Altered Pharmacology of Synaptic and Extrasynaptic GABAA ...

0022-3565/04/3103-1234 –1245$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics JPET 310:1234–1245, 2004

Vol. 310, No. 3 67983/1164611 Printed in U.S.A.

Altered Pharmacology of Synaptic and Extrasynaptic GABAA Receptors on CA1 Hippocampal Neurons Is Consistent with Subunit Changes in a Model of Alcohol Withdrawal and Dependence Jing Liang, Elisabetta Cagetti, Richard W. Olsen, and Igor Spigelman Division of Oral Biology and Medicine, School of Dentistry (J.L., I.S.); and Department of Molecular and Medical Pharmacology, School of Medicine (E.C., R.W.O.), University of California, Los Angeles, Los Angeles, California Received March 5, 2004; accepted May 4, 2004

ABSTRACT Previously, we reported (Cagetti, Liang, Spigelman, and Olsen, 2003) that chronic intermittent ethanol (CIE) treatment leads to signs of alcohol dependence, including anxiety and hyperactivity, accompanied by reduced synaptic ␥-aminobutyric acid (A) receptor (GABAAR) function and altered sensitivity to its allosteric modulators consistent with a measured switch in subunit composition. In this study, we separated the synaptic and extrasynaptic components of GABAAR activation in recordings from pyramidal CA1 cells of hippocampal slices and demonstrated marked differences in the responsiveness of synaptic and extrasynaptic GABAARs to agonists and allosteric modulators in control rats, and in the way they are altered following CIE treatment. Notably, tonic inhibition mediated by extrasynaptic GABAARs was differentially sensitive to the partial agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; THIP) and the allosteric modulator zolpidem, compared with the miniature inhibitory synaptic currents (mIPSCs) in the same cells from saline-treated rats. After CIE treatment, potentiation of tonic cur-

The molecular mechanisms of ethanol tolerance and dependence appear to involve changes in GABAAR function (Buck and Harris, 1990; Morrow et al., 1990; Kang et al., 1996). GABAAR function, including responsiveness to many clinically important drugs, is highly dependent on the subunit composition of native receptors (Olsen et al., 1990; Seeburg et al., 1990; Whiting et al., 2000). Region-specific alterations in GABAAR subunit composition have been observed in several animal models of chronic ethanol consumption This work was supported by National Institutes of Health Grant AA07680. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.067983.

rents by diazepam and zolpidem was lost, whereas potentiation by the ␣4 subunit-preferring benzodiazepine Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) and THIP was only partially reduced. Potentiation of synaptic GABAAR currents by zolpidem was eliminated after CIE, whereas THIP slightly inhibited mIPSCs from control rats and greatly enhanced them after CIE treatment. These results are consistent with ␣1 subunit decreases at synaptic and extrasynaptic GABAARs, whereas ␣4 subunits are increased at synaptic and decreased at extrasynaptic GABAARs. Behaviorally, THIP was active as a hypnotic and anxiolytic but not as an anticonvulsant against pentylenetetrazol seizures in control rats. Only slight tolerance was observed to the sleep time, but not to the anxiolytic, effect of THIP after CIE. Thus, differential alterations in synaptic and extrasynaptic GABAARs appear to play an important role in the brain plasticity of alcohol dependence, and withdrawal signs may be profitably treated with GABAergic drugs such as THIP, which does not show cross-tolerance with ethanol.

(Mhatre et al., 1988, 1993; Buck et al., 1991; Devaud et al., 1995; Mahmoudi et al., 1997; Matthews et al., 1998). The chronic intermittent ethanol (CIE) treatment of rats, which includes multiple (ⱖ60) withdrawal episodes, has recently been validated as a model of human alcohol withdrawal and dependence (Cagetti et al., 2003). CIE treatment leads to a kindling-like state with a persistent decrease in pentylenetetrazol seizure threshold (Kokka et al., 1993), persistently reduced hippocampal GABAA receptor (GABAAR)mediated synaptic function measured 2 to 40 days after the ethanol treatment is ended (Kang et al., 1996, 1998), and decreased sedative/hypnotic responses to positive allosteric modulators of GABAARs such as flurazepam, alphaxalone,

ABBREVIATIONS: CIE, chronic intermittent ethanol; ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; CGP 54626, [S-(R*,R*)][3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl)phosphinic acid hydrochloride; GABAAR, ␥-aminobutyric acid (A) receptor; mIPSC, miniature inhibitory synaptic current; PKC, protein kinase C; PTZ, pentylenetetrazol; Ro15-4513, ethyl 8-azido-6-dihydro-5methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. 1234

