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Institute of Biology, University of Oldenburg, D-26111 Oldenburg, Germany
Submitted 22 March 2004; accepted in final form 28 June 2004
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ABSTRACT |
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-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%. |
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INTRODUCTION |
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; Sivilotti and Nistri 1991), although excitatory actions of GABA have been described that seem especially important for developmental processes (Ben Ari 2002; Owens and Kriegstein 2002). GABA activates ionotropic GABAA and GABAC receptors by opening of an integral ion channel selectively permeable to chloride ions (Bormann and Feigenspan 1995; Bormann et al. 1987). Whereas bicuculline competitively blocks GABAA receptors, it has no effect on GABAC receptors (Feigenspan et al. 1993; Polenzani et al. 1991; Qian and Dowling 1993).
GABAA receptors consist of various combinations of 
14 different subunits, which determine their physiological and pharmacological properties (Barnard et al. 1998; Sieghart 1995). Although the multiplicity of subunits suggests a daunting number of possible combinations, it seems that only some of these combinations are realized. So native receptors contain at least one 
, one , and one 
subunit with the other subunits as possible substitutes for (McKernan and Whiting 1996). The importance of subunit composition for correct targeting of assembled GABAA receptors to synaptic sites has recently been suggested (Moss and Smart 2001).
Horizontal cells are a class of second-order interneurons that modulate the signal transfer between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the vertebrate retina. They are extensively coupled via gap junctions and thereby help establish the antagonistic receptive field structure of bipolar and ganglion cells. Whereas most mammalian species possess two morphologically distinct types of horizontal cell, only one type has been described in the retina of the mouse (Jeon et al. 1998; Peichl and Gonzalez-Soriano 1994). The dendrites of this type synapse exclusively with cones, and the axon terminal system receives synaptic input exclusively from rods. The GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) has been localized to horizontal cells of the cat retina (Sarthy and Fu 1989), pinpointing GABA as a potential neurotransmitter of horizontal cells. The question of whether GABA is released by a vesicular or a nonvesicular mechanism has not been unequivocally answered yet. However, recent data demonstrate the presence of a vesicular GABA transporter in horizontal cells, suggesting that GABA is transported and stored into vesicles, although expression of the vesicular transporter in the plasma membrane cannot be excluded (Cueva et al. 2002).
The physiological properties of mammalian horizontal cells have so far been studied mainly by measuring their light responses using intracellular recording techniques. With the exception of glutamate receptors (Blanco and de la Villa 1999; Rivera et al. 2001), a detailed analysis of their equipment with ligand-gated ion channels is not available. Because the mouse has become increasingly important in the study of retinal neurobiology, we developed a preparation to investigate the physiological fingerprint of horizontal cells by using whole cell and single-channel configurations of the patch-clamp technique. In this paper, we describe the physiological and pharmacological properties of GABAA receptors expressed by solitary mouse horizontal cells, and we determine their single-channel characteristics.
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METHODS |
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Two- to 4-mo-old C57/B mice were deeply anesthetized by intraperitoneal injection of a 0.1 ml solution containing equal parts of 5% ketamine (Ceva, Düsseldorf, Germany) and 1% xylazine (Ceva) and subsequently killed by cervical dislocation. After removal of the cornea, lens, vitreous body, and sclera, the retina was transferred to 1 ml digestion buffer containing 20 U/ml papain (Worthington Biochemical, Freehold, NJ) and 200 U/ml DNase I (Sigma, Deisenhofen, Germany) in Earle's balanced salt solution (EBSS; Sigma). After 40–45 min digestion at 37°C, the retina was transferred to trituration buffer to stop papain activity (5 min, 37°C). This solution contained 1 mg/ml ovomucoid inhibitor (Worthington), 1 mg/ml bovine serum albumin (Sigma), and 100 U/ml DNase I (Sigma) in EBSS. The tissue was centrifuged at 1,000 rpm (5 min, 22°C), and the pellet was resuspended in minimum essential medium (MEM; Sigma). Subsequently, the retina was triturated with fire-polished Pasteur pipettes of decreasing open diameter, and after each trituration step, the cell suspension was carefully checked for the presence of horizontal cells. Those fractions containing horizontal cells were finally pooled, and the cell suspension was plated on glass coverslips, which had been coated with 1 mg/ml concanavalin A (Sigma). The cells were kept in an incubator in 5% CO2-55%O2 at 37°C. After 15–20 min, 1% fetal calf serum (Sigma) was added to improve viability of the cells.
