QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (42) Citing Articles via Google Scholar Google Scholar Articles by Wang, J. Articles by Lu, Y. Search for Related Content PubMed PubMed Citation Articles by Wang, J. Articles by Lu, Y.

 Previous Article  |  Next Article 

The Journal of Neuroscience, February 1, 2003, 23(3):826

Interaction of Calcineurin and Type-A GABA Receptor ₂ Subunits Produces Long-Term Depression at CA1 Inhibitory Synapses

Jian Wang^{1, *}, ShuHong Liu^{1, *}, Ursula Haditsch², WeiHong Tu¹, Kimberley Cochrane¹, Gholamreza Ahmadian³, Linda Tran¹, Jadine Paw¹, YuTian Wang³, Isabelle Mansuy², Michael M. Salter⁴, and YouMing Lu¹

¹ Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Canada, T2N 4N1, ² Institute of Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland, and Departments of ³ Pathology and ⁴ Physiology, Programme in Brain and Behaviour, Hospital for Sick Children, University of Toronto, Toronto, Canada, M5G 1X8

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Long-term depression (LTD) is an activity-dependent weakening of synaptic efficacy at individual inhibitory synapses, a possible cellular model of learning and memory. Here, we show that the induction of LTD of inhibitory transmission recruits activated calcineurin (CaN) to dephosphorylate type-A GABA receptor (GABA_ARs) via the direct binding of CaN catalytic domain to the second intracellular domain of the GABA_AR-₂ subunits. Prevention of the CaN-GABA_A receptor complex formation by expression of an autoinhibitory domain of CaN in the hippocampus of transgenic mice blocks the induction of LTD. Conversely, genetic expression of the CaN catalytic domain in the hippocampus depresses inhibitory synaptic responses, occluding LTD. Thus, an activity-dependent physical and functional interaction between CaN and GABA_A receptors is both necessary and sufficient for inducing LTD at CA1 individual inhibitory synapses.

Key words: GABAA receptors; inhibitory synapses; plasticity; dephosphorylation; calcineurin; hippocampus

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

In CA1 neurons of the hippocampus, field stimulation of Schaffer-collateral fibers evokes diphasic excitatory-inhibitory synaptic currents: a fast EPSC mediated by AMPA receptors followed by a fast IPSC (Lu et al., 2000). A brief high-frequency stimulation (tetanus) of the Schaffer-collateral fibers produces long-term potentiation (LTP) of EPSCs and concomitantly long-term depression (LTD) of IPSCs (Andersen and Lømo, 1968; Stelzer et al., 1987; Lu et al., 2000). These coordinately regulated bidirectional changes of the excitatory and inhibitory synaptic strength are considered to be a cellular model of learning and memory (Bliss and Collingridge, 1993; Paulsen and Moser, 1998). Although we now know a great deal about the molecular steps contributing to the induction of LTP at excitatory synapses (Malenka and Nicoll, 1999; Malinow et al., 2000; Ali and Salter, 2001; Lisman and Zhabotinsky, 2001), the molecular mechanisms underlying the induction of LTD at CA1 individual inhibitory synapses have not been identified (Abraham et al., 1987; Thompson and Gahwiler, 1989; Thompson, 1994; Aizenman et al., 1998).

Fast IPSCs in CA1 neurons are mediated predominantly by type-A GABA receptors (GABA_ARs) (MacDonald and Olsen, 1994). The induction of LTD of GABA_A receptor-mediated IPSCs (GABA_AR-IPSCs) requires activation of excitatory NMDA receptors (Stelzer et al., 1987). The biochemical links between the NMDA receptors and the pathway for inducing LTD of GABA_AR-IPSCs involve calcineurin (CaN) (Lu et al., 2000). CaN, or Ca²⁺/calmodulin-dependent phosphatase 2B, consists of a 61 kDa catalytic domain (CaN-A) and a 19 kDa regulatory subunit (CaN-B) (Cohen, 1989). The enzymatic activity of CaN in CA1 neurons of the hippocampus is increased by activation of NMDA receptors (Lu et al., 2000). Also, there is extensive evidence showing that CaN plays a critical role in the activity-dependent changes of excitatory synaptic transmission in the hippocampus (Winder and Sweatt, 2001). Genetic inhibition of endogenous CaN in the forebrain (CaN transgenic mice), by expressing the auto-inhibitory domain of CaN, showed that LTP at CA1 excitatory synapses induced by subsaturating but not saturating tetanic stimulation was enhanced both in vitro and in vivo (Malleret et al., 2001). In another line of experiments, transgenic mice (CN98 transgenic mice) were generated in which a catalytic domain of CaN (CaN-A) was expressed in the forebrain (Winder et al., 1998). In these animals, NMDA receptor-dependent LTP induced by weak tetanus in the CN98 mutant mice was no different from that seen in control mice, but LTP elicited by stronger tetanus was reduced in CN98 mutant mice. These data suggest that manipulations of CaN activity alter the ability of CA1 excitatory synapses to induce LTP.

Because CaN downregulates GABA_A receptor function (Chen and Wong, 1995), activated CaN may interact with the synaptic GABA_A receptors for NMDA receptor-dependent LTD at CA1 inhibitory synapses. A direct test of this hypothesis has not yet been undertaken. Thus, we used double whole-cell patch-clamp recordings to induce LTD at CA1 individual inhibitory synapses. By taking advantage of a combined molecular genetic and biochemical approach, we demonstrate that NMDA receptor-dependent physical and functional interaction between CaN-A and GABA_AR-₂ subunit (GABA_AR_2S) fulfills necessary and sufficient conditions for inducing LTD of inhibitory transmission.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Double whole-cell patch-clamp recordings and LTD induction. Hippocampal slices (300 µm) were prepared from 30 ±2-d-old CaN transgenic control or mutant mice for Figures 1-4 and from 34 ±3-d-old CN98 transgenic mice for Figures 6 and 7. CaN mutant mice were generated by expressing the autoinhibitory domain in the C terminus of CaN in the forebrain with the doxycycline-dependent reverse tetracycline-controlled transactivator system, resulting in a 35-45% decrease in CaN activity. For all experiments, doxycycline (Mutual Pharmaceutical, Philadelphia, PA) was administered at 6 mg/g food at least 1 week before experimentation. Control mice were treated with dox only, as described in detail previously (Malleret et al., 2001). The slices were prepared as described previously (Lu et al., 1998, 2000). All procedures were in compliance with and approved by the University Animal Care and Use Committee, University of Calgary. For double whole-cell patch-clamp recordings from CA1 interneuron and pyramidal cell pairs, hippocampal slices were visualized with infrared (IR) illumination and a differential interference contrast (DIC) Axioskop 2FS plus equipped with Hamamatsu C2400-07E optics (see Fig. 1A). A whole-cell recording (tight-seal >1 G) with patch electrode (3-5 M) was initially obtained from a CA1 interneuron at the border of stratum radiatum and lacunosum-moleculare (LM). Subsequently, the second whole-cell recordings (tight-seal >10 G) were established from a CA1 pyramidal cell. The synaptic connections between CA1 interneurons and pyramidal cells were also identified by post hoc morphological analysis (see Fig. 1A). Single spikes in interneurons triggered unitary IPSCs in pyramidal cells that were blocked by the GABA_A receptor antagonist bicuculline (10 µM). An extracellular stimulating electrode was placed at the CA1 Schaffer-collateral fibers. LTD was induced by tetanus (two 100 Hz stimuli lasting 1 sec at an intertrain interval of 10 sec) of the Schaffer-collateral fibers. The unitary GABA_AR-IPSCs were filtered at 5 kHz with a low-pass filter. Data were digitized at a frequency of 10 kHz and stored on-line using the pclamp8 system. The input resistance and series resistance in postsynaptic pyramidal cells were monitored using prevoltage steps (2 mV, 100 msec) at 5 min intervals throughout the period of the experiment. Series resistance ranged from 9 to 12 M. Input resistance was 328 ± 29 M. For current-clamp mode, the intracellular solution contained (in mM): 115 K⁺-gluconate, 7.5 K⁺Cl, 27.5 K⁺-methylsulfate, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 guanosine triphosphate, and 0.1% biocytin, pH 7.4, 296 mOsm. For voltage-clamp recordings, the low Cl solution contained (in mM): 142.5 Cs-gluconate, 7.5 CsCl, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, and 0.3 guanosine triphosphate, pH 7.4, 296 mOsm, and the high Cl solution contained (in mM): 150 CsCl, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 guanosine triphosphate, and 0.1% biocytin, pH 7.4, 296 mOsm.

