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J Neurophysiol 77: 994-1002, 1997;
0022-3077/97 $5.00
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The Journal of Neurophysiology Vol. 77 No. 2 February 1997, pp. 994-1002
Copyright ©1997 by the American Physiological Society

GABA-Activated Conductance in Cultured Rat Inferior Colliculus Neurons

Hidenobu Hosomi1, Masahiro Mori1, Mutsuo Amatsu2, and Yasuhiro Okada1

Departments of 1 Physiology and 2 Otorhinolaryngology, Kobe University, School of Medicine, Kobe 650, Japan

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Hosomi, Hidenobu, Masahiro Mori, Mutsuo Amatsu, and Yasuhiro Okada. GABA-activated conductance in cultured rat inferior colliculus neurons. J. Neurophysiol. 77: 994-1002, 1997. With the use of a whole cell voltage-clamp technique and fura-2 fluorescence measurements, the actions of gamma -aminobutyric acid (GABA) on cultured neurons from rat inferior colliculus were investigated. GABA (10-1,000 µM) induced currents in neurons held under voltage clamp that were inhibited by bicuculline (20 µM). Muscimol (100 µM) also evoked the currents, whereas baclofen (100 µM) affected neither the holding currents nor K+ conductance due to depolarizing pulses. The current density-voltage relation of GABA-induced currents, with equal concentrations of Cl- in the internal and external solutions, reversed near 0 mV. Reduction of the internal Cl- concentration shifted the reversal potential in the negative direction as predicted from the Cl- equilibrium potential. Baclofen did not affect Ca2+ conductance due to depolarizing pulses. The extracellular application of 150 mM KCl or 1.0 mM glutamate increased the intracellular Ca2+ concentration ([Ca2+]i) of cultured inferior colliculus neurons only when neurons were bathed in a Ca2+-containing external solution. However, GABA (1.0 mM) failed to increase [Ca2+]i at all concentrations of external Ca2+ used, indicating that GABA neither depolarized the cultured inferior colliculus neurons sufficiently to activate the voltage-dependent Ca2+ conductances nor evoked Ca2+ release from intracellular stores. These results suggest that in cultured rat inferior colliculus neurons, GABAA receptor channels may be predominantly responsible for the membrane conductance evoked by GABA and subsequent hyperpolarization of the neurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The inferior colliculus is a major relay nucleus in the auditory pathway and is closely linked to regulation, recognition, and interaction of auditory inputs. Inferior colliculus neurons respond specifically to the intensity (Rose et al. 1963), the frequency (Merzenich and Reid 1974; Rose et al. 1963), the delay (Rose et al. 1966), and the duration (Casseday et al. 1994) of sound. The inferior colliculus receives complex excitatory and inhibitory inputs. Inhibitory neuronal inputs to the central nucleus of the inferior colliculus are from the dorsal nucleus of the lateral lemniscus (Oliver and Beckius 1992; Shneiderman et al. 1993) and from the inhibitory interneurons in the inferior colliculus itself (Oliver and Beckius 1992; Oliver et al. 1994).

gamma -Aminobutyric acid (GABA) has been thought to play an important role in the inhibitory neuronal functions of the inferior colliculus. Biochemical (Adams 1979; Contreras and Bachelard 1979; Okada 1976) and immunological (Thompson et al. 1985) approaches have revealed the presence of GABA in the inferior colliculus. GABA had an inhibitory effect on the postsynaptic field potentials recorded in the pericentral nucleus of the inferior colliculus (Yamauchi et al. 1989). In the central nucleus of the inferior colliculus, [14C] GABA uptake and release was observed, suggesting the presence of GABAergic synaptic endings (Suneja et al. 1995). A large population of neurons in the central nucleus of the inferior colliculus has GABAergic axonal endings on soma and dendrites (Oliver and Beckius 1992). The presence of GABAA receptors in the inferior colliculus has also been demonstrated by in situ hybridization with an antisense probe for the GABAA receptor subunit mRNA (Gambarana et al. 1991; Miralles et al. 1994; Wisden et al. 1992). The inhibitory action of locally applied GABA on the potentials recorded from the central nucleus of the inferior colliculus was blocked by the systemic administration of picrotoxin, a GABAA antagonist (Hosomi et al. 1995). Application of GABA inhibited the neuronal activity of the central nucleus of the inferior colliculus, and this inhibition was antagonized by bicuculline, a GABAA antagonist (Faingold et al. 1986). However, precise electrophysiological studies such as those in which the patch-clamp technique was used have not yet been reported on the inferior colliculus neurons.