Immunocytochemistry
Horizontal cells plated on glass coverslips were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 min. After several rinses in PB, the coverslips were incubated in a solution containing 5% normal goat serum (NGS) and 0.3% Triton X-100 in PB for 1 h. A polyclonal antibody against the calcium-binding protein calbindin D-28K (SWant, Bellinzona, Switzerland) was diluted 1:500 in a solution containing 3% NGS and 0.3% Triton X-100 in PB for 12–14 h. Binding of the primary antibody was visualized with a goat anti-rabbit Alexa 568 (diluted 1:200; Molecular Probes, Eugene, OR) secondary antibody (2 h). To prevent bleaching, the cells were embedded in VectaShield (Vector Laboratories, Burlingame, CA). Images were taken on a confocal laser scanning microscope (Leica, Nussloch, Germany) using the 568 line of a krypton-argon laser.
Reverse transcriptase-PCR
For reverse transcriptase-PCR, visually identified isolated horizontal cells were harvested with a patch pipette. The intracellular solution contained 140 mM KCl, 0.5 mM EGTA, and 10 mM HEPES, pH 7.4. After seal formation, the cellular contents of individual horizontal cells were carefully aspirated into the pipette by applying negative pressure. The electrode was then lifted from the bath under constant visual control to avoid contamination with neighboring cells or debris. The tip of the pipette was finally broken into an Eppendorf tube containing 20 U RNase inhibitor (RNasin; Promega, Mannheim, Germany). After brief centrifugation, the tube was frozen on dry ice and stored at –80°C.
Contaminating genomic DNA was digested with DNase I (Amplification Grade, Invitrogen) according to the manufacturer's protocol. cDNA synthesis was carried out in a final volume of 25 µl. Each sample contained 1x first-strand buffer (Promega) 0.6 µM oligo(d)T primer (Promega), 0.6 µM random primer (Promega), 0.5 mM of each dNTP (Eppendorf, Hamburg, Germany), and 0.8 U/µl RNasin ribonuclease inhibitor. After primer annealing for 10 min at 72°C, the samples were briefly chilled on ice and incubated for 2 min at 42°C before 0.3 U/µl AMV reverse transcriptase (Promega) was added. cDNA synthesis was carried out for 1 h at 42°C and stopped by incubating the samples for 5 min at 95°C. cDNAs were stored at –20°C.
PCR reactions were carried out in a total volume of 25 µl. This included 6 µl horizontal cell cDNA, 1x reaction buffer (Promega), 1.25 mM MgCl2, 0.2 mM of each dNTP (Eppendorf), 0.8 µM of each primer (MWG), and 1 U Taq-polymerase (Promega). Reactions were overlaid with 35 µl mineral oil (Sigma). The Taq-polymerase was added after incubating the samples for 2 min at 95°C (hot start). The specific primer set for the detection of calbindin included the forward primer (5'-GACGCTGATGGAAGTGGTTAC-3') and the reverse primer (5'-ACGGTCTTGTTTGCTTTCTCT –3'), both of which were designed according to the mouse calbindin coding sequence (GenBank Accession No. NM031984). The predicted size of the amplicon was 340 bp. Amplifications of calbindin transcripts were carried out in a Stratagene Robocycler using the following protocol: 95°C for 2 min, 40 cycles of 95°C for 1 min, 60°C for 1.5 min, 72°C for 3 min, and finally 72°C for 5 min. 20 µl of each amplification product were analyzed on a 2% agarose gel and visualized on a transilluminator after ethidium bromide staining.
