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University of Zurich, Institute of Pharmacology and Toxicology, Zurich, Switzerland
Submitted 10 November 2005; accepted in final form 8 May 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-subunit rendering individual GABAAR subtypes insensitive to diazepam without altering their GABA sensitivity and expression of receptors. Whole cell patch-clamp recordings were performed in hippocampal pyramidal cells from single, double, and triple mutant mice. Comparing diazepam effects in knock-in and wild-type mice allowed determining the contribution of 1, 2, 3, and 5 subunits containing GABAARs to phasic and tonic forms of inhibition. Fast phasic currents were mediated by synaptic 2-GABAARs on the soma and by synaptic 1-GABAARs on the dendrites. No contribution of 3- or 5-GABAARs was detectable. Slow phasic currents were produced by both synaptic and perisynaptic GABAARs, judged by their strong sensitivity to blockade of GABA reuptake. In the CA1 area, but not in the subiculum, perisynaptic 5-GABAARs contributed to slow phasic currents. In the CA1 area, the diazepam-sensitive component of tonic inhibition also involved activation of 5-GABAARs and slow phasic and tonic signals shared overlapping pools of receptors. These results show that the major forms of inhibitory neurotransmission in hippocampal pyramidal cells are mediated by distinct GABAARs subtypes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Mody and Pearce 2004). GABAARs are composed of five subunits, most frequently two , two 
, and one 
subunit. Different subunit isoforms (1–6, 1–4, 1–4, 
, 
, 
, 
) give rise to a considerable diversity of GABAARs (Barnard et al. 1998; Mohler et al. 2002; Sieghart and Sperk 2002) that are differentially expressed in the brain and localized in different cell types and subcellular areas (Fritschy and Mohler 1995; McKernan and Whiting 1996; Pirker et al. 2000). Although much is known about the distribution and subcellular location of major GABAAR subtypes, the functional significance of this diversity is less well understood, chiefly because of a lack of pharmacological tools that distinguish between the different subtypes. The presence of a 2 or 3 subunit is required for the formation of a benzodiazepine-binding site (Knoflach et al. 1991; Pritchett et al. 1989). Additionally, the subunit variant determines activation and deactivation kinetics of GABAARs (Banks and Pearce 2000; Bosman et al. 2002; Hutcheon et al. 2000; Vicini et al. 2001) and their affinity for classical benzodiazepines (Scholze et al. 1996; Smith et al. 2001) such as diazepam (DZ). Mutation of a conserved histidine residue (His 101 in the 1 subunit) into an arginine renders the corresponding GABAAR DZ-insensitive (Benson et al. 1998; Rudolph et al. 1999; Wieland et al. 1992). Using gene targeting to introduce this point mutation in various subunit variants in vivo, it has been shown that different GABAAR subtypes mediate distinct effects of DZ (Rudolph and Mohler 2004). In particular, its sedative properties are mediated by 1-GABAARs (McKernan et al. 2000; Rudolph et al. 1999) and its anxiolytic effects by 2-GABAARs (Low et al. 2000). It can therefore be assumed that specific neuronal circuits use distinct GABAAR subtypes differing in subunit variant.
Two major modes of GABAAR-mediated inhibitory transmission can be observed in mammalian CNS: phasic inhibition mediated by synaptic receptors and tonic inhibition mediated by extrasynaptic receptors (Farrant and Nusser 2005; Mody and Pearce 2004). In the hippocampal formation, phasic inhibitory postsynaptic currents (IPSCs) can further be subdivided into GABAA,fast and GABAA,slow IPSCs based on kinetics and amplitude (Pearce 1993). GABAA,fast IPSCs are mediated mostly by somatic and proximal dendritic synapses (Freund and Buzsaki 1996), whereas GABAA,slow IPSCs are thought to originate at distal dendritic sites (Banks et al. 1998). GABAA,slow IPSCs exhibit on average a larger charge transfer than GABAA,fast IPSCs; their prolonged time course and their sensitivity to GABA reuptake inhibitors (Banks et al. 2000) suggested that they are activated by GABA spillover.
The subunit composition and function of tonically activated GABAARs varies across brain regions and cell types. In cerebellum, dentate gyrus, neocortex, and thalamic relay nuclei, tonic inhibition is mediated by DZ-insensitive GABAARs containing 6 or 4 subunits together with the subunit (Brickley et al. 1996; Cope et al. 2005; Drasbek and Jensen 2005; Mtchedlishvili and Kapur 2006; Nusser and Mody 2002; Porcello et al. 2003; Stell et al. 2003; Sun et al. 2004). In the CA1 area, tonic inhibition is mediated by DZ-sensitive GABAARs in interneurons and, to a lesser extent, pyramidal cells, when ambient GABA concentration is increased (Scimemi et al. 2005; Semyanov et al. 2003). At low GABA concentrations activation of GABAARs containing the 4 subunit was found to dominate (Scimemi et al. 2005). Thus far, however, no single GABAAR subtype has been found to have an exclusive synaptic or extrasynaptic location, but several lines of evidence suggest that specific GABAARs selectively participate in different forms of inhibition. Unraveling this selectivity would help understanding how distinct behaviors and DZ effects are linked with the diverse forms of GABAergic inhibition.
The aim of this study was to investigate whether DZ-sensitive GABAARs differing in subunit composition mediate distinct modes of GABAergic inhibition in the hippocampal formation. Whole cell patch-clamp recordings of CA1 and subicular pyramidal cells were performed on acute slices from knock-in mice carrying a histidine-to-arginine point -mutation in either the 1, 2, or 3 subunit gene to render the respective GABAAR DZ-insensitive. Using this approach in single (1, 2, 3), double (12), and triple (123) mutant mice to pharmacologically isolate GABAARs containing different subunits, we studied the contribution of identified DZ-sensitive GABAAR subtypes to evoked and spontaneous IPSCs and to tonic inhibition in the hippocampus. A particular focus was put on characterization of GABAA,slow IPSCs, because they share elements of both phasic and tonic type of GABAergic inhibition.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Experiments were performed in 129/SvJ knock-in mice carrying DZ-insensitive GABAAR subtypes obtained by a histidine-to-arginine point mutation in the 1, 2, or 3 subunit gene [1(H101R), 2(H101R) and 3(H126R)] (Low et al. 2000; Rudolph et al. 1999) introduced into the mouse genome by homologous recombination in embryonic stem cells. Mice carrying a point mutation in both 1 and 2 subunits (12) were obtained by crossing double heterozygous mice born from homozygous 1(H101R) and 2(H101R) intercrosses. Homozygous 1(H101R)/2(H101R) offspring were identified by PCR analysis from tail biopsy. Likewise, triple mutants, carrying point-mutated 1, 2, and 3 subunits (123) were generated by crossing double homozygous 1(H101R)/2(H101R) and 1(H101R)/3(H126R) mice, which yielded offspring homozygous for 1(H101R) but heterozygous for 2(H101R) and 3(H126R) subunits. These were crossed again with each other to obtain mice homozygous for all three point-mutations, as confirmed by PCR analysis. All experiments were approved by the cantonal Veterinary Office of Zurich and were performed in accordance with the European Community Council Directive (86/609/EEC).