In the present study we established a primary culture of neurons from the central nucleus of the rat inferior colliculus and studied the functional properties of GABA receptors in the cultured inferior colliculus neurons, with the use of the whole cell patch-clamp technique and measurement of intracellular Ca2+ concentration ([Ca2+]i) with the use of fura-2. These data suggest that in the cultured inferior colliculus neurons, GABAA receptors are primarily responsible for the GABA-activated conductance and subsequent hyperpolarization. The role of GABA in the inferior colliculus is discussed. Preliminary results have appeared in abstract form (Hosomi et al. 1996).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell culture

The inferior colliculi of neonatal rats (1 day old; Wistar strain) were cut coronally and removed under ether anesthesia. The central nuclei of the inferior colliculus were collected by trimming the cortices off under a microscope. The tissues were incubated in saline with 0.25% trypsin (Sigma, St. Louis, MO) and 0.01 mg/ml DNase (Sigma) for 10 min at 37°C and then mechanically dissociated by trituration with a Pasteur pipette in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY). The dissociated cells were seeded at low density (1,000-2,000 cells per µl) on poly-L-lysine-coated glass coverslips in Dulbecco's modified Eagle's medium and cultured at 37°C. The pH of the medium was maintained at 7.4 in an atmosphere of humidified air with 5% CO2. The medium was supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco). The bicarbonate concentration of the medium was 20 mM. Neurons that had been cultured on glass coverslips for 1-2 wk were used for the experiments in room air at room temperature (22-24°C).

Patch-clamp recording

GABA was applied with a pressure application pipette, positioned ~50 µm from the soma membrane to give maximal responses under control conditions. Positive pressure of 0.1 kg/cm2 was applied. The consistency of the drug application was achieved within 0.5 s. The duration of the application is indicated by horizontal bars in figures, and was set for 10 s to obtain full response of the neuron to GABA. The cells were continuously perfused with the extracellular solution at a rate of 2.5 ml/min. The volume of the bath was 1.2 ml. The ionic compositions of the extracellular solutions and intracellular pipette solutions are shown in Table 1. To block the generation of Na+ action potentials, tetrodotoxin (1 µM; Sankyo, Tokyo, Japan) was added to the external solutions. For recording K+ conductances, 1 mM CdCl2 was added to the external solution to eliminate the Ca2+ currents. For recording Ba2+ conductances, 90 mM BaCl2 solution containing tetraethylammonium chloride (20 mM) was used instead of the normal external solution, and 150 mM CsCl was used for the pipette solution. The liquid junction potentials between the normal external solution and each of the solutions used were directly measured with a 3 M KCl electrode. The values are listed in Table 1 for each solution. The actual membrane potential was corrected for the calculated liquid junction potential between the external and internal solutions on the assumption that the junction potential between the internal solution and the interior of the cell was negligible (Hagiwara and Ohmori 1982). In this experiment, the external solution did not change between the zero-current potential measurement and the whole cell current measurements under voltage clamp. A capillary filled with 145 mM NaCl agar was used as a reference electrode. Whole cell membrane currents were recorded by the standard patch-clamp method (Hamill et al. 1981) unless indicated otherwise. The nystatin perforated-patch recording technique was also used in some experiments as mentioned in the figure legends (Fig. 3C). Nystatin (5 mg) (Sigma) was first dissolved in 0.1 ml dimethyl sulphoxide (Nacalai Tesque, Kyoto, Japan) and then diluted with the pipette solution (150 mM KCl; Solution A) to a final concentration of 200 µg/ml as previously reported by Armstrong and White (1992). The tip resistance of the patch electrodes filled with 150 mM KCl was 5-7 MOmega . The whole cell membrane currents and potentials were recorded with a patch-clamp amplifier, EPC-9 (HEKA Elektronik, Pfalz, Germany). Data acquisition and analysis were controlled with the use of the Pulse/PulseFit system (HEKA Elektronik) and a Macintosh personal computer. Current traces were filtered at 3 kHz and stored on a hard disk for later analysis. The cell capacitance was measured by integrating the capacity transient with a 5-mV depolarizing voltage step and dividing by the amplitude of the step, 5 mV. The peak amplitude of whole cell currents was normalized by the cell capacitance as a current density (in A/F), and then a normalized conductance (in S/F) was calculated. The value of series resistance during the recording of whole cell currents typically ranged between 6 and 15 MOmega (70-85% compensated). The seal resistance was in the range of 1-10 GOmega .