Electrophysiological recordings
Cells were allowed to settle 30 min before commencement of recordings. Coverslips with retinal neurons were placed in a recording chamber (Luigs and Neumann, Ratingen, Germany) on the stage of an upright microscope (Leica). Horizontal cells were identified as described in the following text using x40 and x63 water-immersion objectives equipped with Nomarski optics (Leica). Whole cell voltage-clamp and outside-out single-channel recordings were performed with an EPC9 double patch-clamp amplifier (Heka, Lambrecht, Germany). Current traces were monitored with a digital oscilloscope (Tektronix, Beaverton, OR) and directly stored to the hard disk of a personal computer. Data were acquired with a sample frequency of 200 Hz in the whole cell mode and with a frequency of 10 kHz for single-channel experiments. Patch pipettes were pulled from borosilicate glass (1.5 mm OD, 1.275 mm ID; Hilgenberg, Malsfeld, Germany) using a horizontal electrode puller (Sutter, Novato, CA). Electrodes with a resistance ranging from 4 to 7 M
were connected to the amplifier with an Ag/AgCl wire. The electrode holder combined with the headstage were mounted on a mechanical, remote-controlled device attached to a three-dimensional micromanipulator (Luigs and Neumann). In whole cell experiments, the series resistance of the electrodes usually ranged between 8 and 15 M and was not compensated for. However, the series resistance was carefully monitored in the time course of an experiment, and only those recordings with a stable series resistance value were considered for analysis. Drugs were applied to horizontal cells or outside-out membrane patches in the extracellular bath solution by the pressure-driven application system DAD-12 Superfusion System (ALA Scientific Instruments, Westbury, NY).
Isolated horizontal cells were continuously superfused (0.5 ml/min) at room temperature with an extracellular solution containing (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose (pH 7.4). The intracellular solution for recordings of whole cell and single-channel currents contained (in mM) 120 CsCl, 20 TEA-Cl, 1 CaCl2, 2 MgCl2, 11 EGTA, and 10 HEPES (pH 7.2). Diazepam, zolpidem, alphaxalone, and methyl 6,7-dimethoxy-4-ethyl--carboxylate (DMCM) (all from Sigma) were prepared as 10-mM stock solutions in DMSO and stored at –20°C. The maximal final concentration of DMSO was 0.03%, which had no effect on GABA-induced currents. Bicuculline methiodide and picrotoxin (both from Sigma) were freshly prepared and added to the GABA containing solution. ZnCl2 (Fluka, Buchs, Switzerland). Pentobarbital (Sigma) was prepared as 10-mM stock solutions in extracellular solution and kept frozen at –20°C.
Data analysis
Current amplitudes were normalized and expressed as the ratio of the GABA-induced peak current in the presence of the drug relative to the control GABA response. For dose-response curves, current amplitudes were normalized to the maximum response, obtained either with saturating concentrations of GABA or, for studies of inhibitory effects, in the absence of antagonists. Data points of dose-response curves were fitted with a sigmoidal logistic function using a Simplex algorithm: I/Imax = 1/[1 + (c/k)n], where c denotes the concentration of agonist or antagonist, k the half-maximally effective concentration, and n the Hill coefficient.
GABA-induced single-channel events in outside-out patches were analyzed after low-pass filtering at 1 kHz (–3 dB, 4-pole Bessel filter). Bursts were constructed by choosing 5 ms as the intraburst interval. Time frames containing bursts of single-channel openings were selected, converted into all-point histograms, and subsequently fitted with multiple Gaussian distributions. Open times, closed times, and open probabilities were determined by half-amplitude threshold analysis. For open time distributions, number of events per burst were binned in 2-ms intervals, and the data were fitted with a first-order exponential probability density function: f(t) = a exp(–t/
), where denotes the mean of the distribution. Desensitizing and deactivating current traces were also fitted with a first-order exponential function. All data analysis was performed with the Pulsefit (Heka), MatLab (MathWorks, Natick, MA) and Origin software packages (Microcal, Natick, MA).
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RESULTS |
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It has been shown previously that only one type of horizontal cell is present in the retina of mice and rats (Jeon et al. 1998; Peichl and Gonzalez-Soriano 1994). This type of horizontal cell typically displays a multipolar morphology, with a long, thin axon, extending within the outer plexiform layer and ramifying in an elaborate axon terminal system. Thus these cells belong to the axon-bearing type or B-type of horizontal cells as described in other mammalian species like cats and primates (Boycott et al. 1978; Kolb et al. 1980). In the mammalian retina, antibodies to the calcium-binding protein calbindin D-28K have been effectively used to label horizontal cells in the rabbit (Röhrenbeck et al. 1987, 1989) and the mouse (Haverkamp and Wässle 2000).