Coimmunoprecipitation, affinity purification ("pull-down"), and Western blotting. The CA1 region was microdissected as described previously (Lu et al., 1998). Four CA1 regions from control or LTD (5 or 30 min after induction of LTD) were pooled and homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 1 mM sodium orthovanadate, and proteinase inhibitor mixture (Sigma, St. Louis, MO) (5 µl/100 mg tissue). After clearing debris by centrifuging at 14,000 × g at 4°C, protein concentration in the extracts was determined by Bradford assay (Bio-Rad, Hercules, CA). The extracts (~500 µg protein) were incubated with nonspecific IgG (2 µg) or polyclonal mouse anti-CaN-A (2 µg; PharMingen, San Diego, CA) with or without 10 µg anti-CaN-A immunizing antigen (peptide I⁴⁵⁷-P⁴⁸²; PharMingen) overnight at 4°C, followed by the addition of 40 µl of Protein G-Sepharose (Sigma) for 3 hr at 4°C. In the immunoprecipitations with polyclonal rabbit antibody against GABA_AR-₁ subunit (anti-₁, 2 µg; Upstate BioTechnology, Lake Placid, NY), the anti-₁ was cross-linked to the protein G-Sepharose. This previous cross-linking of the anti-₁ permits the elution of antigen only to prevent interference of the IgG subunits in blotting (He et al., 1995). Cross-linking was performed with 0.5% glutaraldehyde (30 min, 25°C). The reaction was then terminated and washed four times with lysis buffer. The extracts (~500 µg protein) were then incubated with the cross-linked anti-₁ with or without 10 µg immunizing antigen (a peptide corresponding to residues 1-15 of the ₁ subunit) (Upstate Biotechnology). Immunoprecipitates were washed four times with lysis buffer and denatured with SDS sample buffer and separated by 12% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes using a Bio-Rad mini-protein-III wet transfer unit overnight at 4°C. Transfer membranes were then incubated with blocking solution [5% nonfat dried milk dissolved in TBST buffer (pH 7.5, 10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20)] for 1 hr at room temperature, washed three times, and incubated with monoclonal rabbit primary antibody against CaN-A (1:1000), polyclonal rabbit antibodies against GABA_AR-₁ subunit (anti-₁, 1:1000; Alpha Diagnostic), GABA_AR-₂ subunit (anti-₂, 1:1000; Alpha Diagnostic), or GABA_AR-₂ subunit (anti-₂, 1:2000; Alpha Diagnostic) for 1 hr at room temperature. Membranes were washed three times with TBST buffer and incubated with the appropriate secondary antibodies (1:1000 dilution) for 1 hr followed by washing four times. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences, Arlington, IL). The lanes marked "input" were loaded with 10% of the starting material used for immunoprecipitation. The precipitated bands were semiquantified using "the normalizing method" of the Densitometer Quantity One (see Quantity One User Guide, Bio-Rad). The intensities of the lanes marked input in each gel were normalized as 100%. Each of the other bands in the same gel was then expressed as the percentage of the respective input.

Glutathione S-transferase (GST) fusion proteins of GABA_AR-₁^334-420 (GST-₁), -₂^327-451 (GST-₂), -_2S^317-442 (GST-_2S), -_2S^332-442 (*GST-_2S), and -_2S^317-332 (short form) were prepared from bacterial lysates as described in detail previously (Liu et al., 2000). The extracts (~200 µg of proteins) were incubated with the indicated GST fusion proteins (~100 µg of protein), with or without 200 µg of ₂ peptide, overnight at 4°C, and then for another 3 hr at 4°C after 20 µl of Protein G-Sepharose (Sigma) was added. Beads were washed five times with lysis buffer. Eluted proteins were incubated in sample buffer (final concentration 5% SDS) and subjected to SDS-PAGE (12% gel). Transferred proteins were revealed by Western blot. In the experiments of Figure 3, E and F, the transferred membranes were incubated with monoclonal mouse primary antibody against -adaptin (1:1000; CN Biosciences) and monoclonal mouse antibodies against Src (1:1000; Upstate Biotechnology) for 1 hr at room temperature. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences).

In vitro binding assays.CaN420 (10 µg/ml) or CaN-B subunit (10 µg/ml) was incubated overnight at 4°C in 0.5 ml containing 40 mM Tris-HCl, pH 7.5, 0.5 mM CaCl₂, 150 mM 2-mercaptoethanol, 0.2 mg/ml BSA, 40 µl glutathione-Sepharose beads (Pharmacia), and 10 µg GST-₁, GST-₂, GST-_2S, or *GST-_2S. In some assays, as indicated in Figure 3, 10 µg/ml ₂-peptide or scrambled ₂-peptide (Biosynthesis Inc.) was included. The amino acid sequence of scrambled ₂-peptide was LDHSYFKVNDRDKPKK; it was created by random ordering of the sequence of ₂-peptide, LHYFVSNRKPSKDKDK, corresponding to the 317-332 residues of the GABA_A receptor ₂ subunit. The beads were washed five times with 200 µl PBS containing 0.1% Triton X-100 and eluted twice with 20 µl glutathione elution buffer. Eluted proteins were incubated in sample buffer (final concentration 5% SDS) and subjected to SDS-PAGE (12% gel). Transferred proteins were revealed by Western blot. Expression and purification of CaN420 and CaN-B were described in detail previously (Perrino et al., 1995).

The ₂ subunit phosphorylation assay. Rabbit polyclonal anti-₂pSer³²⁷ antibodies were raised against phospho-₂ peptide (³¹⁷LHYFVSNRKP(p)SKDKDK³³²). The resulting antisera were then affinity purified with phospho-₂ peptide immobilized on Affigel 10 (Bio-Rad). This antibody is competitively blocked with antigen peptide (see Fig. 5B). The GABA_A receptors were immunoprecipitated by previous cross-linked polyclonal rabbit anti-₂ from CA1 hippocampal extracts, as described above. Precipitated GABA_A receptors and the in vitro phosphorylated GST-_2S and the GST-_2S mutant (Ser³²⁷-Ala) (see below) were subjected to SDS-PAGE (12% gel). Transferred proteins were incubated with rabbit polyclonal anti-₂pSer³²⁷ (1:500) for 1 hr at room temperature. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences).

The GST-₁, GST-₂, GST-_2S, or GST-_2S mutant (Ser³²⁷-Ala) or GST alone (200 µg/each), was incubated with 1 µg/ml protein kinase C (PKC), catalytic fragment (BioMol Research Laboratories), protein kinase A catalytic subunit (BioMol Research Laboratories), and 0.4 mM [³²P]-ATP (1000 cmp/pmol), or the same concentration of ATP (see Fig. 5A), in 20 mM HEPES, pH 7.5, 10 mM MgCl₂, 0.5 mM CaCl₂, 10% glycerol, 5 µg/ml diolein, for 5 min at 30°C. The products were incubated with 10 µg of beads for 1 hr at room temperature and washed five times with 200 µl of PBS. The ³²P-labeled GST-₁, GST-₂, GST-_2S beads were then resuspended in phosphatase assay buffer, which contained 40 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 0.4 mg/ml bovine serum albumin, 1 mM dithiothreitol, and 0.45 µM okadaic acid and incubated at 30°C for 1 min in buffer. In Figure 5A, the phosphorylated GST-_2S and the GST-_2S mutant (Ser³²⁷-Ala) were subjected to SDS-PAGE (12% gel).

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Induction of LTD of the unitary GABA_AR-IPSCs at CA1 inhibitory synapses

LTD of the unitary GABA_AR-IPSCs was recorded at CA1 interneuron-pyramidal cell synapses, using double whole-cell patch-clamp recordings with an IR-DIC optic system (Fig. 1A). As shown in Figure 1B, 30 min after tetanus, the amplitude of the unitary GABA_AR-IPSCs decreased to 68.2 ± 7.4% (mean ± SEM) of baseline (n = 10 cells/5 control mice). This decrease was maintained over the course of 1 hr recordings.

View larger version (55K): [in this window] [in a new window]   Figure 1.   Induction of LTD at CA1 individual inhibitory synapses. A, IR-DIC images of double patch pipette tips on a lacunosum-moleculare (LM) interneuron and a pyramidal cell in a hippocampal slice (left). Shown are the morphology (middle) of a synaptically connected LM interneuron and a pyramidal cell pair labeled with biocytin and the arrangement (bottom) of the electrodes at a LM interneuron (i) paired with a pyramidal cell (ii) and an extracellular stimulating electrode at the Schaffer-collateral fibers (iii). B, Induction of LTD of the unitary GABAAR-IPSCs. b1, Single action potentials (top) and 10 consecutive single (center) and averaged GABAAR-IPSCs (bottom) at 60 mV are taken before (i) and after (ii) tetanus. b2, A representative recording (top) and the averaged amplitudes (bottom) of the unitary GABAAR-IPSCs are plotted. b3, Amplitude distribution histograms for the unitary GABAAR-IPSCs before (Baseline) and 20 min after tetanus (LTD) are plotted with bin sizes of 4 pA. Inset, Summarized coefficient variance (CV2 = M2/2; n = 10 cells/5 control mice). C, The number of open channels of the synaptic GABAA receptors was reduced during LTD. c1, Ten to 90% rise time (RT) and time constants () of decay before and after the induction of LTD were unchanged. c2, Current-variance relationships for the unitary GABAAR-IPSCs are plotted before (Baseline) and during LTD. The data points are fitted by a parabolic function (2 = iIm  Im2/No), where 2 is the variance, Im is the mean current, i is the single channel current, and No is the number of synaptically activated channels.