 
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  		TABLE 1. Ionic composition of solutions


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  		FIG. 3. Effects of GABAergic agonists and an antagonist on the membrane conductance. A: GABA (100 µM) did not evoke a current when the standard Ringer solution (Solution 1) contained 20 µM bicuculline, a specific GABAA antagonist. The internal solution was Solution C (150 mM CsCl). Holding potential: -65 mV. B: muscimol (100 µM), a specific GABAA agonist, evoked an inward current at a holding potential of -65 mV. The external solution was the standard Ringer solution (Solution 1). C: effect of GABAB agonist, baclofen (100 µM), on K+ conductance. With nystatin perforated-patch recording, depolarizing pulses of +15 mV for 1 s were applied at 1-s intervals from a holding potential of -65 mV. Baclofen, a GABAB agonist, (100 µM) was applied for 70 s (horizontal bar). K+ conductance and holding currents were not affected by baclofen during and after the application. The external solution was Solution 1 and the internal solution was Solution A containing nystatin (200 µg/ml). D: normalized conductance evoked by application of GABA agonists. Holding potential: -65 mV. Numbers in parentheses: number of neurons tested. Vertical lines: SE.

Fluorescence measurement

[Ca2+]i was recorded by means of fura-2 fluorescence (Grynkiewicz et al. 1985). Inferior colliculus neurons on glass coverslips coated with poly-L-lysine were incubated in a standard Ringer solution with 5 µM fura-2-AM (Molecular Probes, Eugene, OR) for 60 min at 22-24°C. With the use of a diffraction grating spectroscope, CAM 230 (Hamamatsu Photonics, Hamamatsu, Japan), attached to an inverted microscope, Diaphot TMD 300 (Nikon, Tokyo, Japan), the fluorescence signals through an emission filter of 500 nm (bandwidth 11 nm) were measured after alternating excitation wavelengths (bandwidth 10 nm) between 340 and 380 nm every second. The background fluorescence from poly-L-lysine coated on the glass coverslip was negligible. In neurons not loaded with fura-2, application of GABA did not change the fluorescence intensity at any wavelength. In vitro calibration was performed with different free Ca2+ concentrations, ranging from 3.19 nM to 5.03 µM, in a solution composed of 150 mM KCl, 20 mM piperazine-N,N'-bis-2-ethanesulfonic acid, and appropriate amounts of CaCl2 and ethylene glycol-bis-(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) as well as 4 µM fura-2 free acids (Molecular Probes).

Drugs

Bicuculline, muscimol, and nystatin were purchased from Sigma (St. Louis, MO). Tetrodotoxin was from Sankyo (Tokyo, Japan). Fura-2-AM and fura-2 free acid were from Molecular Probes (Eugene, OR). GABA, baclofen, EGTA, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, and other chemicals were from Nacalai Tesque (Kyoto, Japan).

All statistical values are given as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Cell identification

Among cultured cells from the central nucleus of the rat inferior colliculus, small cells of polygonal soma, 10-15 µm diam, with well-defined multiple fine processes (5-8) were identified as inferior colliculus neurons (Fig. 1). Under voltage clamp, the identified neurons showed inward Na+ and outward K+ currents with depolarizing voltage pulses, with a 150 mM KCl internal solution and a normal Ringer external solution (no tetrodotoxin added).