Horizontal cells were obtained from the mouse retina after enzymatical and mechanical dissociation. Because their thin axons are likely to be ruptured, the dissociation process resulted in horizontal cell bodies and axon terminals as separate entities. Horizontal cell somata were characterized by a polygonal-shaped perikaryon that measured 14 µm on average and gave rise to five to eight primary dendrites (Fig. 1, A and B). To avoid confusion, it should be noted that we recorded exclusively from horizontal cell bodies, which will simply be referred to as horizontal cells in the remainder of the text. Although the axon and probably the fine distal dendrites were lost during the dissociation procedure, horizontal cells were viable and readily accessible for patch-clamp electrodes. To confirm their identity, we harvested the cytoplasm of five visually identified horizontal cells after obtaining their electrophysiological fingerprint, and subsequently we performed single-cell reverse transcriptase-PCR with primers specific for calbindin D-28K. In all cases, we obtained a signal at the expected size of 340 bp (Fig. 1B, inset). In addition, immunocytochemistry with polyclonal antibodies directed against calbindin D-28K was carried out on paraformaldehyde-fixed isolated cells. All cells previously identified as horizontal cells showed calbindin-like immunoreactivity (Fig. 1B), thus confirming our PCR results and strongly suggesting that the cells chosen for recordings were horizontal cells. In addition, we performed control experiments on dissociated cells with bipolar morphology, which do not contain calbindin D-28K. Both reverse transcriptase-PCR and immunocytochemistry were negative for these cells (data not shown).
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GABA concentration-response relationships
Morphologically identified horizontal cells were voltage-clamped at a holding potential of –70 mV with equal concentrations of chloride on the intra- and extracellular sides of the membrane. In the whole cell mode of the patch-clamp technique, extracellular application of GABA induced chloride-mediated inward currents in all horizontal cells tested. We obtained successful recordings from a total of 174 horizontal cells. Stable seals with resistances between 2 and 10 G could be established in >90% of the recordings, indicating that the dissociation procedure did not impair the overall structure of the cell membrane. Because GABA concentrations 
1 µM consistently failed to induce measurable currents, GABA was applied at concentrations ranging from 3 to 1,000 µM. Desensitization of GABA receptor-mediated currents was dose-dependent and became apparent at GABA concentrations of 30 µM (Fig. 2A). The maximal current measured at a saturating concentration of GABA (1 mM) was variable and ranged in amplitude from –241 to –1210 pA with a mean value of –633 ± 100 (SE) pA (n = 11).
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Current-voltage (I-V) relationships of GABA-induced currents were obtained by ramping the membrane potential from –70 to 70 mV (100 mV/s) in the presence of 100 µM GABA (Fig. 2D). Nonspecific leak conductances were determined by performing the same ramping protocol in the absence of GABA and eventually subtracting the two current traces. The I-V-curve was linear with no sign of rectification as indicated by fitting the data points with a linear regression function. The current reversed sign close to 0 mV, which is the expected equilibrium potential given symmetrical chloride concentrations on both sides of the membrane.
In addition, we determined the kinetics of activation, desensitization, and deactivation of mouse horizontal cell GABAA receptors. The average rate of activation was measured as the 10–90% rise time of currents elicited by 1 mM GABA. The 10–90% rise time to peak current amplitudes was 89 ± 9 ms (n = 19). Three-second pulses of GABA (1 mM) were used to determine desensitization kinetics. The decline of currents was well fitted using a first-order exponential function with a time constant of 1,142 ± 50 ms (n = 19; Fig. 2E). Similarly, the deactivation properties of GABA receptor channels were measured by fitting a first-order exponential function to the current trace immediately after a 3-s application of GABA (50 µM). Currents decayed slowly with a time constant of 490 ± 24 ms (n = 19; Fig. 2F).
Inhibitory effects of bicuculline and picrotoxin
The plant alkaloid bicuculline has been described as a competitive and reversible blocker of GABAA receptor-mediated currents. Concentrations of bicuculline ranging from 0.1 to 100 µM were co-applied with 50 µM GABA. As shown in Fig. 2C, this GABA concentration induced 
66% of the maximal inward current. Because the application of seven different concentrations took a considerable amount of time, it was crucial to continuously monitor experimental conditions. Therefore control applications of 50 µM GABA without bicuculline were frequently performed in the course of an experiment. A final application of GABA served to indicate that the observed inhibition was indeed caused by the drug and not by rundown of currents or deterioration of the recording conditions. Only those horizontal cells displaying stable GABA-induced inward currents were considered for analysis.