To determine whether LTD was presynaptic or postsynaptic in origin, we estimated the unitary GABA_AR-IPSC variability by computing the inverse of the square of the coefficient variance (CV^-2 = M²/²) (Edwards et al., 1990; Silver et al., 1998), where M is the mean unitary GABA_AR-IPSCs, and  is the variance about M. In accordance with previous observations (Nusser et al., 1998), the distribution of the unitary GABA_AR-IPSCs at the baseline (baseline noise: 3 ± 4.1 pA; n = 10 cells/5 control mice) has several clearly distinguishable peaks. The distribution was fitted by the sum of multiple Gaussian functions with one peak centered at 0 mV (failures) and other skewed peaks. The induction of LTD produced a shift in the distribution of the unitary GABA_AR-IPSCs toward smaller amplitude values, with no change in the number of the failures (Fig. 1B). The mean unitary GABA_AR-IPSC amplitude (M) is reduced after the induction of LTD, whereas the CV obtained by the method of M²/² is unchanged (Fig. 1B). The data indicate that a decrease in the postsynaptic GABA_A receptor function may underlie LTD at CA1 inhibitory synapses.

To determine whether a decrease in the channel conductance () of the synaptic GABA_A receptors or a decrease in the number (N_o) of synaptically activated channels contributes to LTD, we performed nonstationary fluctuation analysis (non-SFA) (Traynelis et al., 1993; De Koninck and Mody, 1994; Otis et al., 1994; Auger and Marty, 1997) for the experiments in Figure 1B (n = 10 cells/5 animals). We first analyzed the kinetic properties of the averaged unitary GABA_AR-IPSCs. As can be seen in Figure 1C, no changes of the unitary GABA_AR-IPSCs rise times or their decay time constants () were observed after the induction of LTD. The average responses were then scaled to the peak and subtracted from individual unitary GABA_AR-IPSCs. The variance of the fluctuation around mean was calculated and plotted against mean currents (Fig. 1C). The data points were fit by a parabolic function (² = iI_m  I_m²/N_o), where ² is the variance, I_m is the mean current, i is the single channel current, and N_o is the number of open channels of synaptic GABA_A receptors. At the baseline, estimated  was 24.2 ± 3.6 pS, which was not significantly different from that (23.6 ± 3.8 pS) during LTD (p > 0.50; n = 10 cells/5 mice). Estimated  is close to previously reported values of 20-32 pS for estimates of  derived from noise analysis in hippocampal granule cells (De Koninck and Mody, 1994; Nusser et al., 1998). In contrast, a significant decrease in N_o during LTD was observed; the synapses have on average 51 ± 7.2 (mean ± SEM; n = 10) open channels on the baseline, and this number decreased to 36 ± 4.8 (mean ± SEM; n = 10 cells) after the induction of LTD. The data suggest that the average number of open channels of the synaptic GABA_A receptor channels is reduced during LTD.

LTD recruits CaN-A to form a complex with GABA_A receptors

To explore the mechanisms underlying reduction in the number of open GABA_A receptor channels during LTD, we explored the physical interaction of endogenous CaN and GABA_A receptors. We immunoprecipitated extracts of control (Fig. 2A) and LTD-CA1 slices using antibodies against either CaN-A (anti-CaN-A) or the GABA_A receptor-₁ subunit (anti-₁). We found that 5 and 30 min after the induction of LTD, GABA_A receptor-₁, -₂, and -₂ subunits, the predominant GABA_A receptor subunits expressed in hippocampus (McKernan and Whiting, 1996), were coimmunoprecipitated with anti-CaN-A. Conversely, CaN-A was immunoprecipitated with anti-₁ (Fig. 2B). A nonspecific IgG did not immunoprecipitate either CaN-A or GABA_A receptors. In control CA1 extracts, however, no coimmunoprecipitation of CaN-A or GABA_A receptors was observed with either antibody (Fig. 2A), indicating that the induction of LTD recruits CaN-A into the GABA_A receptor complex.

View larger version (46K): [in this window] [in a new window]   Figure 2.   LTD recruits CaN-A to form a complex with GABAA receptors. A, B, Immunoprecipitation of the CA1 slices, 30 min after control stimulation (A) or 5 and 30 min after the induction of LTD (B), with nonspecific (N.S.) mouse IgG or a polyclonal mouse anti-CaN-A with (+) or without () 10 µg of immunizing antigen (a peptide corresponding to residues 457-482 of CaN), and a previous cross-linked anti-1 (see Materials and Methods) with (+) or without () 10 µg of immunizing antigen (a peptide corresponding to residues 1-15 of 1 subunit). Blots were probed with monoclonal rabbit anti-CaN-A or rabbit anti-1, anti-2, or anti-2, as indicated. In the lane marked Input, 50 µg of proteins without immunoprecipitation was loaded. The molecular size is marked at the right of the each panel. C, Immunoprecipitates were quantified for anti-CaN-A (filled bars) and anti-1 (open bars). Each immunoprecipitated band was normalized as a percentage of the respective input. Error bars are ±SEM (n = 4).

The major intracellular loops of the GABA_A receptor subunits contain many consensus phosphoserine/threonine residues (Moss et al., 1992; Brandon et al., 1999, 2000), which may be targeted by CaN-A. To investigate this possibility, we constructed GST fusion proteins encoding the second intracellular loops ₁^334-420, ₂^327-451, and _2S^317-442 (short form) of the GABA_A receptors. These fusion proteins precipitated CaN-A from the LTD-CA1 extracts but not from controls (Fig. 3A). The data suggest that synaptic activity drives activated CaN into the GABA_A receptor complex. The GABA_A receptor fusion proteins may bind indirectly to the activated CaN and directly to their respective subunit to pull down CaN-A from the CaN-A-GABA_A receptor complex in the CA1 extracts.

View larger version (51K): [in this window] [in a new window]   Figure 3.   Direct binding of CaN-A to the second intracellular domain of 2 subunit. A, Affinity precipitation of the CA1 extracts, 30 min after control stimulation, or the induction of LTD, with GST-1, GST-2, GST-2S, or GST alone, and blots were probed with anti-CaN-A. B, Direct binding of CaN-A to GST-2. CaN420 (1 µg) or CaN-B (1 µg) was incubated with 10 µg of GST-1 or GST-2, GST-2S, *GST-2S, or GST-2S + 10 µg 2-peptide, or GST-2L (long form) or GST alone, and blots were probed with anti-CaN-A or anti-CaN-B, as indicated. C, CaN-A binds to GST-2317-332. D, Affinity precipitation of the LTD-CA1 extracts with 10 µg of GST-1, GST-2, GST-2S, or GST alone, in the presence of 10 µg of 2-peptide, and blots were probed with anti-CaN-A. E, Affinity precipitation of the CA1 extracts with GST-1, GST-2, GST-2S, or GST alone (top) or in the presence of 2 peptide (bottom), and blots were probed with anti-Src, as indicated. F, Affinity precipitation of the CA1 extracts with GST-1, GST-2, GST-2S, or GST alone (top) or in the presence of 2 peptide (bottom), and blots were probed with anti--adaptin, as indicated. Similar results are observed in each of four experiments (n = 4). The molecular size is marked at the right of the each panel.

We then determined whether CaN-A directly binds to GABA_A receptors. We generated a constitutively expressed recombinant CaN catalytic fragment (CaN420) that exhibits stable Ca²⁺-independent phosphatase activity (Perrino et al., 1995). The GST fusion proteins encoding ₁^334-420, ₂^327-451, _2S^317-442, and _2L^317-446 subunits of the GABA_A receptors were incubated with either CaN420 or CaN-B. As shown in Figure 3B, the CaN420, but not CaN-B, bound to GST-_2S^317-442 and _2L^317-446, but not to GST alone, or ₁- or ₂-peptide fusion proteins. A GST-GABA_A receptor-_2S deletion mutant (GST-_2S^332-442 or *GST-_2S) failed to bind CaN420, indicating the importance of residues 317-332 within the ₂ subunit for direct interaction with CaN-A. This was confirmed with a synthesized ₂-peptide encoding residues 317-332 of the ₂ subunit, which prevented binding of CaN420 to GST-_2S (Fig. 3B). Consistent with this, we also found that CaN-A can bind to GST-₂^317-332 (Fig. 3C) and that ₂-peptide interfered with GABA_A receptor-CaN-A association in the LTD CA1 extracts (Fig. 3D).