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and, to a lesser extent, pentobarbital (Cagetti et al., 2003). Immunoblotting revealed a decrease in ␣1 and ␦ subunit expression and an increase in ␥2 and ␣4 subunits in hippocampus of CIE rats, confirmed by an increase in diazepaminsensitive binding for Ro15-4513, a marker for ␣4␥2 receptors. Analogous changes in ␣1 and ␣4 subunit peptide levels were previously reported in the hippocampus of rats after 40 days of continuous ethanol administration (Matthews et al., 1998). Recordings in hippocampal slices from CIE rats revealed that the decay time of GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) in CA1 pyramidal cells was decreased, and potentiation of mIPSCs by positive modulators of GABAARs was also reduced, compared with controls. However, mIPSC potentiation by the ␣4-preferring benzodiazepine ligands bretazenil and Ro15-4513 was maintained, and increased, respectively (Cagetti et al., 2003). Spillover of GABA released in the synaptic cleft and the presence of ambient GABA (Lerma et al., 1986; Tossman et al., 1986; Attwell et al., 1993) activate extrasynaptic GABAARs. Persistent activation of extrasynaptic GABAARs results in a tonic inhibitory influence on neurons. This small, but significant, GABAergic current has been observed in various brain regions (Otis et al., 1991; Brickley et al., 1996; Salin and Prince, 1996; Bai et al., 2001; Nusser and Mody, 2002). Immunocytochemical studies in these brain regions have provided evidence that the relative densities and subunit composition of extrasynaptic GABAARs are quite different from those of synaptic GABAARs (Soltesz et al., 1990; Nusser et al., 1995, 1998; Devor et al., 2001; Scotti and Reuter, 2001; Brunig et al., 2002). Functional studies determined that extrasynaptic GABAARs activate at lower GABA concentrations and desensitize more slowly than do the synaptic GABAARs (Brickley et al., 1999; Robello et al., 1999; Banks and Pearce, 2000; Hutcheon et al., 2000; Nusser and Mody, 2002). The differences in subunit composition also confer considerable pharmacological differences between the synaptic and extrasynaptic GABAARs (Robello et al., 1999; Hutcheon et al., 2000; Bai et al., 2001; Nusser and Mody, 2002). In the present study, we sought to determine the effect of CIE treatment on different pools of receptors, the extrasynaptic (tonic) and synaptic GABAAR-mediated inhibition in CA1 neurons, and to obtain additional evidence supporting the interpretation that there is a subunit composition switch in this model. Our electrophysiological data demonstrate a loss of the potentiation of tonic GABA current by positive allosteric modulators (diazepam and zolpidem). Furthermore, reduced tonic current potentiation by Ro15-4513 and the directly acting agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP; also known as gaboxadol) suggests reductions in extrasynaptic ␣1 and ␣4 subunit-containing GABAARs. Interestingly, THIP-induced decreases in mIPSC charge transfer in saline-treated rats are “switched” to potentiation in CIE-treated rats, consistent with the evident increases in ␣4 subunits in synaptic GABAARs. In addition, we demonstrate that the soporific effects of THIP are only modestly reduced and its anxiolytic effects are maintained in CIE rats. The novel enhancement by THIP of synaptic GABA receptors tempered by the reduced sensitivity of extrasynaptic receptors after CIE treatment suggests potential usefulness of THIP in ameliorating the symptoms of human alcohol

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withdrawal. Some of these results have been reported previously in abstract form (Liang et al., 2003a,b).