Figure 3A shows a consecutive series of current traces obtained from co-application of 50 µM GABA with increasing concentrations of bicuculline. GABA-induced inward currents were reduced by bicuculline in a dose-dependent manner. The effect of the drug was fully reversible as indicated by the current amplitude under washout conditions. Complete dose-response curves were recorded from six horizontal cells. Low concentrations of bicuculline (1 µM) inhibited GABA-induced currents to 0.64 on average, whereas the current was completely eliminated by 100 µM (Fig. 3A). Low concentrations of bicuculline had a stronger effect on peak amplitudes than on steady-state values, whereas the overall response became nondesensitizing with concentrations >3 µM bicuculline (Fig. 3A). The IC50 values for individual cells ranged from 1.2 to 3.5 µM with a median value of 1.6 µM. Because the inhibitory effect of bicuculline appeared homogeneous across horizontal cells, we calculated the mean of all responses obtained at a given concentration of bicuculline and fitted the data points with a sigmoidal logistic function (Fig. 3B). The IC50 value obtained from the best fit to the data points was 1.7 ± 0.1 µM with a Hill coefficient of 1.1 ± 0.1. These results indicate that horizontal cell GABA responses are sensitive to the inhibitory action of bicuculline. Furthermore, GABA receptor currents are mediated exclusively by GABAA receptors, but not by GABAC receptors, as indicated by the complete block of GABA-induced currents with high concentrations of bicuculline.
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Picrotoxin has been described as an open channel blocker selective for GABA- and glycine-gated chloride channels. To determine its use-dependent mode of inhibition, picrotoxin was applied twice at a given concentration and then thoroughly washed out before the next concentration was tested (Fig. 4A). Consecutive applications of picrotoxin exerted a differential effect on peak and steady-state current values. At 10 µM picrotoxin, 0.60 of the control peak current remained during the first application, whereas the current was reduced to 0.38 during the second application (Fig. 4, A and B). A similar effect was observed at 100 µM concentration with 0.28 and 0.15 of control current values remaining after the first and second application of picrotoxin, respectively. When the steady-state values of the currents were compared, however, two applications of picrotoxin blocked very similar fractions of the GABA-induced current. The fractions measured for the two applications at 10 µM (0.45 and 0.46) and 100 µM picrotoxin (0.13 and 0.16) did not show a significant difference (P < 0.01, t-test). The mean current amplitudes reflecting these findings are summarized in Fig. 4B. This effect was observed in all eight horizontal cells tested. Additional applications of picrotoxin did not further reduce GABA-evoked currents. Interestingly, we consistently found that GABA responses of mouse horizontal cells were not entirely abolished even at high concentrations of picrotoxin. The mean amplitudes of peak and steady-state control responses before and after washout of the drug did not show a statistically significant difference (P < 0.01, t-test), indicating that the inhibitory effect of picrotoxin on GABA receptor-mediated chloride currents was almost fully reversible (Fig. 4B).
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The divalent transition metal cation Zn2+ has been shown to noncompetitively antagonize GABAA receptor-mediated currents (Hosie et al. 2003; Legendre and Westbrook 1991; Westbrook and Mayer 1987). Zn2+ ranging in concentrations from 1 to 1,000 µM was co-applied with 50 µM GABA to eight isolated horizontal cells. Figure 5A shows the inhibitory effect of increasing concentrations of Zn2+. Block of GABA-evoked currents by Zn2+ was fully reversible after complete removal of the blocker from the extracellular solution. It appears that the presence of Zn2+ slowed the onset of the GABA-induced inward currents (Fig. 5A). This effect was consistently observed in all horizontal cells tested.
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Furthermore, we determined the mode of horizontal cell GABAA receptor inhibition by Zn2+. Dose-response curves for GABA were measured under control conditions and in the presence of 5 µM Zn2+ (Fig. 5C). EC50 values calculated from fitting the dose-response curves with a sigmoidal logistic equation were 32.7 ± 3.6 µM (n = 9) for control conditions, and 34.3 ± 2.5 µM (n = 9) in the presence of 5 µM Zn2+. These values were not significantly different (P < 0.01, t-test). In addition, the ratio of GABA-induced currents with and without Zn2+, respectively, could be well fitted with a linear regression line displaying a slope of 0.02 ± 0.04, suggesting that the amount of block exerted by Zn2+ does not depend on the GABA concentration (Fig. 5D). These results indicate that Zn2+ blocks GABAA receptors of mouse horizontal cells in a noncompetitive manner.