Immunohistochemistry
Mice (P21) were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg ip, Abbot Laboratories, Chicago, IL) and were perfused transcardially with 4% paraformaldehyde dissolved in 0.15 M sodium phosphate buffer containing 15% saturated picric acid solution. The brain was extracted, postfixed for 4 h in the same solution, incubated overnight in sodium citrate buffer (pH 4.5), and boiled for 60 s in a microwave oven for antigen retrieval (Fritschy et al. 1998). They were then cryoprotected with 30% sucrose in phosphate-buffered saline, frozen with dry-ice, and cut in 40-µm parasagittal sections using a freezing microtome. Free-floating sections were processed for immunoperoxidase staining using antibodies against the 1, 2, 3, and 5 subunit, as described (Fritschy et al. 1998). For antibody characterization, see Fritschy and Mohler (1995).
Slice preparation
Mice from both sexes (P18-24) were anesthetized with inhaled isoflurane and decapitated. The brain was quickly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF, composition in mM: 125 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2.5 CaCl2, and 11 glucose, oxygenated with 95% O2-5% CO2). The brain was affixed to a vibratome stage (Microm HM 650 V, Microm International AG, Volketswil, Switzerland) with cyanoacrylate and kept in the ice-cold ACSF for slicing. Parasagittal 300- to 350-µm-thick hippocampal slices were prepared and incubated at 33°C for 20 min before being stored at room temperature (25°C) in oxygenated ACSF. For the recording of GABAA,slow IPSCs, mice (P24–P30) were anesthetized with Nembutal and were perfused transcardially with 50 ml ice-cold sucrose-ACSF (composition in mM: 87 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 9 MgCl2, 0.5 CaCl2, 75 sucrose, and 25 glucose). Parasagittal slices (310 µm thick) were cut in sucrose-ACSF, transferred to normal ACSF, and incubated and stored as above.
Electrophysiological recordings and data analysis
Slices were visualized with a CCD camera (PCO Vx45, Till Photonics) mounted on an upright microscope (BX51WI, Olympus), equipped with a long working distance water-immersion objective (Xlumplan FI 20X, 0.95 numerical aperture), a fourfold magnification changer, Nomarski-type differential interference contrast, and infrared illumination. Patch electrodes were pulled from borosilicate glass (GC150TC, Clark Instruments) and had an open tip resistance of 3–4 M
when filled with the internal solution. Kynurenic acid (2 mM) was added to the external ACSF solution to block excitatory synaptic transmission. Recordings were made using a Multiclamp 700A patch-clamp amplifier (Axon Instruments), filtered at 4 kHz, digitized at 20 kHz, stored, and analyzed using IGOR Pro software (Wave Metrics). Access resistance was monitored for all experiments, and they were not included in further analysis if it changed by >20% during the recording.
Evoked IPSCs
Whole cell voltage-clamp recordings from CA1 pyramidal cells of wildtype (WT), 1, 2, 3, 12, and 123 mutant mice were made at room temperature (25°C) with continuous superfusion (1–2 ml/min) of ACSF. External stimulation (0.1–10 µA) was delivered every 10 s with a constant current stimulus isolator (WPI) through a bipolar, custom-made electrode from polytetrafluoroethylene-insulated platinum-iridium wire of 50 µM diam (Advent). Evoked IPSCs (eIPSC) were recorded at a holding potential of 0 mV with a low chloride containing internal solution (in mM: 130 CsGlu, 1 EGTA, 10 HEPES, 5 MgATP, 0.5 NaGTP, and 5 NaCl, pH 7.3, 295 mOsm). Access resistance was 
10–12 M.
To assess the contribution of different GABAAR subtypes to eIPSCs in slices from WT and knock-in mice, DZ (1 µM, dissolved in DMSO) was bath-applied after 15 min of baseline recordings, and eIPSCs were recorded for another 15–30 min. The average eIPSC amplitude after application of DZ was normalized to the peak amplitude of the average baseline eIPSC. No significant difference in the DZ effect was observed between mono- and biexponential fittings; therefore, we chose the monoexponential method. The increase in amplitude and 
after DZ application in each line of mutant mice was compared with WT mice using one-way ANOVA followed by Bonferroni’s post hoc multiple comparisons tests (SPSS 11.5, Lead Technology). In all experiments, n refers to the number of cells recorded. On average, one to two cells were recorded from one animal.
Spontaneous GABAA,fast and GABAA,slow IPSCs
We recorded spontaneous GABAergic events to reliably distinguish between GABAA,slow and GABAA,fast and to facilitate the comparison of results from two hippocampal regions. Whole cell voltage-clamp recordings of spontaneous IPSCs (sIPSC) from CA1 and subiculum pyramidal cells from WT and 123 mutant mice were obtained at room temperature with a holding potential of –60 mV and a high chloride containing internal solution (in mM: 100 CsCl, 2 MgCl2, 1 EGTA, 2 ATP, 0.3 GTP, and 40 HEPES, pH 7,2, 300 mOsm). Experiments with the GABA reuptake inhibitor NO711 (2 µM, dissolved in DMSO) were performed in the presence of the GABAB receptor antagonist CGP 55845 (1 µM, dissolved in DMSO). Continuous recordings started after the holding current had stabilized. Spontaneous events were recorded for a 10- to 15-min baseline period and for the same amount of time in the presence of each drug tested. Banks et al. (2002) reported an increase in frequency of GABAA,slow sIPSCs with age in rats, whereas other parameters such as amplitude and kinetics remained unchanged. We checked for the age dependency of GABAA,slow sIPSCs in WT mice by recording from 34 animals ranging from P15 to P35. As in rats, the frequency of GABAA,slow sIPSCs was higher in older animals. However, we observed a steep increase around P19 (results not shown), whereas in rats, the mean frequency of GABAA,slow sIPSCs was reported to increase gradually with age. Based on this result, all subsequent sIPSC recordings were performed in mice between P20 and P25.
Spontaneous events were detected off-line automatically with the ‘Mini Analysis’ software (Synaptosoft) with the detection threshold being set 5 times higher than the RMS level of baseline noise. All detected events were counted for analysis of frequency. For analysis of kinetics and amplitude only currents without subsequent contaminating events in the decaying phase were considered. Spontaneous events having an onset-to-peak rise time >5 ms were classified as GABAA, slow sIPSCs; remaining events were classified as GABAA,fast sIPSCs (Banks et al. 2000). Amplitude, rise time, and decay time constants for single GABAA,slow sIPSCs were calculated by the Mini Analysis software. Kinetics of GABAA,fast sIPSCs was also calculated individually, whereas their amplitude was determined from an average trace for each recorded cell. This minimized contamination by noise as, in general, the signal-to-noise ratio was smaller for GABAA,fast than for GABAA,slow sIPSCs. The effects of DZ and NO711 on sIPSCs were calculated from the average value of pooled sIPSCs from single experiments before and after drug application. Statistical significance was determined by paired Student’s t-test.