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  		FIG. 1. Single cultured rat inferior colliculus neuron used for the experiments. Morphological features of polygonal soma (10-15 µm diam) with multiple well defined processes were observed. Horizontal bar: 20 µm.

GABA-activated currents in cultured inferior colliculus neurons

The cell capacitance of a single inferior colliculus neuron was 9.6 ± 0.4 (SE) pF (n = 47). The resting membrane potential was -59.3 ± 1.9 (SE) mV (n = 47) 1 min after patched membrane was broken when neurons were bathed in a standard Ringer solution (Solution 1) and the intracellular pipette solution was Solution A (150 mM KCl). Figure 2, A and B, shows typical current responses of a single inferior colliculus neuron to extracellularly applied GABA (100 µM) for 60 and 10 s, respectively. GABA evoked inward currents at a holding potential of -65 mV, with Solution A (150 mM KCl) in the pipette. Desensitization developed within seconds. The time to peak was 1.1 ± 0.1 s(n = 11). The current was inactivated with a half-decay time of 3.8 ± 0.5 s (n = 11). With application of GABA (100 µM) at 30-s intervals, sustained desensitization of the GABA response was observed. Longer intervals between the applications led to the recovery from the desensitization, and finally a 90-s interval led to full recovery (Fig. 2C). The normalized conductance-concentration relationship for the currents activated at a holding potential of -65 mV by pressure applications of GABA is illustrated in Fig. 2D. The half-maximal concentration was 49 µM.


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  		FIG. 2. Responses of membrane conductance to pressure application of gamma -aminobutyric acid (GABA) in cultured rat inferior colliculus neurons. Horizontal bars: period of application of GABA (100 µM). The external solution was a standard Ringer solution (Solution 1) and the pipette solution was Solution A (150 mM KCl). Holding potential: -65 mV. A: typical time course of the current activated by application of GABA for 60 s. GABA rapidly induced an inward current. Cell capacitance: 7.4 pF. B: typical example of the current activated by application of GABA for 10 s. Cell capacitance: 9.6 pF. C: desensitization and recovery of GABA response. GABA (100 µM) was applied at 30 s intervals (top). The 2nd and 3rd applications evoked smaller responses due to desensitization. Application of GABA at 60-s intervals produced responses that were slightly smaller than the 1st (middle). With 90-s intervals, neurons recovered between applications and each application of GABA produced identical responses (bottom). The GABA-activated currents had not run down 5 min after the conventional whole cell recording had been achieved. Nystatin perforated-patch recording was also performed. The GABA-activated currents were stable up to 30 min (data not shown). D: influence of the concentration of GABA on the normalized conductance. Holding potential: -65 mV. Each point represents the mean of 4-5 neurons. Vertical lines: SE. The change in the open pipette currents due to the junction potential was recorded on application of the solution containing 25% of the control Cl- concentration, with the pressure application system. The current has reached the plateau level within 0.5 s. However, it is possible that GABA applied from the pipette was diluted in the bath solution before reaching the receptors on the neuron, because of the distance of the pipette tip from the neuron (50 µm). Thus, when GABA initially reached the neuron, its concentration may have been less than that in the pipette.

GABA receptor subtype in the cultured inferior colliculus neuron

In the presence of 20 µM bicuculline, a specific GABAA receptor antagonist, in the normal external solution (Solution 1), GABA (100 µM) did not evoke currents at a holding potential of -65 mV, with Solution C (150 mM CsCl) in the pipette (Fig. 3A). In the absence of bicuculline, muscimol (100 µM), a specific GABAA agonist, evoked an inward current (Fig. 3B), which was similar to that evoked by GABA. Next we studied the effects of baclofen, a specific GABAB agonist, on the membrane permeability in cultured inferior colliculus neurons. Neurons were bathed in the normal external solution containing 1 mM CdCl2, with a nystatin-containing internal solution (150 mM KCl; Solution A). Voltage pulses of +15 mV for 1 s were applied every other second from a holding potential of -65 mV. Application of baclofen (100 µM) affected neither holding currents nor K+ conductance due to the depolarizing pulses (n = 10) (Fig. 3C). Baclofen (100 µM) was also applied to the dendritic region of the neuron to determine whether GABAB receptors are located on dendrites rather than the soma (Newberry and Nicoll 1985). Baclofen, however, did not affect the holding currents and the K+ conductance by depolarization.

