A). However, at +50 mV current deactivation occurred equally with both components (af = 51.2% and as = 48.8%), whereas at -50 mV current deactivation occurred primarily with the fast component (af = 72.9% and as = 27.1%). The net result was that the current deactivated more rapidly at -50 mV (average deactivation time constant of 47.6 ms) than at +50 mV (average deactivation time constant of 36.3 ms at -50 mV). On average (n = 5 patches), when outside-out patches were clamped to -50 mV, the fast phase of deactivation occurred with a time constant 10.1 ± 1.9 ms and contributed 66 ± 3.9% of total desensitization. When patches were clamped to +50 mV the fast phase of deactivation was 6.9 ± 1.2 ms but only contributed 47 ± 6.2% of total deactivation. When patches were clamped to -50 mV, the slow phase of deactivation occurred with a time constant of 88.7 ± 13.6 ms and contributed 34 ± 3.9% of total deactivation, whereas when patches were clamped to +50 mV the slow phase of deactivation occurred with a time constant of 73.9 ± ms and contributed 56.0 + 8.0% of total deactivation. The weighted sum of deactivation time constants when macropatches were voltage clamped to -50 mV was 35.9 ± 6.8 ms and at +50 mV was 42.8 ± 6.8 ms (P < 0.05, paired t-test) (Fig. 4B).

ATP dependence of currents

We determined the ATP dependence of dentate granule cell GABAA receptor currents. When whole cell GABAA receptor currents were recorded from acutely isolated dentate granule cells with pipettes that did not contain ATP (n = 8), two response patterns were noted. In one-half of the neurons studied, stable GABAA receptor currents in response to repeated application of 30 µM GABA were recorded for <= 30 min (Figs. 5, top panel, and 6, black-triangle), whereas in the remaining neurons GABAA receptor current began to decline (run-down) within 10 min of establishing access and continued to so steadily thereafter (Figs. 5, bottom panel, and 6, ). There were no obvious morphological or physiological differences between the neurons in which currents ran down and those they did not. When 2 mM ATP was included in the recording pipette, steady GABAA receptor currents were recorded for 30 min (n = 7), (Fig. 6, black-square).



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Fig. 5. Presence and absence of run-down of hippocampal dentate granule cell GABAA receptor currents recorded with no ATP in the pipette. GABAA receptor currents were recorded from acutely isolated dentate granule cells with a recording pipette containing no ATP. In this and all subsequent figures, the cell was voltage clamped to 0 mV, and the transmembrane chloride gradient was such that EGABA was at -65 mV, and GABA elicited outward currents; 30 µM GABA was repeatedly applied at 5-min intervals. Top traces: example stable recordings of GABAA receptor currents with no ATP in the pipette; bottom traces: steady run-down of current in similar recording conditions.



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Fig. 6. Run-down of hippocampal dentate granule cell GABAA receptor currents. The data recorded with pipettes that did not contain ATP were separated into 2 groups, one in which steady currents were recorded for >30 min (black-triangle, n = 4) and a second one where gradual run-down started within 10 min of establishing access (, n = 4). When GABAA receptor currents were recorded with pipettes containing 2 mM ATP, they remained stable for >30 min (black-square, n = 7). The current amplitude was normalized to the maximal current elicited after access; 30 µM GABA was applied at 5-min intervals.

To determine if the run-down in GABAA receptor currents during the recording period with recording pipettes that did not contain ATP resulted from a reduction in the affinity and/or efficacy of GABA for GABAA receptors, two GABA concentration-response relationships were determined in a single neuron before (Fig. 7A, top traces) and after (Fig. 7A, bottom traces) the onset of run-down. In this dentate granule cell, run-down resulted in a decrease in maximal GABAA receptor current and increase in EC50 (Fig. 7B). However, because it took ~20 min to complete a concentration-response relationship, this experiment could not be repeated a sufficient number of times to quantify the effect. Therefore to determine the effect of run-down in GABAA receptor an alternate approach was used. GABA concentration-GABAA receptor-current relationships were determined in 13 dentate granule cells recorded with 2 mM ATP in the pipette (Fig. 8A) and in another 9 cells were recorded without ATP (Fig. 8B). In the neurons recorded with ATP in the pipette (Fig. 8A), the concentration-response data were recorded after two currents evoked by 10 µM GABA were identical. In neurons recorded without ATP in the pipette (Fig. 8B), if there was an initial increase in current data collection was started after two applications of 10 µM GABA elicited identical currents. Data collection was started immediately if run-down was apparent. Because run-down occurred as the concentration-response relationship was being studied, application of GABA in ascending concentrations might have resulted in the currents elicited by high GABA concentration that were smaller than if they had been recorded earlier. To compensate for this potential run-down, GABA was applied in ascending or descending concentration steps ranging from 1 to 1,000 µM in alternate neurons. Compared with granule cells recorded with 2 mM ATP, in granule cells recorded without ATP the threshold for evoking GABAA receptor currents was higher, and smaller peak GABAA receptor currents were recorded at all GABA concentrations (Fig. 8, A and B).