Tonic inhibition recordings
Tonic inhibition was determined by the change in holding current after application of the GABAAR antagonist picrotoxin (2 or 100 µM dissolved in DMSO). Sensitivity to DZ (1 µM) or L-655,708 (5 µM) was measured by applying the drug to the bath solution and assessing the effect of picrotoxin. Whole cell recordings from CA1 pyramidal cells in WT and 123 mutant mice were made at a holding potential of –60 mV with a high chloride containing internal solution. GABAB receptors were blocked with the antagonist CGP 55845 (1 µM). Recordings were analyzed with the Mini Analysis software with the detection threshold set 5 times higher than the level of baseline noise. The holding current was measured during 10-ms segments preceding spontaneous events to avoid contamination with phasic currents. The average holding current was calculated in recordings taken after chloride equilibration (baseline), 200 s after DZ application, 120–150 s after picrotoxin application (t1), and 240–300 s after picrotoxin application (t2). For each experiment, changes in holding current after drug application were statistically compared with baseline with paired Student’s t-test. Differences between genotypes were analyzed with one-way ANOVA and Bonferroni’s post hoc multiple comparisons tests.
Drugs
Chemicals were purchased from Sigma/Fluka or Tocris, and DZ was provided by Hoffmann-La Roche. We tested DMSO alone at the appropriate concentrations on IPSCs to rule out direct effects when it was used as a solvent.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The distribution of the four subunit variants (1, 2, 3, 5) contributing to DZ-sensitive GABAARs was analyzed in the hippocampal formation by immunoperoxidase staining in P21 WT mice, revealing clear-cut differences in staining intensity and regional distribution (Fig. 1). Although staining intensity cannot be compared across antibodies, comparisons can be made relative to other brain regions. Thus the 2 and 5 subunit exhibited the strongest immunoreactivity in the hippocampal formation, with 2 being more intense in the dentate gyrus than in CA1, whereas the 5 subunit was strongest in CA1, especially in the pyramidal cell layer. Staining for both subunits was homogeneous across dendritic and cell body layers of the hippocampal formation, suggesting the presence of the corresponding receptors on the soma and dendrites of principal cells. As reported previously (Brünig et al. 2002), the 1 subunit antibody strongly labeled a population of interneurons throughout the hippocampal formation and produced only a moderate staining in the dendritic layers of CA1, CA3, and the dentate gyrus, whereas the cell body layers appeared almost devoid of staining. The 3 subunit was expressed at low levels in CA1 and was present in a few isolated interneurons, mainly found in the stratum oriens and in the hilus. The boundary between CA1 and subiculum was particularly evident for the 5 subunit, which is almost not detectable in the latter region.
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) and the cerebral cortex (Fagiolini et al. 2004).
Differential contribution of 1- and 2-GABAA receptors to evoked synaptic inhibition in CA1 pyramidal cells
To assess the major GABAAR subtypes mediating phasic inhibition, the effects of DZ on eIPSCs were compared in WT mice and five lines of mutant mice carrying DZ-insensitive GABAA receptors (1, 2, 3, 12, and 123 knock-in mice). Current pulses were delivered every 10 s through an extracellular stimulus electrode and their intensity was adjusted to generate eIPSCs of similar amplitude in all experiments (Table 1). There was no difference in rise time and decay time constants of eIPSCs in the different genotypes investigated (Table 1).
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3 knock-in mice compared with WT (Fig. 2, B and C), whereas in 12 and 123 knock-in mice, it was abolished (amplitude: 6 ± 5%, n = 7 and 6 ± 2%, n = 7; Fig. 2A, right). In contrast, DZ produced a significant increase in peak amplitude (1: 33 ± 5%, n = 15 and 2: 27 ± 8%, n = 10) and decay time in recordings from 1 and 2 knock-in mice (Fig. 2, B and C). Therefore no contribution of either 3- or 5-GABAARs to evoked phasic inhibition could be resolved in these mice, whereas both 1- and 2-GABAARs mediate the bulk of eIPSCs in CA1 pyramidal cells.
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1 knock-in mice similar to WT (50 ± 10%, n = 7; P < 0.02), whereas in 2 knock-in mice, the increase was much smaller (8 ± 4%, n = 5; P < 0.002). The opposite effect was observed in recordings with distal stimulation (Fig. 3): in 2 knock-in mice, the increase in amplitude after DZ application (46 ± 16%, n = 5, P < 0.002) was similar to WT mice, whereas in 1 knock-in mice, it was only 20 ± 5% (n = 8, P < 0.02). Therefore 2-GABAARs preferentially mediate phasic synaptic inhibition on the soma and 1-GABAA receptor on the dendrites of CA1 pyramidal cells, in line with the subcellular distribution of these subunits (Fig. 1). The fact that the activation of distinct GABAAR subtypes can be resolved in these knock-in mice makes them a suitable tool for further study of GABAergic transmission in the hippocampal formation.
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GABAA,fast and GABAA,slow IPSCs represent two distinct modes of phasic synaptic inhibition in CA1 pyramidal cells, best distinguished in recordings of spontaneous events. GABAA,slow IPSCs have been suggested to involve a mixture of synaptic and perisynaptic or extrasynaptic receptors and to be mediated by a specialized, yet not identified, type of interneuron (Banks et al. 2000). Although 5-GABAARs do not seem to be much involved in eIPSCs (Fig. 2), a contribution to GABAA,slow IPSCs is conceivable. Therefore to determine whether different GABAARs mediate GABAA,fast and GABAA,slow sIPSCs, the effects of DZ were assessed in 123 knock-in and WT mice, using continuous whole cell voltage-clamp recordings from CA1 and subicular pyramidal cells.
Baseline properties of sIPSCs (peak amplitude, rise time, and decay time constant) did not differ significantly between genotypes and hippocampal regions (CA1, subiculum; Tables 2 and 3). Examples of recordings from a CA1 pyramidal cell are shown in Fig. 4, A and D. sIPSCs were analyzed and subdivided into GABAA,slow (Fig. 4, B and E) and GABAA,fast sIPSCs (Fig. 4, C and F).
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123 knock-in mice from –83.5 ± 6.1 to –111.2 ± 9.8 pA (P < 0.01, n = 9) and in WT mice from –117.0 ± 17.4 to –229.3 ± 46.7 pA (P < 0.05, n = 7). In addition, a significant increase in decay time constant was observed in WT mice, from 40.7 ± 4.0 to 63.0 ± 9.6 ms after DZ application (P < 0.05, n = 7), whereas the increase in decay time in 123 knock-in mice just failed to reach significance. The rise time of GABAA,slow sIPSCs did not change significantly after DZ application in either genotype.