The normalized conductances evoked at a holding potential of -65 mV by GABA, muscimol, and baclofen were 1,000 ± 118, 1,648 ± 173, and 0 ± 0 S/F, respectively(n = 8-11) (Fig. 3D).

Ionic mechanism of the GABA-activated current

The current density-voltage relationships for GABA-activated currents were studied. The current density-voltage relation of the GABA-induced currents was obtained by measuring the GABA-induced currents at different holding potentials (-80 to +80 mV). The extracellular solution was Solution 1 and the intracellular pipette solution was Solution C (150 mM CsCl). The reversal potential was -4.4 mV, corresponding to the equilibrium potential of Cl- (-0.8 mV) (Fig. 4A).


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  		FIG. 4. Whole cell currents activated by GABA at different voltages. A: current density-voltage relationship of GABA-activated currents at different holding potentials. GABA (100 µM) was applied to a neuron after the holding voltage was fixed at a certain level. Each point is the mean of 4-6 neurons obtained from the total of 42 neurons. Vertical lines: SE. Reversal potential: -4.4 mV. Data points of the current density-voltage relation was fitted by 3rd-order polynominals, from which the interpolated reversal potential was calculated. B: current density-voltage relationship of GABA-activated currents with Solution B (25% Cl- solution) in the pipette and Solution 1 in the bath. Reversal potential shifted to -33.8 mV. Each point represents the mean of 5-6 neurons obtained from the total of 46 neurons. Vertical lines: SE.

When 75% of Cl- in the KCl internal solution was replaced with isomolar gluconate, an impermeable anion (Barker and Harrison 1988; Fatima-Shad and Barry 1993) (Solution B), reversal potential of GABA-activated current was -33.8 mV (Fig. 4B). This internal Cl- substitution with gluconate changed the equilibrium potential of Cl- from -0.8 to -36.4 mV. Therefore it is suggested that GABA significantly altered the membrane permeability to Cl- through GABAA receptor channels.

Effect of a GABAB agonist on Ca2+ conductance

The effects of baclofen on voltage-activated Ca2+ conductance in cultured inferior colliculus neurons were studied to test the possibility of the GABAB receptor-mediated modulation of the voltage-activated Ca2+ conductance. Barium was used as a major external divalent cation with Solution 2 (90 mM BaCl2, 20 mM tetraethylammonium chloride) in the bath, and 150 mM CsCl solution (Solution C) was used for the pipette solution. In cultured inferior colliculus neurons, Ba2+ currents were rarely recorded with depolarizing voltage steps. Even if successfully recorded, the inward Ba2+ currents were small in amplitude. Application of 100 µM baclofen did not affect the Ba2+ currents, maximally activated by the voltage pulses of -44 mV from a holding potential of -89 mV (n = 2) (Fig. 5).


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  		FIG. 5. Effect of baclofen on voltage-activated Ca2+ conductance. Depolarizing pulses of -44 mV for 100 ms were applied at 1-s intervals for maximal activation of the Ba2+ conductance from a holding potential of -89 mV. Ba2+ currents were not affected by application of baclofen (100 µM) (n = 2). Top: example of current traces of Ba2+ currents recorded. Each trace was recorded at the time point indicated by (1), (2), and (3) in the bottom. Bottom: amplitude of the Ba2+ current is plotted. Horizontal bar: duration of the application. Solution 2 (90 mM BaCl2, 20 mM tetraethylammonium chloride) was in the bath and Solution C (150 mM CsCl) was in the pipette.

Effect of GABA on [Ca2+]i

Activation of GABA receptors increased [Ca2+]i because of Ca2+ entry from the extracellular solution, release of Ca2+ from intracellular pools, or a combination of both (Horváth et al. 1993; Lorsignol et al. 1994; Nilsson et al. 1993; Parramón et al. 1995; Walton et al. 1993). To examine the possibility that GABA receptors of the cultured inferior colliculus neuron would mediate increase in [Ca2+]i, we performed [Ca2+]i measurement with the use of fura-2-AM.