It is known that synaptic GABA_A receptor function is regulated by tyrosine kinase Src and adaptin  and  subunits of AP2 (Kittler et al., 2000; Brandon et al., 2001) and that Src- and adaptin-₂ subunit interaction increases the synaptic GABA_A receptor activity. Thus, CaN may have acted as a competitive inhibitor of these molecules to downregulate the GABA_A receptor function. To investigate this, we examined whether ₂-peptide interferes with Src- and adaptin-GABA_A receptor association. Consistent with previous studies, we found that GABA_A receptor fusion proteins precipitated the endogenous Src (Fig. 3E) and -adaptin (Fig. 3F) in the CA1 extracts. In the presence of ₂ peptide, -adaptin but not Src can still be precipitated. The data suggest that Src may also bind to residues 317-332 of the ₂ subunit.

CaN-A-GABA_A receptor complex formation is necessary for LTD

To test whether this activity-dependent interaction between CaN-A and GABA_A receptors is essential for the induction of LTD at CA1 individual inhibitory synapses, we examined the consequence of blocking CaN-A-GABA_A receptor complex formation. First, we blocked endogenous CaN by expressing a peptide corresponding to the autoinhibitory domain in the C terminal of CaN-A in the hippocampus of transgenic mice (Malleret et al., 2001). No interaction between CaN-A and GABA_A receptors could be observed 5 and 30 min after tetanus in the CaN mutant mice (Fig. 4A), and the effect of the tetanus on the unitary GABA_AR-IPSCs was completely abolished in mutant mice in that the unitary GABA_AR-IPSCs 30 min after tetanus was 93 ± 10.6% of baseline (Fig. 4B) (n = 10 cells/5 animals). Second, blockade of the CaN-A-GABA_A receptor complex formation by an NMDA receptor antagonist, AP-5 (Fig. 4C), prevented the induction of LTD of the unitary GABA_AR-IPSCs (Fig. 4D). Third, we applied 10 µM ₂-peptide directly into the CA1 postsynaptic pyramidal cells and found that it abolished LTD of the unitary GABA_AR-IPSCs (Fig. 4E). A peptide with the same amino acid composition, but in random order, scrambled ₂-peptide, served as a control, and did not prevent induction of LTD. Thus, NMDA receptor-dependent interaction of activated CaN and GABA_A receptors was required for induction of LTD at CA1 inhibitory synapses.

View larger version (44K): [in this window] [in a new window]   Figure 4.   Blockade of CaN-A-GABAA receptor complex formation prevents the induction of LTD. A, Immunoprecipitation of the CaN mutant CA1 slices, 5 and 30 min after tetanus, with an N.S. IgG or anti-CaN-A or anti-1. Blots were probed with anti-CaN-A or anti-2. In the lane marked Input, 50 µg of proteins without immunoprecipitation was loaded. Similar results are observed in each of the four experiments. B, LTD is abolished in the CaN mutant mice. b1, Single action potentials (top) and averaged unitary GABAAR-IPSCs at 60 mV (bottom) are taken before (i) and after (ii) tetanus (arrowhead). b2, The averaged amplitudes of the unitary GABAAR-IPSCs are plotted for the experiments with CaN control (open circles; the same as in Fig. 1B) or CaN mutant mice (filled circles). C, Immunoprecipitation of the CaN control mice CA1 slices, after tetanus in the presence of 50 µM AP-5, with an N.S. IgG or anti-CaN-A or anti-1. Blots were probed with anti-CaN-A or anti-2. D, Induction of LTD depends on NMDA receptors. d1, Single action potentials (top) and averaged unitary GABAAR-IPSCs at 60 mV (bottom) are taken before (i) and after (ii) tetanus (arrowhead). d2, Normalized unitary GABAAR-IPSCs are plotted for the recordings with the control (filled circles; n = 5) or AP-5 (open circles; n = 6). E, 2-peptide blocks the induction of LTD. e1, Single action potentials (top) and averaged unitary GABAAR-IPSCs at 60 mV (bottom) are taken before (i) and after (ii) tetanus (arrowhead). e2, Normalized unitary GABAAR-IPSC amplitudes are plotted for the experiments with 2-peptide (open circles; n = 8) or scrambled 2-peptide (filled circles; n = 7).

CaN-A and LTD dephosphorylates GABA_A receptor ₂ subunits

The CaN-A-GABA_A receptor complex formation may strategically position the CaN catalytic domain to dephosphorylate synaptic GABA_A receptors. It is now known that CaN-A directly interacts with residues 317-332 within the ₂ subunit that contains a phosphoserine (pSer³²⁷) residue. We therefore developed a phosphospecific antibody to pSer³²⁷ ₂ peptide (anti-₂pSer³²⁷) to analyze LTD-dependent changes in GABA_A receptor ₂ subunit phosphorylation in CA1 neurons. Anti-₂pSer³²⁷ was specific for pSer³²⁷ in ₂ subunit, reacting with phosphorylated wild-type GST-_2S but not with the mutant GST-_2S (Ser³²⁷-Ala) (Fig. 5A). Next, we immunoprecipitated GABA_A receptors from the CA1 extracts. Blot analysis of the immunoprecipitates with anti-₂pSer³²⁷ detected multiple reactive bands, but only the one corresponding to the 51 kDa was selectively blocked by preabsorption with the pSer³²⁷-₂-peptide antigen (Fig. 5B), demonstrating that GABA_A receptor ₂ subunit is phosphorylated under basal conditions. After induction of LTD of the unitary GABA_AR-IPSCs in the CA1 slices, we found that the immunoreactivity to anti-₂pSer³²⁷ was significantly decreased at 5 min (74.2 ± 13.2% over control; p < 0.05; n = 4) and 30 min (70.9 ± 10.1% over control; p < 0.05; n = 5; normalized by immunoreactivity to a general anti-₂) (Fig. 5C). This net dephosphorylation of GABA_A receptor ₂ subunit was not caused by a tetanus-induced decrease in total protein, because blot analysis showed that the amount of ₂ subunit was unchanged (Fig. 4D).

View larger version (49K): [in this window] [in a new window]   Figure 5.   CaN-A and LTD dephosphorylate GABAA receptors. A, Protein immunoblot of phosphorylated GABAA receptors using anti-2pS327. Top, The GST-2S (lanes 1, 3) or the mutant GST-2S (Ser327-Ala) (lanes 2, 4) were subjected to in vitro phosphorylation for 1 min (lanes 1, 3) or 5 min (lanes 2, 4). Bottom, Blot of SDS-PAGE by anti-GST antibody. B, Immunoblot of SDS-PAGE after precipitation by previous cross-linked polyclonal rabbit anti-2 (see Materials and Methods) from CA1 slices with either a rabbit polyclonal anti-2, as indicated (lane 1), or anti-2pS327 without (lane 2) or with phosphopeptide antigen (lane 3) or nonphosphopeptide (lane 4). C, Anti-2pS327 immunoblots of SDS-PAGE after precipitation by N.S. IgG (lane 1) or anti-2 from CA1 slices 5 and 30 min after control stimulation (lane 2) or the induction of LTD (lane 3). The precipitates were quantitated for 2 subunit phosphorylation. The levels of 2 subunit phosphorylation after induction of LTD (filled bars) were normalized to their respective lane 2 (control stimulation; open bars). Error bars are ±SEM (n = 4; *p < 0.01). D, The anti-2pS327 (top) and anti-2 (bottom) immunoblots of SDS-PAGE after precipitation by N.S. IgG (lane 1) or anti-2 from the CaN mutant slices without tetanus (lane 2) or with tetanus (lane 3) or the CaN control mice with tetanus in the presence of AP-5 (lane 4) or calyculin-A (lane 5). Data were normalized to lane 2 from the same gel in bar graph. *p < 0.01 (n = 5); paired Student's t test.

To assess whether activated CaN is responsible for LTD-dependent dephosphorylation of the GABA_A receptor ₂ subunit, we examined phosphorylation of the ₂ subunits in the CaN mice. No change in the basal level of ₂ subunit phosphorylation was observed in either the CaN control or the mutant mice. However, tetanic stimulation failed to reduce the immunoreactivity to anti-₂pSer³²⁷ in the CaN mutants, as shown in Figure 5D, suggesting that CaN-A is necessary for the GABA_A receptor dephosphorylation in situ. Moreover, blockade of NMDA receptors by AP-5 inhibited tetanus-induced dephosphorylation of the GABA_A receptors in CaN control mice. We subsequently studied the consequence of blocking other protein phosphatases (PPs) by applying 1 µM calcyculin A, an inhibitor of PP1/2A but not of CaN (Cohen and Cohen, 1989). We found that calyculin A had no effect on the decrease in the immunoreactivity to anti-₂pSer³²⁷ in CaN control mice (Fig. 5D).