The effect of DZ in 123 knock-in mice indicates that 5-GABAARs participate in GABAA,slow sIPSCs. However, the increase in GABAA,slow sIPSCs in CA1 pyramidal cells was significantly larger in WT than in 123 knock-in mice (2-tailed, unpaired Student’s t-test, nWT = 7, n123 = 8), suggesting that 1- and/or 2-GABAARs also contribute to GABAA,slow sIPSCs. In contrast, GABAA,fast sIPSCs were not affected by DZ in 123 knock-in mice, unlike in WT (Fig. 4, N and O). These findings are consistent with the observations made on eIPSCs and suggest that the contribution of 5-GABAARs to phasic inhibition is restricted to GABAA,slow IPSCs.
As shown in Fig. 1, 5 subunit immunoreactivity is almost undetectable in subiculum, just adjacent to the CA1 area, allowing to characterize GABAA,slow sIPSCs in neurons lacking 5-GABAARs and to validate the findings obtained in 123 knock-in mice. Experiments were performed applying the same protocol used for the CA1 area (Fig. 4, G and K). GABAA,slow sIPSCs were observed in baseline recordings of subicular pyramidal cells, albeit at a lower frequency than in CA1 (fSubiculum = 0.039 ± 0.005/s; fCA1 = 0.099 ± 0.012/s). Bath-application of DZ (1 µM) increased the amplitude and decay time constant of both GABAA,slow and GABAA,fast sIPSCs, in cells from WT mice (Fig. 4, H, J, N, and O), whereas in 123 knock-in mice, these values remained unchanged (Fig. 4, L–O). In addition, application of the inverse agonist at the benzodiazepine binding site L-655,708 (5 µM) only affected the amplitude of GABAA,slow in WT CA1 pyramidal cells (–74.1 ± 12.4 to –58.1 ± 9.5 pA, n = 7, P < 0.05), but not in subicular pyramidal cells (n = 5). Amplitude and kinetics of GABAA,fast sIPSCs in CA1 and subiculum (n = 7 and n = 5, respectively) remained unchanged. L-655,708 exhibits 
50 times higher affinity for 5-GABAARs over GABAARs containing other subunits (Quirk et al. 1996). These results further confirmed that 5-GABAARs are absent in the subiculum and thus are not required for the generation of GABAA,slow and that these receptors do not contribute to GABAA,fast sIPSCs.
Because of their slow time-course and sensitivity to GABA reuptake inhibitors, GABAA,slow IPSCs have been proposed to involve, at least in part, extrasynaptic receptors activated by GABA spillover (Banks et al. 2000). To compare the subunit profile of synaptic and extrasynaptic receptors contributing to GABAA,slow sIPSCs in CA1 pyramidal cells, we enhanced the spillover component using the selective GABA reuptake inhibitor NO711. Recordings from CA1 pyramidal cells from WT and 123 knock-in mice (Fig. 5, A and D) were obtained under baseline condition, in presence of 2 µM NO711 (Table 4), and finally after application of DZ (1 µM). To prevent tonic activation of GABAB receptors (Le Feuvre et al. 1997; Scanziani 2000), 1 µM of the GABAB antagonist CGP55845 was present throughout the experiment.
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123 knock-in mice, the average amplitude increased from –96.2 ± 8.6 to –175.0 ± 32.2 pA and the decay time constant changed from 27.85 ± 18.2 to 47.44 ± 2.26 ms (P < 0.05, n = 8). In WT mice, the average amplitude increased from –73.1 ± 6.2 to –121.0 ± 19.2 pA and the decay time constant changed from 21.43 ± 1.99 to 38.69 ± 2.50ms (P < 0.05, n = 10). There was also a significant increase in rise time in both genotypes. In contrast, NO711 had no effect on the amplitude and kinetics of GABAA,fast sIPSCs (Fig. 5, C and F). Subsequent application of DZ (1 µM) further enhanced amplitude, rise and decay time constant of GABAA,slow sIPSCs in both genotypes (Fig. 5, B and E). As expected, DZ also enhanced the amplitude and decay time constant of GABAA,fast sIPSCs in WT but not in 123 knock-in mice (Fig. 5, G and H). In the presence of NO711, no difference between the relative changes in GABAA,slow sIPSCs amplitudes in WT (1.67 ± 0.20, n = 10) and 123 knock-in mice (1.49 ± 0.16, n = 8) could be observed after DZ application.
Diazepam-activated tonic inhibition in CA1 pyramidal cells is mediated by 5-GABAARs
GABAA,slow sIPSCs share elements of tonic GABAergic transmission such as involving perisynaptic GABAARs. Therefore we assessed which DZ-sensitive GABAAR subtypes contribute to tonic inhibition in CA1 pyramidal cells. Whole cell voltage-clamp recordings were made in slices from WT and 123 knock-in mice using a high chloride-containing intracellular solution. Using a perfusion rate of 1 ml/min, tonic inhibition was measured as the change in holding current after the application 100 µM picrotoxin in the presence of the GABAB antagonist CGP 55845 (1 µM). No GABA reuptake inhibitor was used. Under these conditions, application of 100 µM picrotoxin produced an outward shift in the baseline current of 33 ± 4 pA (n = 7) at room temperature and 35 ± 9 pA (n = 5) at physiological temperature. The tonic conductance was mediated by GABAARs because it was also blocked by bicuculline (10 µM; data not shown). Because it has been reported that low doses of picrotoxin selectively (e.g., more rapidly) block tonic inhibition (Semyanov et al. 2003), we performed the same experiments using 2 µM picrotoxin and measured the outward shift of the holding current during two distinct time windows: at 120–150 (t1) and 240–300 s (t2) after picrotoxin application. The holding current at t1 changed by 22 ± 5 pA (n = 6; Fig. 6, A and B, middle, and C) and by 33 ± 4 pA at t2 (n = 6; Fig. 6, A and B, right, and C). Root mean square noise decreased from 5.4 ± 0.27 to 4.4 ± 0.27 pA (P < 0.05) at t1 and to 3.9 ± 0.18 pA (not significant) at t2. The amplitude of fast events remained constant at t1, whereas a tendency for smaller GABAslow amplitudes was observed at that time-point. At t2, both fast and slow events were significantly reduced in amplitude (–44.8 ± 1.1 to –28.6 ± 0.65 pA, n = 6, P < 0.01 in fast sIPSCs and –61.1 ± 7.5 to –34.4 ± 1.9 pA, n = 6, P < 0.01 in slow sIPSCs; Fig. 6D). At t1, the frequency and decay time constant of GABAA,fast sIPSCs were unaltered compared with baseline (3.8 ± 0.26 to 4.0 ± 0.17 Hz, n = 6; and 21.7 ± 0.7 to 23.1 ± 0.9 ms, respectively; Fig. 6, E and F, left trace), whereas the frequency and decay time of GABAA,slow sIPSCs were significantly reduced (from 0.09 ± 0.01 to 0.06 ± 0.01 Hz; n = 6, P < 0.05 and from 27.6 ± 2.3 to 20.2 ± 2.8 ms, P < 0.05, respectively; Fig. 6, E and F, right). The selective change in decay time and frequency of GABAA,slow sIPSCs that correlated with the change in holding current provides further indication that slow events and tonic inhibition share a common pool of extrasynaptic and thus most likely 5-containing GABAARs (Caraiscos et al. 2004a).