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Fig. 7. Run-down resulted in diminished potency and efficacy of GABA. A: GABAA receptor currents were recorded from an acutely dissociated dentate granule cell with a pipette containing no ATP by application of 3-1,000 µM GABA before (top) and after (bottom) the onset of run-down. Initially GABA was applied in an ascending order of concentrations, and after the onset of run-down it applied in a descending order. After run-down 1 mM GABA elicited smaller peak current than 300 µm GABA before run-down. B: data from cell in A were fit to an equation for sigmoidal curve to derive the maximal current and EC50 before and after run-down.



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Fig. 8. Smaller GABAA receptor peak currents were recorded from dentate granule cells when recording pipettes contained no ATP compared with currents recorded with recording pipettes containing 2 mM ATP. GABAA receptor currents were recorded from dentate granule cells isolated from a 28- to 35-day-old rats with pipettes that contained 2 mM ATP (A) or no ATP (B). Traces were from 2 different neurons. The durations of GABA applied were indicated by the bar below each trace, and concentrations were indicated below the bottom trace.

In dentate granule cells recorded with ATP in the pipette, the peak currents obtained in response to GABA concentrations ranging from 1 to 1,000 µM were fitted to a sigmoid function, and the equation for the best fit was used to derive EC50 and maximal GABAA receptor currents elicited by GABA (Fig. 9, black-square). The mean EC50 for GABA for these neurons was 39 ± 6 µM, and the maximal current was 961 ± 102 pA (n = 13). In dentate granule cells recorded without ATP in the pipette, the mean GABA EC50 was 117 ± 30 µM, and the maximal current was 377 ± 115 pA (n = 9, Fig. 9, ). The EC50 (P < 0.05) was significantly higher in neurons recorded with no ATP in the pipette compared with those recorded with 2 mM ATP in the pipette. Maximal currents (P < 0.05) were significantly smaller when obtained with no ATP in the recording in the pipette compared with those obtained with 2 mM ATP in the recording pipette. These findings were similar to the changes in GABA EC50 and maximal current caused by run-down observed in a neuron recorded with no ATP in the pipette.



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Fig. 9. GABA efficacy and potency were lower when currents were recorded with no ATP in the pipette. GABA concentration-GABAA receptor peak current relationships were plotted for groups of hippocampal dentate granule cells acutely isolated from 28- to 35-day-old rats recorded with 2 mM ATP in the pipette (n = 13, black-square) and recorded with no ATP in the pipette (n = 9, ). Each point represented the mean of peak currents, and error bars showed SEs. The line was the best fit of data to a sigmoid function. The maximal current (Imax) and EC50 were derived from the equation for the sigmoid function that best fit the data. Note lower efficacy and potency of GABA when granule cells were recorded with no ATP in the pipette.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation and deactivation properties

Current models of GABAergic synapses terminating on the somata of dentate granule cells postulate that mIPSCs result from saturation of a small number of postsynaptic receptors by a high concentration of GABA (Busch and Sakmann 1990; De Koninck and Mody 1994; Edwards et al. 1990). The rapid rise time of mIPSCs on dentate granule cell somata was only partially replicated by application of 1 mM GABA for 2 ms to macropatches containing small numbers of receptors because the 10-90% rise time of GABAA receptor activation current was submillisecond but slower than the 290-µs rise time reported for mIPSCs (De Koninck and Mody 1994). One explanation of faster rise time mIPScs on soma and proximal dendrites of dentate granule cells compared with the activation rate of GABAA receptors on macropatches was that our application system was not sufficiently fast to replicate conditions in the synaptic cleft and test the saturation hypothesis. Alternately, rapid rise times of mIPSCs are possible even for nonsaturating GABA concentrations if the time course of GABA in the synaptic cleft is rate limiting (Galarreta and Hestrin 1997). The deactivation of GABAA receptor currents on removal of saturating concentration of GABA was quantitatively and qualitatively distinct from the decay of mIPSCs recorded from the somata of dentate granule cells. Edwards et al. (1990) fitted the decay of mIPSCs with the sum of two exponential functions, with time constants of 2.0 ± 0.38 ms and 54 ± 18 ms. Several other studies (De Koninck and Mody 1994; Otis and Mody 1992; Soltesz et al. 1995) described the decay of mIPSCs by a single exponential function with a time constant of 3.7-7.2 ms. The deactivation of GABAA receptor currents from macropatches excised from dentate granule cells was fitted best to two exponential functions with time constants of 9.9 ± 1.8 and 95.8 ± 4.5 ms. There are several possible explanations for the discrepancy between the deactivation of macropatch GABAA receptor currents and mIPSC decay. In addition to the deactivation kinetic properties of the GABAA receptors, the mIPSC decay is likely to be effected by cable filtering of dendritic mIPSCs (Soltesz et al. 1995), rate of reuptake of GABA in the synaptic cleft, and unbinding and rebinding of GABA to the postsynaptic receptors. Additionally, we determined the deactivation properties in outside-out membrane patches pulled from the somata of dentate granule cells that may have included synaptic and extrasynaptic GABAA receptors; in contrast mIPSC decay largely depends on the properties of subsynaptic receptors.