To identify further specific ₂ subunit dephosphorylation, but not other subunits of the GABA_A receptors, we labeled the GST-₁, -₂, or -₂ subunit with ³²P in vitro. CaN420 specifically caused a decrease in the level of ³²P labeling of the GST-_2S only, and CaN-A immunoprecipitated from LTD-CA1 slices (CaNltd) produced similar results (Fig. 6). In addition, in the presence of ₂-peptide, neither CaN420 nor CaNltd affects ³²P labeling of the GST-_2S (Fig. 6), indicating that GABA_A receptor _2S residues 317-332 represent the interacting site of the endogenous CaN-A. Taken together, the above results indicate that activity-dependent CaN-A-GABA_A receptor complex formation enables CaN-A to dephosphorylate the GABA_A receptor _2S subunit that leads to the induction of LTD of the unitary GABA_AR-IPSCs.

View larger version (55K): [in this window] [in a new window]   Figure 6.   CaN-A and LTD dephosphorylates GABAA receptor 2 subunit. 32P-labeled GABAA receptor GST fusion proteins (see below) were exposed to 10 µg/ml iCaN420 (heat-inactivated control; lane 1) or CaN420 (lane 2), CaNctr (control; lane 3), or CaNltd (lane 4) and analyzed by SDS-PAGE and autoradiography. The extent of 32P labeling for the experiments with iCaN420 (black bars), CaN420 (open bars), CaNctr (cross bars), or CaNltd (hatched bars) were quantitated, and normalized to respective controls (n = 4; *p < 0.01).

Overexpression of CaN-A reduces mGABA_AR-IPSCs

We next explored whether the CaN-A-GABA_A receptor complex formation is sufficient for the induction of LTD. We expressed the CaN catalytic domain, CaN-A, in the hippocampus of transgenic mice (CN98 mutant mice) (Mansuy et al., 1998; Winder et al., 1998). We affinity precipitated extracts from the CN98 mutant and control CA1 slices using GST-₁^334-420, -₂^327-451, and -_2S^317-442. We observed that these fusion proteins precipitated CaN-A from the CN98 mutant CA1 extracts but not from control (Fig. 7A), showing that overexpressed CaN-A physically interacts with GABA_A receptors.

View larger version (39K): [in this window] [in a new window]   Figure 7.   Interaction of CaN-A and GABAA receptors reduces the number of open channels of the synaptic GABAA receptors. A, Affinity precipitation of the CN98 control, or the mutant CA1 slices, with GST-1, GST-2, GST-2S, or GST alone and blots were probed with anti-CaN-A. In the lane marked Input, 50 µg of proteins without immunoprecipitation was loaded. Similar results are observed in each of the four experiments. B, The number of open channels of the synaptic GABAA receptors was reduced in CN98 mutant mice. b1, Sample traces of mIPSCs in the presence of 1 µM TTX are taken from the experiments with CN98 control or mutant mice. b2, The number of events in CN98 control and mutant mice is binned at 4 pA. Inset, Summarized coefficient variance (CV-2; n = 10 cells). b3, Ten to 90% rise time and time constants () of decay in mutant mice were unchanged. b4, Current-variance relationships for the mIPSCs are plotted for the experiments with CN98 control or mutant mice.

We then examined whether an interaction of the overexpressed CaN-A and synaptic GABA_A receptor causes a depression of the GABA_AR-IPSCs. Pharmacologically isolated (in the presence of 10 µM CNQX and 1 µM TTX), spontaneously miniature GABA_AR-IPSCs (mGABA_AR-IPSCs) in CA1 pyramidal cells were measured. A significant decrease in mean amplitude of mGABA_AR-IPSCs, but not in their frequency, was observed in the CN98 mutant mice (Fig. 7B) (63.9 ± 6.6% of the controls; n = 10 cells/10 animals; p < 0.05). Mean intervals of mGABA_AR-IPSCs in the CN98 mutant and control mice were 49.2 ± 7.2 and 52.6 ± 6.9 msec (n = 10 cells/5 animals; p > 0.05), respectively. The distribution of the unitary GABA_AR-IPSCs shifted to smaller-amplitude values in the CN98 mutant mice, whereas the CV was unchanged (Fig. 7B). Using peak-scaled, non-SFA, the properties of synaptically activated GABA_A receptor channels in CN98 mice were determined. A mean  of 23.6 ± 4.3 pS (mean ± SEM; n = 10 cells/10 animals) was obtained in CN98 control mice. This value of  shows no difference from that in CN98 mutant mice. The synapses in CN98 mice have on average 48 ± 6.1 (mean ± SEM; n = 10 cells/5 animals) open channels, and this number decreased to 32 ± 4.2 (mean ± SEM; n = 10 cells/10 animals) in CN98 mutant mice, indicating that a decrease in the number of the synaptically activated GABA_A receptor channels is responsible for the reduced mGABA_AR-IPSCs in CN98 mutant mice.

CaN-A-induced responses and LTD occlude each other

If a reduction of the GABA_AR-IPSCs by CaN-A mimics the features of tetanus-induced LTD, then LTD and the GABA_AR-IPSC reduction by CaN-A may mask each other. This was initially investigated by comparing the current-variance relationship of spontaneously occurring IPSCs (sIPSCs; without TTX) 30 min after tetanus in CN98 control mice with that in mutant mice (Fig. 8A). Estimated sIPSC rise times in control and the CN98 mutant mice were 0.22 ± 0.02 and 0.24 ± 0.03 msec, respectively. The decay time constants were 3.18 ± 0.21 msec in control compared 3.21 ± 0.18 msec in the CN98 mutant mice. The channel conductance () of the synaptic GABA_A receptors in the CN98 mutant mice was 24.1 ± 3.2 pS, similar to that (22.9 ± 2.8 pS) in control mice (Fig. 8A). In contrast, the number (N_o) of open channels of the synaptic GABA_A receptors was reduced in CN98 mutant mice; the synapses in control mice have on average 53 ± 8.2 (mean ± SEM) open channels, and this number decreased to 39 ± 6.4 (mean ± SEM) in the CN98 mutant mice. No reduction of the N_o after tetanus was observed in CN98 mutant mice, whereas tetanus did produce a decrease in the number of synaptically activated GABA_A receptor channels in CN98 control mice. Thus, CaN-A caused a decrease in the number of the synaptic GABA_A receptor channels that appeared to occlude the tetanus effect.

View larger version (36K): [in this window] [in a new window]   Figure 8.   CaN-A-induced depression and LTD occluded each other. A, Effect of tetanus on spontaneous IPSCs. Single (a1) or averaged (20 consecutive responses) (a2) traces are taken from the experiments with the CN98 control or mutant mice. a3, Current-variance relationships for spontaneous IPSCs are plotted before (filled symbols) and 20 min after tetanus (open symbols) from the experiments with the CN98 control (circles) or the mutant mice (triangles). The data points are fitted by a parabolic function. B, CaN420 reduces amplitudes of the unitary GABAAR-IPSCs. b1, The traces are single action potentials (top) and averaged 10 consecutive unitary GABAAR-IPSCs (bottom) taken at the time indicated by the letters. Normalized unitary GABAAR-IPSCs for the recordings with 10 µg/ml iCaN420 (filled circles; n = 5) or 10 µg/ml CaN420 (open circles; n = 6) are plotted. b2, Single action potential and superimposed unitary GABAAR-IPSCs at 60 to +60 mV (40 mV increment) recorded with high Cl (top). Current-voltage relationships (n = 4) are taken from 5 min (Baseline; open circles) and 20 min after starting recording (filled circles). C, Effects of tetanus on CaN420 action. Single action potentials (top) and averaged 10 consecutive unitary GABAAR-IPSCs (bottom) are taken at the time indicated by the letters. Normalized amplitudes of the unitary GABAAR-IPSCs when tetanus (arrowhead) was delivered 10 min after the start of recording (c1) or without tetanus (c2) are plotted. CaN420 (10 µg/ml) was actively perfused during the period indicated by the horizontal bars.

Second, we examined the dependence of LTD on the concentration of exogenous CaN-A. A consistent threshold for the depression of the unitary GABA_AR-IPSCs was detected by application of CaN420 (2 µg/ml), with a maximum depression near 10 µg/ml. CaN420 (10 µg/ml) was then applied directly into the CA1 pyramidal cells through the patch electrodes. The amplitude of the unitary GABA_AR-IPSCs decreased to 58.4 ± 6.7% of baseline (n = 6 recordings). In contrast, heat-inactivated CaN420 (iCaN420) had no effect (Fig. 8B). Moreover, application of CaN420 did not alter the reversal potentials, as shown by the current-voltage curves for peak amplitude of the unitary GABA_AR-IPSCs in Figure 8B. The data demonstrate that activated CaN reduces the peak amplitude of unitary GABA_AR- IPSCs, with no change in driving force.