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123 knock-in mice, a similar inward shift in baseline holding current was also observed after the application of 1 µM DZ (8 ± 2 pA, n = 5; Fig. 7, B and C), showing that 5-GABAARs mediate the DZ-sensitive component of tonic inhibition in CA1 pyramidal cells. The application of 5 µM L-655,708 instead of DZ in on pyramidal cells of the CA1 area from WT mice did not significantly alter the holding current (n = 4; Fig. 7C), indicating that DZ application increased the affinity of previously silent 5-GABAARs to the point that they were activated by ambient GABA. As no reliable electrophysiological data on the affinity and activity of L-655,708 on GABAARs carrying point-mutated -subunits is available we only used this substance in WT mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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subunit composition in the hippocampal formation. In CA1 pyramidal cells, fast synaptic inhibition is mediated selectively by 1- and 2-GABAARs, with a spatial segregation of the two different receptor subtypes in distal and proximal compartments, respectively. Slow phasic inhibition involves both synaptic and extrasynaptic receptors, the latter being principally 5-GABAARs. Finally, DZ-sensitive tonic inhibition, which can be observed in the absence of GABA reuptake inhibitors, is mediated by 5-GABAARs. In subiculum, where the 5 subunit is expressed at very low levels, GABAA,slow sIPSCs can be readily detected, indicating that these synaptic events do not depend on 5-GABAARs. Nevertheless, a mixture of synaptic and extrasynaptic receptors also mediates them. Altogether, these results indicate that the distinct modes of synaptic inhibition in hippocampal neurons involve different GABAAR subtypes, organized in a region-specific manner and precisely targeted to distinct synaptic, perisynaptic, and extrasynaptic sites. Technical considerations
The conclusions of this study are derived from a pharmacological distinction of GABAAR subtypes in knock-in mutant mice. There are two major prerequisites to validate these conclusions. First, the point-mutation should not alter the organization and functional properties of GABAARs in mutant mice. Several lines of evidence indicate that this prerequisite is fulfilled: Animals containing one or more point-mutations develop normally, have no overt phenotype and the cellular and subcellular location of GABAARs is unchanged (Benke et al. 2004; Mohler et al. 2004; Rudolph and Mohler 2004; Rudolph et al. 1999; Yee et al. 2004). The functional properties of receptors containing mutated subunits are unaltered in vivo (Bacci et al. 2003; Fagiolini et al. 2004; Marowsky et al. 2004). The present results reveal no difference across genotypes in the kinetics of sIPSCs or eIPSCs, and in the stimulus strength required to evoke IPSCs of similar amplitude, confirming that functional properties of GABAARs are normal in mutant mice. Second, the effects of DZ on peak amplitude and/or decay time constant should be detectable on all sensitive GABAAR subtypes and should not be masked by agonist saturation. At least for the 1, 2, and 5-GABAARs, these conditions were fulfilled, confirming that mouse CA1 pyramidal cells have an incomplete postsynaptic GABAAR occupancy at room temperature (Hajos et al. 2000; Perrais and Ropert 1999, 2000). Failure to detect a contribution of 3-GABAARs to eIPSCs likely reflects the low abundance of these receptors in CA1 pyramidal cells (Laurie et al. 1992) (Fig. 1). Therefore with the knock-in strategy, the function of GABAARs containing specific subunits can be analyzed with great selectivity (Rudolph and Mohler 2004) and without inducing compensatory changes, as occurs in gene deletion experiments. To date, no drugs are available to discriminate between 1-, 2-, and 3-GABAARs in vivo with a comparable degree of selectivity. One obvious limitation of the approach lies in the fact that it is limited to DZ-sensitive GABAARs.
Somatic and dendritic inputs are mediated differentially by 2- and 1-GABAARs
The results from the analysis of eIPSCs are largely in agreement with the known subcellular distribution of GABAAR subtypes in CA1 pyramidal cells. 2-GABAARs located predominantly on the axon-initial segment and on the soma, facing terminals from chandelier cells and from basket cells expressing cholecystokinin, respectively (Nusser et al. 1996; Nyiri et al. 2001). The 1-GABAARs on the soma are contacted by parvalbumin-positive basket cell terminals, and they predominate in the dendritic layers of CA1, as seen by immunohistochemistry (Fig. 1) (Brunig et al. 2002). This segregation of different GABAARs to different subcellular compartments is of functional relevance, as shown in vivo with the contribution of distinct interneurons signaling through these receptors during specific behavioral states (Klausberger 2003; Klausberger et al. 2002). Additionally, Pouille and Scanziani (2001, 2004) have recently shown that feedforward and feedback inhibition are mediated predominantly by somatic and dendritic receptors, respectively. The differential modulation by DZ of IPSCs evoked by proximal and distal stimulation of inhibitory inputs in 1 and 2 knock-in mice confirms this functional segregation of 2- and 1-GABAARs, thereby validating the use of this knock-in model to pharmacologically isolate specific GABAAR subtypes. The agreement between the present results and the known segregation of 1- and 2-GABAARs in CA1 neurons makes it unlikely that the difference in the effects of DZ on amplitude of eIPSCs in 1 and 2 knock-in mice are caused by differential occupancy of receptors located on the soma and dendrites. To explain these results, the degree of occupancy of these receptors should be opposite on the soma and dendrites (1 high on soma and low on dendrites, and vice versa for 2), which seems rather unlikely. Finally, it is well established that 2-GABAARs deactivate more slowly than 1-GABAARs, as shown in recombinant systems (McClellan and Twyman 1999) and in vivo (Bosman et al. 2005; Goldstein et al. 2002; Vicini et al. 2001). This distinction was not apparent with eIPSCs, reflecting the detection of compound, nonsynchronous events and the variable effects of dendritic filtering on proximal and distal stimulation.
GABAA,slow IPSCs involve activation of synaptic and perisynaptic receptors
GABAA,slow IPSCs in CA1 pyramidal cells have been observed in evoked, spontaneous, and miniature IPSCs (Banks et al. 1998; Pearce 1993) GABAA,slow eIPSCs exhibit the same properties as GABAA,slow sIPSCs and probably emanate from the same synapse (Banks et al. 1998). Our data extend the previous findings by showing the presence of GABAA,slow sIPSCs also in the subiculum. It was not possible to determine whether GABAA,slow IPSCs in CA1 and subicular pyramidal cells are produced by similar mechanisms. The question whether GABAA,slow IPSCs are produced by one specific cell type common to both brain areas therefore remains unresolved.