Effects of benzodiazepines, barbiturates, and steroids
GABAA receptors are known to be modulated by benzodiazepines like diazepam or flunitrazepam, which potentiate GABA-induced currents by increasing the frequency of channel opening (Study and Barker 1981). Furthermore, it has been shown that the 2 subunit is required to confer benzodiazepine sensitivity to GABAA receptors (Pritchett et al. 1989). To study the effects of benzodiazepines on horizontal cell GABAA receptors, we applied diazepam in concentrations ranging from 1 to 10 µM together with 50 µM GABA. Diazepam potentiated the peak current in all eight horizontal cells tested with a mean enhancement of 2.51 ± 0.16 at the highest concentration of 10 µM (Fig. 6A). A dose-response curve was obtained by calculating the normalized mean responses of all cells and fitting the data points with a logistic function (Fig. 6B). The EC50 value for diazepam was 6.6 µM with a Hill coefficient of 0.8. The potentiating effects diazepam were fully reversible after washout of the drug.
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; Pritchett et al. 1989) in the concentration range of 1–10 µM. Like diazepam, zolpidem augmented the inward currents induced by 50 µM GABA with a maximal enhancement of 2.48 ± 0.13 at 10 µM (Fig. 6C). Again, a dose-response curve was generated by normalizing the current amplitudes to the control response to 50 µM GABA and plotting the normalized values against the concentration of zolpidem (Fig. 6D). The EC50 value was 85 nM with a Hill coefficient of 0.7, indicating high affinity of horizontal cell GABAA receptors for the BZ1-selective drug zolpidem.
In addition, GABA-induced currents were uniformly inhibited by the inverse benzodiazepine agonist DMCM. At a concentration of 1 µM, currents were reduced to 0.67 ± 0.02 of control values. Barbiturates have been described to modulate GABAA receptor function by increasing the mean open duration time of the chloride channels (Macdonald et al. 1989), whereas the synthetic neuroactive steroid alphaxalone acts by increasing the average open time and opening frequency (Twyman and Macdonald 1992). Both compounds enhanced GABAA receptor-mediated currents of horizontal cells. Pentobarbital (50 µM) increased current amplitudes to 2.68 of control values (n = 11), and alphaxalone (1 µM) augmented the GABA response to 5.1 (n = 10). The pharmacological properties of mouse horizontal cell GABAA receptors are summarized in Table 1.
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We determined the single-channel conductance of GABAA receptors by recording from outside-out patches pulled from the cell bodies of isolated horizontal cells. Channel openings induced by 10 µM GABA (10 s) were recorded at a holding potential of –70 mV. Because of the low density of GABAA receptors on the surface of horizontal cell bodies, single-channel events could be recorded in <50% of all membrane patches. In those patches containing GABAA receptors, extracellular application of GABA induced single-channel openings to multiple conductance levels (Fig. 7A).
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In addition, we measured the main conductance and both subconductance states by recording GABA-induced single channel currents at holding potentials ranging from –70 mV to 70 mV. The resulting single-channel current amplitudes were plotted against voltage and fitted with a linear regression line. The conductances measured from the slope of the I-V relationships were 29.8 ± 0.7 pS for the main level, and 20.2 ± 0.3 and 10.8 ± 0.1 pS for the two subconductance states, respectively (Fig. 7E).
Finally, we determined the open time distribution of GABA-induced single-channel events to the main conductance level (Fig. 7F). The majority of openings took place within 1–3 ms, whereas open times >10 ms occurred very infrequently. The distribution was fitted by an exponential probability density function with a mean of 2.43 ± 0.04 ms. The mean open time, the mean closed time, and the open probability (Po) for the main conductance and the two subconductance states are summarized in Fig. 7G. The main conductance (M) and the second subconductance state (S2) showed similar open times (2.23 ± 0.15 ms for M, 2.14 ± 0.32 ms for S2), and closed times (2.43 ± 0.23 ms for M, 2.30 ± 0.25 ms for S2). Thus open probabilities for M and S2 were not significantly different (44.6 ± 1.9% for M, 42.4 ± 3,2% for S2; P < 0.01, t-test). In contrast, the first subconductance state (S1) was characterized by a significantly increased value for Po (Fig. 7G). Although open times are slightly smaller compared with M and S2, closed times are even more reduced (Fig. 7G). The single-channel properties of mouse horizontal cell GABAA receptors are summarized in Table 2.