In other experiments, tetanus produced a reduction of the unitary GABA_AR-IPSCs, but there was no further decrease when CaN420 was applied intracellularly (Fig. 8C). On the other hand, in cells not conditioned by tetanus, perfusion of CaN420 at the same time after beginning recording caused a decrease in the unitary GABA_AR-IPSCs that reached a stable level at 63.8 ± 7.9% of the baseline (n = 6 cells). Thus, the CaN-A-induced reduction of the unitary GABA_AR-IPSCs and LTD occluded each other, indicating an overlapping mechanism of action.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Our analysis of the molecular mechanisms contributing to LTD of the unitary GABA_AR-IPSCs revealed a novel postsynaptic event: an activity-dependent physical and functional interaction between CaN-A and the GABA_A receptor-₂ subunit permits a rapid and sustained reduction of inhibitory synaptic strength at individual CA1 synapses. Furthermore, our results indicate that the CaN-GABA_A receptor complex formation occurs only at synapses conditioned by activation of postsynaptic NMDA receptors. We determined that activated endogenous CaN was recruited into the GABA_A receptors at CA1 inhibitory synapses via a direct binding with the _2S subunit, because a synthesized ₂-peptide that encodes residues 317-332 of the _2S subunit prevented CaN interaction with GABA_A receptors. Because intracellular application of the ₂-peptide directly into the postsynaptic neurons inhibited the induction of LTD at CA1 individual inhibitory synapses, the most parsimonious explanation for our results is that activation of NMDA receptors results in increased Ca²⁺ entry, which activates CaN, and drives the activated CaN to dephosphorylate postsynaptic GABA_A receptors, leading to a downregulation of GABA_A receptor function.

Although we did not directly rule out presynaptic release properties during LTD, similar values of the coefficient variance of the evoked unitary IPSCs and spontaneous IPSCs before versus after the induction of LTD were observed. Using peak-scaled, non-SFA, our data showed that the reduced number of the synaptically activated GABA_A receptor channels accounts for LTD at CA1 inhibitory synapses. Thus, taken together, our biochemical and electrophysiological data are not consistent with the presynaptic locus of NMDA receptor-dependent LTD of the unitary GABA_AR-IPSCs in CA1 neurons.

CaN is known to play a role in the induction of LTD of excitatory transmission in CA1 neurons (Kirkwood and Bear, 1994; Mulkey et al., 1994). Consistent with these pharmacological studies, a recent study in the knock-out mice that lack CaN regulatory domain B1 showed that the induction of LTD at CA1 excitatory synapses was impaired (Zeng et al., 2001). The mechanisms by which the CaN-dependent signaling pathway participates in LTD at CA1 excitatory synapses are thought to involve the CaN-dependent activation of PP1 by inactivating I-1, a PP1 inhibitor (Lisman and Zhabotinsky, 2001). Consistent with this idea, the postsynaptic injection of I-1 peptides that mimics the phosphorylated, activated state of I-1 blocks the induction of LTD of excitatory transmission in CA1 pyramidal cells (Mulkey et al., 1994). From our results using GST fusion proteins of the subunit intracellular domains, we suggest that activated CaN is directly targeted to the phospho-Ser³²⁷ of the _2S subunit. Therefore, low-frequency stimulation activates the CaN-dependent PP1 pathway that leads to the induction of LTD of excitatory transmission, whereas a brief high-frequency stimulation drives an activated CaN directly onto the GABA_A receptors, leading to the induction of LTD at CA1 inhibitory synapses.

Among the mechanisms proposed for modification of GABA_A receptor activity, one of the simplest is a change in the number of GABA_A receptors in the postsynaptic membrane. There is extensive evidence showing that the GABA_A receptor ₂ subunit plays a critical role in postsynaptic membrane trafficking as well as in synaptic targeting of GABA_A receptors. For example, the number of synaptic GABA_A receptors was decreased in cerebral cortex of mice lacking the ₂ subunit (Essrich et al., 1998). The ₂ subunits of GABA_A receptors interact differentially with diverse intracellular molecules including GABARAP (Nymann-Andersen et al., 2002), -adaptin (Kittler et al., 2000), and Src (Brandon et al., 2001), all of which have been implicated in synaptic targeting of GABA_A receptors. For example, in cultured hippocampal neurons, the -adaptin-₂ subunit association disrupts the clathrin-dependent GABA_A receptor endocytosis. Therefore, it is possible that CaN-A interferes with the -adaptin-₂ subunit interaction to regulate the number of functional GABA_A receptors in the inhibitory postsynaptic sites. However, our observation show that the ₂-peptide that prevents CaN-A-GABA_A receptor complex formation did not change the -adaptin-GABA_A receptor association, indicating that CaN acts via direct CaN-A-₂ subunit interactions rather than as an adaptin substrate. Interestingly, clathrin-mediated endocytosis is involved in the expression of cerebellar LTD at excitatory synapses (Wang and Linden, 2000). It will be of importance to explore whether CaN regulation of GABA_A receptors is mediated by the clathrin-dependent GABA_A receptor internalization. The internal versus surface expression of GABA_A receptors in the postsynaptic sites needs to be examined to determine CaN-mediated GABA_A receptor trafficking to and from the cell surface that is therefore likely to be an important mechanism for expression of LTD at inhibitory synapses. In addition to adaptin, there has been a report that tyrosine kinase Src modulates neuronal as well as recombinant GABA_A receptors to enhance receptor channel activity (Moss et al., 1995). Src kinase also phosphorylates both Y³⁶⁵ and Y³⁶⁷ residues of the _2L (long form) subunit (Moss et al., 1995). By specifically blocking the association of Src with the GABA_A receptor ₂ subunit (Fig. 3E), CaN may act as a competitive inhibitor of Src to downregulate GABA_A receptor function. Clearly, further studies will be needed to clarify the interaction of CaN and Src kinase with regard to GABA_A receptor function.

There is extensive evidence showing that PKC phosphorylates GABA_A receptors and increases the peak amplitude of mIPSCs recorded from hippocampal granule cells (Poisbeau et al., 1999) (but see Connolly et al., 1999). PKC also enhances the channel activity of recombinant GABA_A receptors expressed in the L929 cell line (Lin et al., 1994, 1996). A recent study shows that activation of PP1 decreases in phosphorylation of Ser^657/660 on the catalytic domain of PKC and PKC_II (Thiels et al., 2000). This decrease in PKC phosphorylation is associated with a decrease in PKC activity. Thus, CaN may act through the PP1 pathway to downregulate PKC leading to the downregulation of GABA_A receptor function for inducing LTD at CA1 inhibitory synapses. Because the GABA_A receptor subunit combination and the receptor density expressed in CA1 pyramidal cells are heterogeneous (Pettit and Augustine, 2000), the effect of PKC phosphorylation of the GABA_A receptor on its channel activity may depend on the receptor types under study. Therefore, whether the CaN-PKC pathway is involved in LTD at CA1 inhibitory synapses needs to be clarified in hippocampal slices. Further studies will use the strategies and protocols established in this study to determine how protein kinases are targeted to the synaptic GABA_A receptors in synaptic plasticity. The final illustration of the cellular and molecular mechanisms underlying bidirectional regulation of GABA_A receptors mediated by kinases and phosphatases in synaptic plasticity can teach us much about what kinds of molecules are needed to build arrays of the GABA_A receptor regulations.

CaN is reported to modulate the channel kinetics of the GABA_A receptors in cultured neurons (Jones and Westbrook, 1997). However, we found that there were no changes of the unitary GABA_AR-IPSC rise times or their decay time constants after activation of CaN. The different observations could be attributed to the fact that in previous studies, reduced decay times of IPSC were caused by inhibition of endogenous CaN, which is generally dependent on the basal level of CaN activity. In hippocampal slices, however, we found that there were no physical and functional interactions of CaN and GABA_A receptors at basal CA1 inhibitory synapses. Therefore, it is possible that enzymatic regulation of synaptic receptors in cultured neurons is different from that in acute isolated neurons in the slices. Consistent with this idea, two other previous studies show that the activated but not basal level of CaN reduces the peak amplitude of whole-cell GABA_A receptor currents in acute isolated hippocampal neurons (Stelzer and Shi, 1994; Chen and Wong, 1995).