Slow events have been proposed to involve phasic activation of perisynaptic or even extrasynaptic GABAARs on GABA spillover from the synaptic cleft (Banks et al. 1998). The sensitivity of GABAA,slow to DZ in 123 knock-in mice and their depression by L-655,708 in WT mice show that 5-GABAARs contribute to GABAA,slow sIPSCs. More importantly, this contribution is amplified and likely dominates the current in conditions of enhanced spillover, because the DZ-induced increase in GABAA,slow currents became indistinguishable between WT and 123 knock-in mice in presence of a GABA uptake inhibitor. An enhanced diffusion volume covered by the neurotransmitter and the prolonged gating kinetics of the receptors by the elevated concentration of GABA can explain the change in rise and decay time constant of GABAA,slow in the presence of a GABA uptake inhibitor. Which particular mechanism prevails would most likely be determined by the amount of spilled out GABA.
Our results clearly show that the spillover component of slow phasic currents is mediated by 5-GABAARs and nicely agree with the predominantly extrasynaptic distribution of 5-GABAARs (Brunig et al. 2002; Caraiscos et al. 2004a,b; Crestani et al. 2002). Interestingly, however, GABAA,slow sIPSCs also occurred in subiculum where the 5 subunit is virtually absent. The potentiation by DZ in WT mice provides direct evidence that GABAA,slow sIPSCs there are also mediated by other DZ-sensitive GABAAR subtypes. These IPSCs are otherwise indistinguishable from those recorded in CA1, suggesting that the function of 5-containing GABAARs is taken over by other GABAARs with similar peri- and extrasynaptic localization in subicular neurons.
The stronger effect of DZ on GABAA,slow sIPSCs in WT compared with 123 knock-in mice in CA1 pyramidal cells and the partial effect of L-655,708 (Fig. 4) implicate the involvement of additional GABAAR subtypes in slow currents, with 1-GABAARs as likely coparticipants, because this subunit has been shown sometimes to colocalize with the 5 subunit in the same clusters (Hutcheon et al. 2004). The lack of DZ effect on GABAA,fast sIPSCs in 123 knock-in mice (Fig. 4) indicates that 5-GABAARs do not participate in fast phasic inhibition. We did not detect any spillover component in fast GABAergic sIPSCs in the CA1 region and even the combination of NO711 and DZ on cells from 123 mice revealed no contribution of 5-GABAARs to GABAA,fast. Therefore these GABAARs are targeted selectively to sites mediating GABAA,slow sIPSCs in CA1 pyramidal cells, where they are probably located both in the synaptic cleft and perisynaptically. Using paired intracellular recordings; Thomson et al. (2000) provided indirect pharmacological evidence for the existence of 5-GABAARs mediating fast phasic inhibition from bistratified cells, which innervate distal pyramidal cell dendrites. The failure to detect these receptors in point-mutated mice might reflect the minor contribution of these events to the overall number of sIPSCs received by pyramidal cells. Banks et al. (1998) provided evidence for blockade of GABAA,fast sIPSCs by furosemide, suggesting a contribution of 4-GABAARs to these events. We did not assess the contribution of 4-GABAARs, which are DZ-insensitive, but their low expression level in CA1 area (Pirker et al. 2000) makes it unlikely that they play a major role in mediating GABAA,fast sIPSCs.
Our findings emphasize the proposed role of 5-GABAARs as detectors for extrasynaptic GABA (Caraiscos et al. 2004a; Glykys and Mody 2006), and reveal their dual assignment to the generation of slow GABAergic events and the mediation of DZ-sensitive tonic inhibition. In both cases, GABA is not confined to synaptic clefts and the extent to which the neurotransmitter release underlying GABAA,slow sIPSCs contributes to ambient GABA remains to be examined.
Tonic inhibition
Thus far we have shown that in the CA1 area, GABAA,slow events partially arise from the activation of 5-GABAARs receptors that are predominantly located extrasynaptically. Because GABAA,slow sIPSCs are rare, their early block by the activity-dependent antagonist picrotoxin, which parallels the early decrease of tonic GABAergic currents, most likely reflects crosstalk between the receptor populations mediating these two types of GABAergic currents. The pronounced sensitivity of tonic inhibition and GABAA,slow for picrotoxin is in line with a higher opening probability of tonically activated, extrasynaptic receptors.
The increase in holding current after application of DZ in both WT and 123 knock-in mice confirms that 5-GABAARs can generate both tonic inhibition and slow events in CA1 pyramidal cells. Several lines of evidence indicate that most 5-GABAARs contain the 3 subunit in pyramidal neurons and 3-containing GABAARs are present in extrasynaptic regions (Pirker et al. 2000). It would be of major interest to know whether these receptors have the required high affinity for GABA. The fact that in our experiments the inverse agonist L-655,708 in contrast to DZ failed to exert any effect on the holding current of CA1 pyramidal cells raises questions about the role of 5-GABAARs detecting ambient GABA concentrations in the absence of DZ. DZ increases the probability of GABAARs to be activated after GABA binding. This can lead to tonic opening of receptors under low ambient GABA concentration. Consequently, an inverse agonist fails to produce any effect on previously not activated receptors. Scimemi et al. (2005) have been the first to address the dependency of 5-GABAARs activation on GABA concentrations. The authors found that L-655,708 decreases the holding current only when ambient GABA concentrations are elevated. Under physiological conditions, synchronous neural network activity might lead to such heightened ambient GABA concentration and subsequent activation of extracellular 5-GABAARs (Towers et al. 2004). Nevertheless our findings are relevant to explain the physiological effects of DZ, because this drug experiences widespread pharmaceutical use.
Significance of the work
We have shown that the subunit composition of GABAARs not only affects their subcellular distribution but also dictates their functional role. The clear segregation of GABAAR subtypes demonstrated here shows the complex organization of the inhibitory system at the molecular level and provides a compelling explanation for the specific effects of different GABAARs in distinct behaviorally significant signaling pathways.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Address for reprint requests and other correspondence: K. E. Vogt, Inst. of Pharmacology and Toxicology, Univ. of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland (E-mail: kvogt{at}pharma.unizh.ch)
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|---|
Banks MI, Hardie JB, and Pearce RA. Development of GABAA receptor-mediated inhibitory postsynaptic currents in hippocampus. J Neurophysiol 88: 3097–3107, 2002.
Banks MI, Li TB, and Pearce RA. The synaptic basis of GABAA,slow. J Neurosci 18: 1305–1317, 1998.
Banks MI and Pearce RA. Kinetic differences between synaptic and extrasynaptic GABAA receptors in CA1 pyramidal cells. J Neurosci 20: 937–948, 2000.
Banks MI, White JA, and Pearce RA. Interactions between distinct GABAA circuits in hippocampus. Neuron 25: 449–457, 2000.[CrossRef][Web of Science][Medline]
Barnard EA, Skolnick P, Olsen RW, Mohler H, Sieghart W, Biggio G, Braestrup C, Bateson AN, and Langer SZ. International Union of Pharmacology. XV. Subtypes of -aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 50: 291–313, 1998.