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DISCUSSION |
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). In contrast, a high percentage of immature rabbit retinal cells grown in monolayer culture express TTX-sensitive sodium channels (Löhrke and Hofmann 1994). Thus the presence of voltage-dependent sodium channels appears to differ between species, cell types, and preparations.
It has been shown previously that exposure to proteases like papain does not affect the properties of GABA-gated chloride channels of dopaminergic amacrine cells of the mouse retina (Gustincich et al. 1997). Similarly, other studies have also reported no alterations of the GABA response when comparing digested tissue and cultured neurons (Kapur and Macdonald 1996).
Physiological properties of horizontal cell GABAA receptors
In summary, GABA responses were characterized by high-threshold doses of 3 µM of the agonist, variable but generally small amplitudes even at saturating concentrations of GABA, and rather low affinities. Although current amplitudes differed within half an order of magnitude, values normalized to the maximum current were remarkably similar. The EC50 value measured for mouse horizontal cells was 30.1 µM, which is in about the same range than that of acutely dissociated pyramidal neurons, adult cortical neurons, and thalamic neurons (Celentano and Wong 1994; Oh et al. 1995). Individual EC50 values were evenly distributed around the mean. These results suggest expression by horizontal cells of a variable number of GABAA receptors but very similar affinities of these receptor molecules for GABA.
Pharmacological properties and receptor subunit composition
It has been a common observation that GABA-induced currents of all horizontal cells tested were affected by the various modulators and inhibitory substances in a very similar manner. Thus given the physiological similarity described in the preceding text, it is tempting to speculate that all horizontal cells express the same or a highly related GABAA receptor subunit composition. The effect of subunit composition on the affinity of GABAA receptors for GABA has been studied in detail using heterologous expression systems (Ebert et al. 1994; Saxena and Macdonald 1996).
GABA-induced currents were completely and reversibly blocked by the competitive GABAA receptor antagonist bicuculline, indicating that mouse horizontal cells express exclusively GABAA receptors. In contrast, horizontal cells of teleost fish have been shown to contain both GABAA and GABAC receptors (Qian and Dowling 1993). The measured IC50 value is very similar to that obtained for acutely isolated dopaminergic amacrine cells of the mouse retina and cultured amacrine cells of the rat retina (Feigenspan and Bormann 1998; Feigenspan et al. 2000).
Benzodiazepine receptors are usually subdivided into two different pharmacological types, BZ1 and BZ2. Whereas BZ1 sites show high affinity for CL-218872, zolpidem, and -carbolines, BZ2 sites are characterized by a lower affinity for these substances and a high affinity for flunitrazepam. GABA responses of all horizontal cells were modulated by the BZ1-preferring agonist zolpidem as well as the benzodiazepine agonist diazepam and the inverse agonist DMCM. The EC50 value for zolpidem was 85 nM, whereas the affinity of the GABA receptors for diazepam was about an order of magnitude lower (6.6 µM), indicating BZ1-like pharmacological properties. However, maximal enhancement of GABA-induced currents was very similar (2.51 for diazepam and 2.48 for zolpidem). Studies in heterologous expression systems have suggested that sensitivity to benzodiazepines is conferred by the presence of the 2 subunit (Pritchett et al. 1989). The augmentation by diazepam in all horizontal cells tested could therefore be explained by the expression of the 2 subunit. In addition, the characteristics of a BZ1 binding site are consistent with the presence of the 1 subunit. Because the presence of the 4 subunit in combination with one and the 2 subunit confers insensitivity to benzodiazepines (Wisden et al. 1991), we exclude the possibility of 4 expression in horizontal cell GABAA receptors.
With an IC50 value of 7.3 µM, GABA receptor currents of horizontal cells displayed a moderate to high sensitivity to the divalent metal cation Zn2+. The effects of Zn2+ on GABA-induced currents are determined by the receptor isoforms (Draguhn et al. 1990; Smart et al. 1991). The presence of 1 or x (x = 1–3) confers high sensitivity to Zn2+, whereas on addition of a subunit, the inhibitory effect of Zn2+ is lost. However, it is reasonable to assume that the presence of a subunit causes a decrease in the susceptibility to Zn2+ rather than a total loss (Saxena and Macdonald 1996). Therefore the observed sensitivity of horizontal GABAA receptors to the inhibitory action of Zn2+ is compatible with the presence of the 2 subunit in all receptors.