In the normal external solution without tetrodotoxin, neither bath application nor focal pressure application of GABA (1.0 mM) increased [Ca2+]i (Fig. 6A), whereas application of 150 mM KCl or 1.0 mM glutamate increased [Ca2+]i (Fig. 6, B and C). In a Ca2+-free external solution with 0.1 mM EGTA, application of GABA, 150 mM KCl, or glutamate did not increase [Ca2+]i (data not shown).


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  		FIG. 6. Effects of application of GABA, high-KCl solution, and glutamate on intracellular Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. The changes in [Ca2+]i after application of (A) 1.0 mM GABA, (B) 150 mM KCl solution, or (C) 1.0 mM glutamate are shown with the normal external solution in the bath. Horizontal bars: duration of the application. Each point represents the mean of 6 cells. SE is shown every 10 s as a vertical line.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In cultured rat inferior colliculus neurons, application of GABA activated membrane conductance, which was not observed in the presence of bicuculline. Muscimol also induced the currents. The GABA-activated currents reversed around 0 mV when the internal and external Cl- concentration was the same. Moreover, internal Cl- substitution with gluconate, an impermeable anion (Barker and Harrison 1988; Fatima-Shad and Barry 1993), shifted reversal potential as predicted from theoretical Cl- equilibrium potentials. These results indicate that GABA activated the membrane conductance primarily through GABAA receptor channels. Recently, another subtype of GABA receptors, a GABAC receptor channel, which is insensitive to bicuculline and baclofen, has been reported in neurons from the frog tectum (Nistri and Sivilotti 1985; Sivilotti and Nistri 1989) and in those from the retina of tiger salamander (Lukasiewicz and Werblin 1994; Lukasiewicz et al. 1994). In this experiment, bicuculline completely blocked the GABA-activated currents. Thus GABAC receptors were unlikely to be involved in the GABA-activated currents in the cultured inferior colliculus neurons.

Through GABAB receptor activation, GABA has been reported to enhance K+ conductance in rat hippocampal pyramidal cells (Gähwiler and Brown 1985; Newberry and Nicoll 1984) and inhibit voltage-activated Ca2+ conductance in embryonic chick sensory neurons (Dunlap and Fischbach 1981) and in rat hippocampal neurons (Scholz and Miller 1991). In this experiment, however, baclofen applied onto the soma or the dendritic region did not affect either K+ or Ca2+ conductance activated by depolarizing pulses, suggesting that GABA may not significantly affect the membrane conductance through GABAB receptors in cultured rat inferior colliculus neurons. It is suggested that GABAB receptors might not be dense enough to affect the membrane conductance in cultured inferior colliculus neurons. However, the distribution of GABAB receptors in the inferior colliculus has been reported. Autoradiographic study has indicated that, in the central nucleus of the rat inferior colliculus, GABAB receptors occur with intermediate binding density among the brain as well as GABAA receptors (Chu et al. 1990). A moderate density of GABAB receptors was also reported in the pigeon inferior colliculus (Veenman et al. 1994). In the rat inferior colliculus, application of baclofen produced inhibition of the neuronal response to acoustic stimuli (Faingold et al. 1989). In a binding study in which slice preparations of the rat inferior colliculus were used, the GABAB binding value was about a third of that of GABAA (Chu et al. 1990). The binding assays of GABAB receptors in the inferior colliculus have not yet been reported on 1-day-old rats. Other brain regions including the hippocampus and the medial geniculate showed substantial density of GABAB receptors in 1-day-old rats (Turgeon and Albin 1994). Therefore it is presumed that GABAB receptors were present in the inferior colliculus of 1-day-old rats, from which the neurons were obtained for culture and the culture conditions in this experiment resulted in the reduction of the expression of GABAB receptors. Moreover, it could be speculated that GABAB receptors need some sort of trophic support that is not necessary for the expression of GABAA receptors. This possibility needs to be studied further.