Much of the difficulty in assigning changes at the level of individual inhibitory synapses is that the IPSCs, originating from feed-forward (Alger and Nicoll, 1982) as well as feed-back activation of the CA1 interneurons (Lacaille and Schwartzkroin, 1988) by the Schaffer-collateral stimulation, are polysynaptic and thus obscure any link between properties of IPSCs and the efficacy of individual inhibitory synapses. Conventionally, the GABA_AR-IPSCs in CA1 pyramidal neurons are studied under conditions in which excitatory glutamate receptors are blocked. This manipulation, however, fails to account for NMDA receptor-dependent intracellular events that are essential for both GABA_A receptor regulation and the initiation of sustained change in inhibitory synaptic strength. Here, we established double whole-cell patch-clamp recordings by which we monitored the unitary GABA_AR-IPSCs between identified pairs of CA1 interneurons and the innervated pyramidal cells from hippocampal slices. Using both genetic manipulations and biochemical assays, we determined the mechanisms for excitatory activity-dependent LTD at inhibitory synapses. These data demonstrate a previously unknown molecular mechanism by which individual GABAergic synapses alter their efficacy in CA1 neurons of the hippocampus. Because inhibitory synaptic strength is critical for the control of networks within the brain (Ben-Ari and Represa, 1990), our results may suggest a CaN-dependent cellular substrate of learning and memory.

	FOOTNOTES

Received Aug. 16, 2002; revised Nov. 14, 2002; accepted Nov. 15, 2002.

^* J.W. and S.L. contributed equally to this work.

This work was supported by the Canadian Institute for Health Research (Y.M.L), Heart and Stroke Foundation, Canada (Y.M.L), Alberta Heritage Foundation for Medical Research (Y.M.L), Canada Foundation for Innovation (Y.M.L), and Alberta Foundation for Innovation and Science (Y.M.L). We thank Dr. Brian Perrino for the construct of CaN420. We thank Drs. John F. MacDonald, Wayne Giles, Keith Sharkey, and Brian MacVicar, for critical comments on this manuscript.

Correspondence should be addressed to Dr. YouMing Lu, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Canada, T2N 4N1. E-mail: luy{at}ucalgary.ca.

	References

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

• Abraham WC, Gustafsson B, Wigstrom H (1987) Long-term potentiation involves enhanced synaptic excitation relative to synaptic inhibition in guinea-pig hippocampus. J Physiol (Lond) 394:367-380[Abstract/Free Full Text].
• Aizenman CD, Manis PB, Linden DJ (1998) Polarity of long-term synaptic gain change is related to postsynaptic spike firing at a cerebellar inhibitory synapse. Neuron 21:827-835[Web of Science][Medline].
• Alger BE, Nicoll RA (1982) Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro. J. Physiol (Lond) 328:105-123[Abstract/Free Full Text].
• Ali DW, Salter MW (2001) NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity. Curr Opin Neurobiol 11:336-342[Web of Science][Medline].
• Andersen P, Lømo T (1968) Inhibitory synapses. In: Structure and function of inhibitory neuronal mechanisms (von Euler C, Skoglund S, Soderberg U, eds), pp 335-342. Oxford: Pergamon.
• Auger C, Marty A (1997) Heterogeneity of functional synaptic parameters among single release sites. Neuron 19:139-150[Web of Science][Medline].
• Ben-Ari Y, Represa A (1990) Brief seizure episodes induce long-term potentiation and mossy fiber sprouting in the hippocampus. Trends Neurosci 13:312-318[Web of Science][Medline].
• Bliss TV, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39[Medline].
• Brandon NJ, Uren JM, Kittler JT, Wang H, Olsen R, Parker PJ, Moss SJ (1999) Subunit-specific association of protein kinase C and the receptor for activated C kinase with GABA type A receptors. J Neurosci 19:9228-9234[Abstract/Free Full Text].
• Brandon NJ, Delmas P, Kittler JT, McDonald BJ, Sieghart W, Brown DA, Smart TG, Moss SJ (2000) GABA_A receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway. J Biol Chem 275:38856-38862[Abstract/Free Full Text].
• Brandon NJ, Delmas P, Hill J, Smart TG, Moss SJ (2001) Constitutive tyrosine phosphorylation of the GABA_A receptor 2 subunit in rat brain. Neuropharmacology 41:745-752[Web of Science][Medline].
• Chen QX, Wong RK (1995) Suppression of GABA_A receptor responses by NMDA application in hippocampal neurons acutely isolated from the adult guinea-pig. J Physiol (Lond) 482:353-362[Abstract/Free Full Text].
• Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58:453-508[Web of Science][Medline].
• Cohen P, Cohen PTW (1989) Protein phosphatases come of age. J Biol Chem 264:21435-21438[Free Full Text].
• Connolly CN, Kittler JT, Thomas P, Uren JM, Brandon NJ, Smart TG, Moss SJ (1999) Cell surface stability of gamma-aminobutyric acid type A receptors. Dependence on protein kinase C activity and subunit composition. J Biol Chem 274:36565-36572[Abstract/Free Full Text].
• De Koninck Y, Mody I (1994) Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABA_A receptor channels. J Neurophysiol 71:1318-1334[Abstract/Free Full Text].
• Edwards FA, Konnerth A, Sakmann B (1990) Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. J Physiol (Lond) 430:213-249[Abstract/Free Full Text].
• Essrich C, Lorez M, Benson JA, Fritschy JM, Luscher B (1998) Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat Neurosci 1:563-571[Web of Science][Medline].
• He TC, Jiang N, Zhuang H, Wojchowski DM (1995) Erythropoietin-induced recruitment of Shc via a receptor phosphotyrosine-independent, Jak2-associated pathway. J Biol Chem 270:11055-11061[Abstract/Free Full Text].
• Jones MV, Westbrook GL (1997) Shaping of IPSCs by endogenous calcineurin activity. J Neurosci 17:7626-7633[Abstract/Free Full Text].
• Kirkwood A, Bear MF (1994) Homosynaptic long-term depression in the visual cortex. J Neurosci 14:3404-3412[Abstract].
• Kittler JT, Delmas P, Jovanovic JN, Brown DA, Smart TG, Moss SJ (2000) Constitutive endocytosis of GABA_A receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J Neurosci 20:7972-7977[Abstract/Free Full Text].
• Lacaille JC, Schwartzkroin PA (1988) Stratum lacunosum-moleculare interneurons of hippocampal CA1 region. II. Intrasomatic and intradendritic recordings of local circuit synaptic interactions. J Neurosci 8:1411-1424[Abstract].
• Lin YF, Browning MD, Dudek EM, MacDonald RL (1994) Protein kinase C enhances recombinant bovine 112L GABAA receptor whole-cell currents expressed in L929 fibroblasts. Neuron 13:1421-1431[Web of Science][Medline].
• Lin YF, Angelotti TP, Dudek EM, Bowing MD, MacDonald RL (1996) Enhancement of recombinant 112L -aminobutyric acid A receptor whole-cell currents by protein kinase C is mediated through phosphorylation of both 1 and 2L subunits. Mol Pharmacol 50:185-195[Abstract].
• Lisman JE, Zhabotinsky AM (2001) A model of synaptic memory: a CaMKII/PP1 switch that potentiates transmission by organizing an AMPA receptor anchoring assembly. Neuron 31:191-201[Web of Science][Medline].
• Liu F, Wan Q, Pristupa Z, Wang YT, Niznik HB (2000) Direct protein-protein binding enables reciprocal dopamine D5 and GABA_A receptor cross-talk. Nature 403:274-280[Medline].
• Lu YM, Roder JC, Davidow J, Salter MW (1998) Src activation in the induction of long-term potentiation in CA1 hippocampal neurons. Science 279:1363-1367[Abstract/Free Full Text].
• Lu YM, Mansuy IM, Kandel ER, Roder J (2000) Calcineurin-mediated LTD of GABAergic inhibition underlies the increased excitability of CA1 neurons associated with LTP. Neuron 26:197-205[Web of Science][Medline].
• MacDonald RL, Olsen RW (1994) GABA_A receptor channels. Annu Rev Neurosci 17:569-602[Web of Science][Medline].
• Malenka RC, Nicoll RA (1999) Long-term potentiationa decade of progress? Science 285:1870-1874[Abstract/Free Full Text].
• Malinow R, Mainen ZF, Hayashi Y (2000) LTP mechanisms: from silence to four-lane traffic. Curr Opin Neurobiol 10:352-357[Web of Science][Medline].
• Malleret G, Haditsch U, Genoux D, Jones MW, Bliss TV, Vanhoose AM, Weitlauf C, Kandel ER, Winder DG, Mansuy IM (2001) Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin. Cell 104:675-686[Web of Science][Medline].
• Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39-49[Web of Science][Medline].
• McKernan RM, Whiting PJ (1996) Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19:139-143[Web of Science][Medline].
• Moss SJ, Doherty CA, Huganir RL (1992) Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor. J Biol Chem 267:14470-14476[Abstract/Free Full Text].
• Moss SJ, Gorrie GH, Amato A, Smart TG (1995) Modulation of GABA_A receptors by tyrosine phosphorylation. Nature 377:344-348[Medline].
• Mulkey RM, Endo S, Shenolikar S, Malenka RC (1994) Involvement of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression. Nature 369:486-488[Medline].
• Nusser Z, Hajos N, Somogyi P, Mody I (1998) Increased number of synaptic GABA_A receptors underlies potentiation at hippocampal inhibitory synapses. Nature 395:172-177[Medline].
• Nymann-Andersen J, Wang H, Chen L, Kittler JT, Moss SJ, Olsen RW (2002) Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors. J Neurochem 80:815-823[Web of Science][Medline].
• Otis TS, De Koninck Y, Mody I (1994) Lasting potentiation of inhibition is associated with an increased number of gamma-aminobutyric acid type A receptors activated during miniature inhibitory postsynaptic currents. Proc Natl Acad Sci USA 91:7698-7702[Abstract/Free Full Text].
• Paulsen O, Moser EI (1998) A model of hippocampal memory encoding and retrieval: GABAergic control of synaptic plasticity. Trends Neurosci 21:273-278[Web of Science][Medline].
• Perrino B, Ng LY, Soderling TR (1995) Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain. J Biol Chem 270:340-346[Abstract/Free Full Text].
• Pettit DL, Augustine GJ (2000) Distribution of functional glutamate and GABA receptors on hippocampal pyramidal cells and interneurons. J Neurophysiol 84:28-38[Abstract/Free Full Text].
• Poisbeau P, Cheney MC, Browning MD, Mody I (1999) Modulation of synaptic GABA_A receptor function by PKA and PKC in adult hippocampal neurons. J Neurosci 19:674-683[Abstract/Free Full Text].
• Silver RA, Momiyama A, Cull-Candy SG (1998) Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses. J Physiol (Lond) 510:881-902[Abstract/Free Full Text].
• Stelzer A, Shi H (1994) Impairment of GABAA receptor function by N-methyl-D-aspartate-mediated calcium influx in isolated CA1 pyramidal cells. Neuroscience 62:813-828[Web of Science][Medline].
• Stelzer A, Slater N, Bruggencate G (1987) Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy. Nature 326:698-701[Medline].
• Thiels E, Kanterewicz BI, Knapp LT, Barrionuevo G, Klann E (2000) Protein phosphatase-mediated regulation of protein kinase C during long-term depression in the adult hippocampus in vivo. J Neurosci 20:7199-7207[Abstract/Free Full Text].
• Thompson SM (1994) Modulation of inhibitory synaptic transmission in the hippocampus. Prog Neurobiol 42:575-609[Web of Science][Medline].
• Thompson SM, Gahwiler BH (1989) Activity-dependent disinhibition. I. Repetitive stimulation reduces IPSP driving force and conductance in the hippocampus in vitro. J Neurophysiol 61:501-511[Abstract/Free Full Text].
• Traynelis SG, Silver RA, Cull-Candy SG (1993) Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse. Neuron 11:279-289[Web of Science][Medline].
• Wang YT, Linden DJ (2000) Expression of cerebellar long-term depression requires postsynaptic calthrin-mediated endocytosis. Neuron 25:635-647[Web of Science][Medline].
• Winder DG, Sweatt JD (2001) Roles of serine/threonine phosphatases in hippocampal synaptic plasticity. Nat Rev 2:461-474.
• Winder DG, Mansuy IM, Osman M, Moallem TM, Kandel ER (1998) genetic and pharmacological evidence for a novel intermediate phase of long-term potentiation suppressed by calcineurin. Cell 92:25-37[Web of Science][Medline].
• Zeng H, Chattarji S, Barbarosie M, Rondi-Reig L, Philipot BD, Miyakawa T, Bear MF, Tonegawa S (2001) Forebrain-specific calcineurin knockout selectively impair bi-directional synaptic plasticity and working/episodic-like memory. Cell 107:617-629[Web of Science][Medline].