Even high concentrations of the open channel blocker picrotoxin did not entirely block GABA-mediated inward currents. Picrotoxin has been shown to directly activate chloride channels by interacting with the 1 subunit (Sigel et al. 1989), suggesting the presence of this subunit in mouse horizontal cells.
It has been shown that the neuromodulatory effects of steroids are at least partially determined by subunit composition (Gee and Lan 1991; Korpi and Luddens 1993; Lan et al. 1991; Puia et al. 1990, 1993). A large potentiating effect has been associated with expression of the 3 subunit (Lambert et al. 1995; Lan et al. 1991), whereas the presence of the 
subunit inhibits neurosteroid modulation (Zhu et al. 1996). In addition, recombinant receptors containing 1 are more sensitive to neurosteroids than those containing 6, but the identity of the subunit apparently does not play a crucial role (Zhu et al. 1996). In our hands, the neuroactive steroid alphaxalone caused a large potentiation of GABA-induced currents. The magnitude of this effect is very similar to the augmentation of GABAA receptor currents observed in dopaminergic amacrine cells (Feigenspan et al. 2000), and it is in good agreement with a GABA receptor containing 1, but lacking the subunit.
The kinetic properties of mouse horizontal cell GABAA receptors are similar to those of heterologously expressed combinations of 132L (Haas and Macdonald 1999). The slow time constants obtained by fitting the current traces with first-order exponential functions range within the same order of magnitude.
Finally, preliminary evidence indicates that GABA-induced currents of horizontal cells are modulated by extracellular dopamine. This effect is most likely mediated by activation of cAMP-dependent protein kinase A (PKA) (Feigenspan and Bormann 1994), and biochemical work has identified the 3 subunit as the main target for PKA (Browning et al. 1993).
In summary, the physiological and pharmacological data indicate that GABAA receptors expressed by mouse horizontal cells are composed of the 1, 1, 3, and 2 subunits. Although the combination 122 is the most abundant in the brain (McKernan and Whiting 1996) and considered to represent the BZ1 subtype, the nature of the subunit does not appear to be relevant for determining benzodiazepine pharmacology (Benke et al. 1994; Hadingham et al. 1993). Because 19% of the GABAA receptors in the rat cerebral cortex contain both 1 and 3 subunits (Li and De Blas 1997), 1132 also seems a likely combination for horizontal cell GABAA receptors. Concerning the insensitivity to picrotoxin, it is also reasonable to assume two different types of GABAA receptor with those lacking the 1 subunit being resistant to the antagonist.
GABA transporters
We currently do not know whether or not mouse horizontal cells express GABA transporters as described in lower vertebrates (Schwartz 1982, 1987). Immunocytochemical studies have demonstrated the presence of the GABA transporters GAT-1, -2, and -3 in amacrine cells, displaced amacrine cells, interplexiform cells, pigment epithelium, and Müller cells of the rat retina. Surprisingly, however, horizontal cells in the same preparation appeared negative for the known GABA transporters (Brecha and Weigmann 1994; Johnson et al. 1996). In contrast, a vesicular GABA transporter is expressed in horizontal cells (Cueva et al. 2002), indicating the possibility of vesicular GABA release in the mammalian retina or expression of a vesicular transporter in the plasma membrane.
We found no evidence for the presence of a GABA transporter current in mouse horizontal cells. The GABA-induced response was completely blocked by the selective GABAA receptor antagonist bicuculline, indicating that the current is entirely mediated by GABAA receptors. The picrotoxin-resistant fractional current, which served as evidence for a GABA transporter in a lower vertebrate retinal preparation (Dong et al. 1994), is most likely caused by a differential subunit composition of GABAA receptors.
To our knowledge, there is no ultrastructural evidence for the existence of GABAergic synapses between horizontal cells. Thus it is tempting to speculate that GABA receptors of horizontal cells are extrasynaptic as described for GABAA receptors elsewhere in the nervous system (Fritschy et al. 1992; Nusser et al. 1995, 1996). A possible function of these receptors would then be the continuous monitoring of the GABA concentration in the outer retina, which in turn affects the signal processing properties on the level of horizontal cells.
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ACKNOWLEDGMENTS |
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Address reprint requests and other correspondence to: A. Feigenspan (E-mail: andreas.feigenspan{at}uni-oldenburg.de).
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INTRODUCTION
, steady-state currents, which are plotted as fractions of the peak currents (
). Steady-state currents were measured at the end of the 5-s application. Each column represents the mean ± SE of 8 horizontal cells.