In cultured rat inferior colliculus neurons, extracellular application of a solution of high concentration of K+ (150 mM) increased [Ca2+]i in the presence of but not the absence of extracellular Ca2+, suggesting that depolarization might increase membrane permeability to Ca2+, presumably by activating voltage-dependent Ca2+ channels. However, GABA did not increase [Ca2+]i regardless of the extracellular Ca2+ concentration. Thus GABA neither depolarized the cultured rat inferior colliculus neurons sufficiently to activate the voltage-dependent Ca2+ channels nor evoked Ca2+ release from intracellular stores. GABA increased [Ca2+]i through GABAA receptor activation in pituitary cells of neonatal rats (Horváth et al. 1993), in embryonic rat spinal cord cells (Walton et al. 1993), and in rat lactotrophs (Lorsignol et al. 1994). Moreover, GABA induced depolarization through GABAA receptors in neonatal rat hippocampal neurons (Ben-Ari et al. 1989), in nerve endings of rat pituitary (Zhang and Jackson 1993), and in dendrites of rat hippocampal pyramidal neurons (Staley et al. 1995). GABAA-receptor-mediated depolarization will lead to activation of voltage-dependent Ca2+ channels and subsequent increase in [Ca2+]i. Whether GABA depolarizes or hyperpolarizes a single neuron through GABAA receptor Cl- channels depends on the Cl- concentration gradient (Cherubini et al. 1991; Nicoll et al. 1990; Zhang and Jackson 1993). In cultured inferior colliculus neurons, the intracellular Cl- concentration would be low so that Cl- flows into the neurons through GABAA receptor channels, which subsequently hyperpolarizes them. The inhibitory effects of GABA on the inferior colliculus have been reported. Iontophoretic studies have shown that GABAergic drugs have inhibitory effects on the rat inferior colliculus neurons (Faingold et al. 1986). The amplitude of the collicular auditory evoked potentials was enhanced after microinjection of bicuculline into the inferior colliculus, which was inhibited after the microinjection of 4,5,6,7-tetrahydroisoxasolo-5,4 pyridin-2,3-ol, a GABAA agonist (Bagri et al. 1989). Electrically stimulated postsynaptic depolarizing field potential of the inferior colliculus was abolished by local application of GABA (Hosomi et al. 1995). In inferior colliculus neurons, it is more likely that GABA hyperpolarizes the neurons rather than depolarizes them, although the possibility that GABA could depolarize the inferior colliculus neurons to the extent not to activate the voltage-dependent Ca2+ conductance has not been excluded.

The inferior colliculus is a critical site for inducing audiogenic seizure (Kesner 1966; McCown et al. 1984; Wada et al. 1970). The GABAergic system in the inferior colliculus has been reported to be involved in the initiation of audiogenic seizure. Microinjection of GABA into the inferior colliculus blocked audiogenic seizure (Duplisse et al. 1974). Focal blockade of GABAA receptors by bicuculline in the inferior colliculus of normal rats resulted in susceptibility to audiogenic seizure (Bagri et al. 1989; Millan et al. 1986). Reduction in GABA-mediated inhibition (Faingold et al. 1986) and a significant increase in GABA (Ribak et al. 1988) have been reported in the inferior colliculus of the genetically epilepsy-prone rat, in which reuptake of GABA released is possibly enhanced. Thus GABAergic suppression appears to regulate the audiogenic seizure susceptibility. Our results suggest that GABA would hyperpolarize cultured neurons from the central nucleus of the rat inferior colliculus primarily through GABAA receptors. Along with the function of regulating auditory transmission, these GABA-sensitive neurons could relate to the initiation of audiogenic seizure, although this possibility needs to be studied further.

    ACKNOWLEDGEMENTS

  We thank Dr. Dermot Cox for careful reading of the manuscript and for correcting the English.

  This work was supported by grants from the Ministry of Education, Science, and Culture of Japan and was performed during the tenure of a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.

    FOOTNOTES

  Address for reprint requests: M. Mori, Dept. of Physiology, Kobe University, School of Medicine 7-5-1, Kusunoki-Cho, Chuo-Ku, Kobe 650, Japan.

  Received 26 January 1996; accepted in final form 7 October 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References
0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society



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