Benke D, Fakitsas P, Roggenmoser C, Michel C, Rudolph U, and Mohler H. Analysis of the presence and abundance of GABAA receptors containing two different types of subunits in murine brain using point-mutated subunits. J Biol Chem 279: 43654–43660, 2004.
Benson JA, Low K, Keist R, Mohler H, and Rudolph U. Pharmacology of recombinant -aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated -subunits. FEBS Lett 431: 400–404, 1998.[CrossRef][Web of Science][Medline]
Bosman LW, Heinen K, Spijker S, and Brussaard AB. Mice lacking the major adult GABAA receptor subtype have normal number of synapses, but retain juvenile IPSC kinetics until adulthood. J Neurophysiol 94: 338–346, 2005.
Bosman LW, Rosahl TW, and Brussaard AB. Neonatal development of the rat visual cortex: synaptic function of GABAA receptor subunits. J Physiol 545: 169–181, 2002.
Brickley SG, Cull-Candy SG, and Farrant M. Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol 497: 753–759, 1996.
Brunig I, Scotti E, Sidler C, and Fritschy JM. Intact sorting, targeting, and clustering of -aminobutyric acid A receptor subtypes in hippocampal neurons in vitro. J Comp Neurol 443: 43–55, 2002.[CrossRef][Web of Science][Medline]
Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D, Newell JG, Jackson MF, Lambert JJ, Rosahl TW, Wafford KA, MacDonald JF, and Orser BA. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by 5 subunit-containing -aminobutyric acid type A receptors. Proc Natl Acad Sci USA 101: 3662–3667, 2004a.
Caraiscos VB, Newell JG, You-Ten KE, Elliott EM, Rosahl TW, Wafford KA, MacDonald JF, and Orser BA. Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane. J Neurosci 24: 8454–8458, 2004b.
Cope DW, Hughes SW, and Crunelli V. GABAA receptor-mediated tonic inhibition in thalamic neurons. J Neurosci 25: 11553–11563, 2005.
Crestani F, Keist R, Fritschy JM, Benke D, Vogt K, Prut L, Bluthmann H, Mohler H, and Rudolph U. Trace fear conditioning involves hippocampal 5 GABAA receptors. Proc Natl Acad Sci USA 99: 8980–8985, 2002.
Drasbek KR and Jensen K. THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex. Cereb Cortex Oct 2005; 10.1093/ceicol/bhj055.
Fagiolini M, Fritschy JM, Low K, Mohler H, Rudolph U, and Hensch TK. Specific GABAA circuits for visual cortical plasticity. Science 303: 1681–1683, 2004.
Farrant M and Nusser Z. Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat Rev Neurosci 6: 215–229, 2005.[CrossRef][Web of Science][Medline]
Freund TF and Buzsaki G. Interneurons of the hippocampus. Hippocampus 6: 347–470, 1996.[CrossRef][Web of Science][Medline]
Fritschy JM and Mohler H. GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359: 154–194, 1995.[CrossRef][Web of Science][Medline]
Fritschy JM, Weinmann O, Wenzel A, and Benke D. Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry. J Comp Neurol 390: 194–210, 1998.[CrossRef][Web of Science][Medline]
Glykys J and Mody I. Hippocampal network hyperactivity following selective reduction of tonic inhibition in GABAA receptor 5 subunit deficient mice. J Neurophysiol Feb 1, 2006; 10.1152/jn. 01122.2005.
Goldstein PA, Elsen FP, Ying SW, Ferguson C, Homanics GE, and Harrison NL. Prolongation of hippocampal miniature inhibitory postsynaptic currents in mice lacking the GABAA receptor 1 subunit. J Neurophysiol 88: 3208–3217, 2002.
Hajos N, Nusser Z, Rancz EA, Freund TF, and Mody I. Cell type- and synapse-specific variability in synaptic GABAA receptor occupancy. Eur J Neurosci 12: 810–818, 2000.[CrossRef][Web of Science][Medline]
Hutcheon B, Fritschy JM, and Poulter MO. Organization of GABAA receptor -subunit clustering in the developing rat neocortex and hippocampus. Eur J Neurosci 19: 2475–2487, 2004.[CrossRef][Web of Science][Medline]
Hutcheon B, Morley P, and Poulter MO. Developmental change in GABAA receptor desensitization kinetics and its role in synapse function in rat cortical neurons. J Physiol 522: 3–17, 2000.
Klausberger T. Brain-state- and cell-type-specific firing of hippocampal interneurons in vivo. Nature 421: 844–848, 2003.[CrossRef][Medline]
Klausberger T, Roberts JD, and Somogyi P. Cell type- and input-specific differences in the number and subtypes of synaptic GABAA receptors in the hippocampus. J Neurosci 22: 2513–2521, 2002.
Knoflach F, Rhyner T, Villa M, Kellenberger S, Drescher U, Malherbe P, Sigel E, and Mohler H. The 3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands. FEBS Lett 293: 191–194, 1991.[CrossRef][Web of Science][Medline]
Laurie DJ, Wisden W, and Seeburg PH. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci 12: 4151–4172, 1992.[Abstract]
Le Feuvre Y, Fricker D, and Leresche N. GABAA receptor-mediated IPSCs in rat thalamic sensory nuclei: patterns of discharge and tonic modulation by GABAB autoreceptors. J Physiol 502: 91–104, 1997.
Low K, Crestani F, Keist R, Benke D, Brunig I, Benson JA, Fritschy JM, Rulicke T, Bluethmann H, Mohler H, and Rudolph U. Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290: 131–134, 2000.
Marowsky A, Fritschy JM, and Vogt KE. Functional mapping of GABAA receptor subtypes in the amygdala. Eur J Neurosci 20: 1281–1289, 2004.[CrossRef][Web of Science][Medline]
McClellan AM and Twyman RE. Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors. J Physiol 515: 711–727, 1999.
McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S, Myers J, Cook G, Ferris P, Garrett L, Bristow L, Marshall G, Macaulay A, Brown N, Howell O, Moore KW, Carling RW, Street LJ, Castro JL, Ragan CI, Dawson GR, and Whiting PJ. Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAA receptor 1 subtype. Nat Neurosci 3: 587–592, 2000.[CrossRef][Web of Science][Medline]
McKernan RM and Whiting PJ. Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19: 139–143, 1996.[CrossRef][Web of Science][Medline]
Mody I and Pearce RA. Diversity of inhibitory neurotransmission through GABAA receptors. Trends Neurosci 27: 569–575, 2004.[CrossRef][Web of Science][Medline]
Mohler H, Fritschy JM, Crestani F, Hensch T, and Rudolph U. Specific GABAA circuits in brain development and therapy. Biochem Pharmacol 68: 1685–1690, 2004.[CrossRef][Web of Science][Medline]
Mohler H, Fritschy JM, and Rudolph U. A new benzodiazepine pharmacology. J Pharmacol Exp Ther 300: 2–8, 2002.