Desensitization and rectification

Compared with CA1 pyramidal neuron GABAA receptor currents, dentate granule cell GABAA receptor currents had a slow desensitization rate and large residual current (Celentano and Wong 1994; Jones and Westbrook 1995). One possible explanation for this slow rate of desensitization may have been the presence of a delta  subunit in these receptors. The mRNA for the delta  subunit is well expressed in dentate granule cells, and delta  subunit incorporation into recombinant GABAA receptors was shown to slow desensitization of the receptor currents (Fisher and Macdonald 1997; Haas and Macdonald 1998; Saxena and Macdonald 1994). Other possibilities include the potential contribution of alpha 4 subtype-containing isoforms to the currents and the modulation of desensitization kinetics by phosphorylation.

Dentate granule cell GABAA receptor currents did not rectify at positive holding potentials when intracellular and extracellular chloride concentrations were symmetrical. There was a linear current-voltage relationship for unitary mIPSCs recorded from the somata of dentate granule cells (De Koninck and Mody 1994). In contrast outward rectification of GABAA receptor single channel currents present on cultured hippocampal neurons (Segal and Barker 1984), hippocampal neurons in slices (Gray and Johnston 1985), and whole cell currents recorded from acutely isolated CA1 neurons (Burgard et al. 1996) have been reported. Burgard et al. (1996) demonstrated that the degree of rectification depended on the subunit composition of GABAA receptors; for example, alpha 1beta 3gamma 2 receptor currents rectified far less than alpha 5beta 3gamma 2 receptor currents. It was likely that the linear current-voltage relationship of the dentate granule cell GABAA receptor currents was a consequence of its distinct subunit composition.

The deactivation of GABAA receptor currents from macropatches after brief, rapid application was slower at +50 mV than at -50 mV. This was primarily due to greater participation of the slower phase of deactivation at +50 mV compared with -50 mV. This might explain the voltage dependence of mIPSC decay in dentate granule cells (Otis and Mody 1992). Slower deactivation of GABAA receptor currents at depolarized potentials was described in cultured cerebellar granule cells (Mellor and Randall 1998).

Run-down of dentate granule cell GABAA receptor current

Progressive loss or run-down of GABAA receptor currents under whole cell recording conditions has been reported in many types of neurons, including acutely isolated CA1 pyramidal neurons (Stelzer et al. 1988), cultured chick spinal cord neurons (Gyenes et al. 1994), and recombinant receptors expressed in fibroblasts (Lin et al. 1994). Run-down of currents became apparent within 10 min in native neurons, and a large reduction of current occurred within 30 min. In contrast, in acutely isolated dentate granule cells in the absence of ATP in the recording pipette, either no run-down occurred for 30 min or run-down started in first 10 min of recording and then proceeded slowly. Clearly run-down and ATP dependence of acutely dissociated dentate granule cells and CA1 neurons was different where GABAA receptor currents ran down rapidly in the absence of ATP in the recording pipette (Chen et al. 1990). The greater susceptibility of GABAA receptor currents to run-down in acutely dissociated CA1 pyramidal neurons may have been due to their larger volume and greater metabolic needs, greater susceptibility to trauma associated with dissociation, or different GABAA receptor isoform expressed by these cells.

This study suggested that the reduction in GABAA receptor currents observed (when recorded with no ATP in the pipette) was due to reductions in both potency and efficacy of GABA. However this conclusion was limited because the current data were obtained during the development of run-down. Additionally, because the concentration-response curve recorded within the pipettes containing no ATP was not generated with sufficiently high GABA concentration to obtain a plateau current amplitude, the reported maximal current was likely underestimated, and a large reduction in GABA potency may have falsely appeared as a reduction in GABA efficacy. In another study a reduction in efficacy but increased potency of GABA in cultured chick neuron GABAA receptors was reported (Gyenes et al. 1994). This probably reflected differences in isoforms or neurons used for the study.


    ACKNOWLEDGMENTS

We thank N. Esmaiel for assistance with the experiments.

This work was supported by a grant from the Epilepsy Foundation of America, by National Institute of Neurological Disorders and Stroke Grants KO8 NS-01748 and KO2 NS-02081 to J. Kapur and RO1 NS-33300 to R. L. Macdonald, and by a grant from the Lucille P Markey Charitable Trust Fund.

Present address of J. Kapur: Dept. of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908.


    FOOTNOTES

Address for reprint requests: R. L. Macdonald, Neuroscience Laboratory Building, 1103 East Huron, Ann Arbor, MI 48104-1687.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 May 1998; accepted in final form 29 January 1999.


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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society