---

Copyright © 2003 Society for Neuroscience  0270-6474/03/233826-11$05.00/0

This article has been cited by other articles:

				S. J. Clapcote, S. Duffy, G. Xie, G. Kirshenbaum, A. R. Bechard, V. Rodacker Schack, J. Petersen, L. Sinai, B. J. Saab, J. P. Lerch, et al. Mutation I810N in the {alpha}3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS PNAS, August 18, 2009; 106(33): 14085 - 14090. [Abstract] [Full Text] [PDF]

				K. C. Marsden, J. B. Beattie, J. Friedenthal, and R. C. Carroll NMDA Receptor Activation Potentiates Inhibitory Transmission through GABA Receptor-Associated Protein-Dependent Exocytosis of GABAA Receptors J. Neurosci., December 26, 2007; 27(52): 14326 - 14337. [Abstract] [Full Text] [PDF]

				Z.-H. Qi, M. Song, M. J. Wallace, D. Wang, P. M. Newton, T. McMahon, W.-H. Chou, C. Zhang, K. M. Shokat, and R. O. Messing Protein Kinase C{epsilon} Regulates {gamma}-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of {gamma}2 Subunits J. Biol. Chem., November 9, 2007; 282(45): 33052 - 33063. [Abstract] [Full Text] [PDF]

				N. A. Addy, E. F. Fornasiero, T. R. Stevens, J. R. Taylor, and M. R. Picciotto Role of Calcineurin in Nicotine-Mediated Locomotor Sensitization J. Neurosci., August 8, 2007; 27(32): 8571 - 8580. [Abstract] [Full Text] [PDF]

				N. Wanaverbecq, A. Semyanov, I. Pavlov, M. C. Walker, and D. M. Kullmann Cholinergic Axons Modulate GABAergic Signaling among Hippocampal Interneurons via Postsynaptic {alpha}7 Nicotinic Receptors J. Neurosci., May 23, 2007; 27(21): 5683 - 5693. [Abstract] [Full Text] [PDF]

				J. O. McNamara, Y. Z. Huang, and A. S. Leonard Molecular Signaling Mechanisms Underlying Epileptogenesis Sci. Signal., October 10, 2006; 2006(356): re12 - re12. [Abstract] [Full Text] [PDF]

				H.-S. Sun, Z.-P. Feng, T. Miki, S. Seino, and R. J. French Enhanced Neuronal Damage After Ischemic Insults in Mice Lacking Kir6.2-Containing ATP-Sensitive K+ Channels J Neurophysiol, April 1, 2006; 95(4): 2590 - 2601. [Abstract] [Full Text] [PDF]

				D. E. Naylor, H. Liu, and C. G. Wasterlain Trafficking of GABAA Receptors, Loss of Inhibition, and a Mechanism for Pharmacoresistance in Status Epilepticus J. Neurosci., August 24, 2005; 25(34): 7724 - 7733. [Abstract] [Full Text] [PDF]

				M. J. Alldred, J. Mulder-Rosi, S. E. Lingenfelter, G. Chen, and B. Luscher Distinct {gamma}2 Subunit Domains Mediate Clustering and Synaptic Function of Postsynaptic GABAA Receptors and Gephyrin J. Neurosci., January 19, 2005; 25(3): 594 - 603. [Abstract] [Full Text] [PDF]

				I. Mody Aspects of the homeostaic plasticity of GABAA receptor-mediated inhibition J. Physiol., January 1, 2005; 562(1): 37 - 46. [Abstract] [Full Text] [PDF]

				E. I. Charych, W. Yu, R. Li, D. R. Serwanski, C. P. Miralles, X. Li, B. Y. Yang, N. Pinal, R. Walikonis, and A. L. De Blas A Four PDZ Domain-containing Splice Variant Form of GRIP1 Is Localized in GABAergic and Glutamatergic Synapses in the Brain J. Biol. Chem., September 10, 2004; 279(37): 38978 - 38990. [Abstract] [Full Text] [PDF]

				J. Meier and R. Grantyn Preferential accumulation of GABAA receptor {gamma}2L, not {gamma}2S, cytoplasmic loops at rat spinal cord inhibitory synapses J. Physiol., September 1, 2004; 559(2): 355 - 365. [Abstract] [Full Text] [PDF]

				C. Patenaude, C A. Chapman, S. Bertrand, P. Congar, and J.-C. Lacaille GABAB receptor- and metabotropic glutamate receptor-dependent cooperative long-term potentiation of rat hippocampal GABAA synaptic transmission J. Physiol., November 15, 2003; 553(1): 155 - 167. [Abstract] [Full Text] [PDF]

				L. Deng and G. Chen Cyclothiazide potently inhibits {gamma}-aminobutyric acid type A receptors in addition to enhancing glutamate responses PNAS, October 28, 2003; 100(22): 13025 - 13029. [Abstract] [Full Text] [PDF]

				Y. Li, L.-J. Wu, P. Legendre, and T.-L. Xu Asymmetric Cross-inhibition between GABAA and Glycine Receptors in Rat Spinal Dorsal Horn Neurons J. Biol. Chem., October 3, 2003; 278(40): 38637 - 38645. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (42) Citing Articles via Google Scholar Google Scholar Articles by Wang, J. Articles by Lu, Y. Search for Related Content PubMed PubMed Citation Articles by Wang, J. Articles by Lu, Y.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help