Mtchedlishvili Z and Kapur J. High-affinity, slowly desensitizing GABAA receptors mediate tonic inhibition in hippocampal dentate granule cells. Mol Pharmacol 69: 564–575, 2006.
Nusser Z and Mody I. Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J Neurophysiol 87: 2624–2628, 2002.
Nusser Z, Sieghart W, Benke D, Fritschy JM, and Somogyi P. Differential synaptic localization of two major -aminobutyric acid type A receptor subunits on hippocampal pyramidal cells. Proc Natl Acad Sci USA 93: 11939–11944, 1996.
Nyiri G, Freund TF, and Somogyi P. Input-dependent synaptic targeting of 2-subunit-containing GABAA receptors in synapses of hippocampal pyramidal cells of the rat. Eur J Neurosci 13: 428–442, 2001.[CrossRef][Web of Science][Medline]
Pearce RA. Physiological evidence for two distinct GABAA responses in rat hippocampus. Neuron 10: 189–200, 1993.[CrossRef][Web of Science][Medline]
Perrais D and Ropert N. Effect of zolpidem on miniature IPSCs and occupancy of postsynaptic GABAA receptors in central synapses. J Neurosci 19: 578–588, 1999.
Perrais D and Ropert N. Altering the concentration of GABA in the synaptic cleft potentiates miniature IPSCs in rat occipital cortex. Eur J Neurosci 12: 400–404, 2000.[CrossRef][Web of Science][Medline]
Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, and Sperk G. GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101: 815–850, 2000.[CrossRef][Web of Science][Medline]
Porcello DM, Huntsman MM, Mihalek RM, Homanics GE, and Huguenard JR. Intact synaptic GABAergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking subunit. J Neurophysiol 89: 1378–1386, 2003.
Pouille F and Scanziani M. Enforcement of temporal fidelity in pyramidal cells by somatic feed-forward inhibition. Science 293: 1159–1163, 2001.
Pouille F and Scanziani M. Routing of spike series by dynamic circuits in the hippocampus. Nature 429: 717–723, 2004.[CrossRef][Medline]
Price CJ, Cauli B, Kovacs ER, Kulik A, Lambolez B, Shigemoto R, and Capogna M. Neurogliaform neurons form a novel inhibitory network in the hippocampal CA1 area. J Neurosci 25: 6775–6786, 2005.
Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H, Schofield PR, and Seeburg PH. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338: 582–585, 1989.[CrossRef][Medline]
Quirk K, Blurton P, Fletcher S, Leeson P, Tang F, Mellilo D, Ragan CI, and McKernan RM. [3H]L-655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit. Neuropharmacology 35: 1331–1335, 1996.[CrossRef][Web of Science][Medline]
Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, and Mohler H. Benzodiazepine actions mediated by specific -aminobutyric acidA receptor subtypes. Nature 401: 796–800, 1999.[CrossRef][Medline]
Rudolph U and Mohler H. Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annu Rev Pharmacol Toxicol 44: 475–498, 2004.[CrossRef][Web of Science][Medline]
Scanziani M. GABA spillover activates postsynaptic GABAB receptors to control rhythmic hippocampal activity. Neuron 25: 673–681, 2000.[CrossRef][Web of Science][Medline]
Scholze P, Ebert V, and Sieghart W. Affinity of various ligands for GABAA receptors containing 432, 42, or 132 subunits. Eur J Pharmacol 304: 155–162, 1996.[CrossRef][Web of Science][Medline]
Scimemi A, Semyanov A, Sperk G, Kullmann DM, and Walker MC. Multiple and plastic receptors mediate tonic GABAA receptor currents in the hippocampus. J Neurosci 25: 10016–10024, 2005.
Semyanov A, Walker MC, and Kullmann DM. GABA uptake regulates cortical excitability via cell type-specific tonic inhibition. Nat Neurosci 6: 484–490, 2003.[Web of Science][Medline]
Semyanov A, Walker MC, Kullmann DM, and Silver RA. Tonically active GABAA receptors: modulating gain and maintaining the tone. Trends Neurosci 27: 262–269, 2004.[CrossRef][Web of Science][Medline]
Sieghart W and Sperk G. Subunit composition, distribution and function of GABAA receptor subtypes. Curr Top Med Chem 2: 795–816, 2002.[CrossRef][Medline]
Smith AJ, Alder L, Silk J, Adkins C, Fletcher AE, Scales T, Kerby J, Marshall G, Wafford KA, McKernan RM, and Atack JR. Effect of subunit on allosteric modulation of ion channel function in stably expressed human recombinant -aminobutyric acidA receptors determined using 36Cl ion flux. Mol Pharmacol 59: 1108–1118, 2001.
Stell BM, Brickley SG, Tang CY, Farrant M, and Mody I. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by subunit-containing GABAA receptors. Proc Natl Acad Sci USA 100: 14439–14444, 2003.
Sun C, Sieghart W, and Kapur J. Distribution of 1, 4, 2, and subunits of GABAA receptors in hippocampal granule cells. Brain Res 1029: 207–216, 2004.[CrossRef][Web of Science][Medline]
Thomson AM, Bannister AP, Hughes DI, and Pawelzik H. Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus. Eur J Neurosci 12: 425–436, 2000.[CrossRef][Web of Science][Medline]
Towers SK, Gloveli T, Traub RD, Driver JE, Engel D, Fradley R, Rosahl TW, Maubach K, Buhl EH, and Whittington MA. 5 subunit-containing GABAA receptors affect the dynamic range of mouse hippocampal kainate-induced gamma frequency oscillations in vitro. J Physiol 559: 721–728, 2004.
Vicini S, Ferguson C, Prybylowski K, Kralic J, Morrow AL, and Homanics GE. GABAA receptor 1 subunit deletion prevents developmental changes of inhibitory synaptic currents in cerebellar neurons. J Neurosci 21: 3009–3016, 2001.
White JA, Banks MI, Pearce RA, and Kopell NJ. Networks of interneurons with fast and slow -aminobutyric acid type A (GABAA) kinetics provide substrate for mixed gamma-theta rhythm. Proc Natl Acad Sci USA 97: 8128–8133, 2000.
Wieland HA, Luddens H, and Seeburg PH. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J Biol Chem 267: 1426–1429, 1992.
Yee BK, Hauser J, Dolgov VV, Keist R, Mohler H, Rudolph U, and Feldon J. GABA receptors containing the 5 subunit mediate the trace effect in aversive and appetitive conditioning and extinction of conditioned fear. Eur J Neurosci 20: 1928–1936, 2004.[CrossRef][Web of Science][Medline]
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E. D. Eggers and P. D. Lukasiewicz Receptor and transmitter release properties set the time course of retinal inhibition. J. Neurosci., September 13, 2006; 26(37): 9413 - 9425. [Abstract] [Full Text] [PDF]
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