Materials and Methods Production of CIE Rats. The Institutional Animal Care and Use Committee approved all animal experiments. Male Sprague-Dawley rats (170 –190 g) were housed in the vivarium under a 12-h light/ dark cycle and had free access to food and water. Intoxicating doses of ethanol (Pharmco Products, Brookfield, CT) were administered by oral intubation on a chronic regimen: for the first five doses, rats received 5 g/kg of body weight as a 25% (w/v) solution in saline once every other day, and for the following 55 doses, 6 g/kg ethanol 30% (w/v) once every day. The control group received saline (20 ml/kg of body weight). This ethanol regimen led rats to experience multiple cycles of intoxication and withdrawal phases. The blood ethanol concentrations peaked (300 – 400 mg/dl) at 1 h after gavage and declined rapidly such that they were undetectable at 8 h after treatment (Kang et al., 1998). Although saline-treated rats initially tended to gain weight faster than ethanol-treated rats, the weights of the two groups after 60 doses were not significantly different. After the treatment and 2 days of withdrawal, rats were subjected to behavioral experiments. Alternatively, rats were withdrawn for 2 to 5 days and euthanized, and tissues were prepared for experiments. Since it was reported that certain effects of chronic alcohol administration revert to control levels within 24 h of alcohol withdrawal, whereas others persist for more than a week (Buck and Harris, 1990; Buck et al., 1991; Devaud et al., 1996), we compared the properties of GABAAR activation at 2 and at 5 days after the last CIE dose. There were no significant differences in the kinetics of mIPSCs or the tonic GABAAR-mediated currents at 2 days (n ⫽ 10 cells, 4 rats) or at 5 days (n ⫽ 5 cells, 2 rats) after alcohol withdrawal. Furthermore, the effects of THIP (0.3 and 3 ␮M) application on synaptic and extrasynaptic currents did not differ between the 2-day and 5-day withdrawal groups. Therefore, results combine the electrophysiological data from CIE rats at 2 to 5 days of withdrawal. Electrophysiology. Transverse slices (400 ␮m thick) of dorsal hippocampus were obtained using standard techniques (Spigelman et al., 1992; Kang et al., 1996). Whole-cell patch clamp recordings were obtained from cells located in the CA1 pyramidal layer at 34 ⫾ 0.5°C during perfusion with artificial cerebrospinal fluid (ACSF) composed of 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 26 mM NaHCO3, and 10 mM D-glucose. The ACSF was continuously bubbled with a 95/5% mixture of O2/CO2 to ensure adequate oxygenation of slices and a pH of 7.4. Patch pipettes contained 135 mM cesium gluconate, 2 mM MgCl2, 1 mM CaCl2, 11 mM ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid, 10 mM HEPES, 2 mM K2ATP, 0.2 mM Na2GTP; pH adjusted to 7.25 with CsOH. GABAAR-mediated mIPSCs were pharmacologically isolated by adding tetrodotoxin (0.5 ␮M), D-(⫺)-2-amino-5-phosphonopentanoate (40 ␮M), 6-cyano-7-nitroquinoxaline-2,3-dione (10 ␮M), and CGP 54626 (1 ␮M) to the ACSF from stock solutions. Stock solutions of 6-cyano-7-nitroquinoxaline-2,3-dione, bretazenil, diazepam, zolpidem, and Ro15-4513 were made with pure dimethyl sulfoxide. The final concentration of dimethyl sulfoxide did not exceed 42 ␮M in the recording chamber. Signals were recorded in voltage-clamp mode with an amplifier (Axoclamp 2B, Axon Instruments Inc., Union City, CA). Whole-cell access resistances were in the range of 2.5 to 15 M⍀ before electrical compensation by about 90%. During voltage-clamp recordings, access resistance was monitored by measuring the size of the capacitative transient in response to a 5-mV step command, and experiments were abandoned if changes ⬎20% were encountered. At least 10 min was allowed for equilibration of the pipette solution with the intracellular milieu before commencing mIPSC recordings. Data were acquired with pClamp 8 software (Axon Instruments Inc.), digitized at 20 kHz (Digidata 1200B; Axon Instruments Inc.), and analyzed using the Clampfit software (Axon Instruments Inc.)

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and the Mini Analysis Program (versions 5.2.2 and 5.4.8; Synaptosoft, Decatur, GA). Detection and Analysis of mIPSCs and Tonic Currents. The recordings were low-pass filtered off-line (Clampfit software) at 2 kHz. The mIPSCs were detected (Mini Analysis Program) with threshold criteria of 5 pA amplitude and 20 pA 䡠 ms area. Frequency of mIPSCs was determined from all automatically detected events in a given 100-s recording period. For kinetic analysis, only single-event mIPSCs were chosen during visual inspection of the recording trace. This included mIPSCs with a stable baseline, sharp rising phase, and exponential decay. Double- and multiple-peak mIPSCs were excluded. The mIPSC kinetics were obtained from analysis of the averaged chosen single events (⬎120 events/100-s recording period) aligned with half-rise time in each cell. Decay time constants were obtained by fitting a double exponential to the falling phase of the averaged mIPSC in each neuron. The tonic current magnitudes were obtained from the mean baseline current during the 100-s recording periods. The investigator performing the recordings and mIPSC analysis was blind to the treatment (saline or CIE) that the rats received. All comparisons of group differences in mIPSC kinetics and drug effects were made with ANOVA (Sigmastat; SPSS Inc., Chicago, IL). Sleep Time Assay. The hypnotic effect of THIP (30 mg/kg i.p.; H. Lundbeck A/S, Copenhagen, Denmark) was tested on CIE- and saline-treated rats (n ⫽ 11). THIP was dissolved in 0.9% saline. Injection volume was 2 ml/kg. The latency time (the time required after drug injection to lose the righting reflex) and sleep time were measured. Sleep time was determined as follows. After drug injection and loss of righting reflex, rats were placed on their backs in a V-shaped trough and a timer was started. The sleep time period ended when the animal was able to flip over three times in 30 s. Latency and sleep time are reported as mean (minutes) ⫾ S.E.M. Statistical significance was calculated by ANOVA. Pentylenetetrazol (PTZ) Seizure Threshold and Anticonvulsant Effect of THIP. After 2 days of treatment withdrawal, a group of CIE- and saline-treated rats were tested for the anticonvulsant effect of THIP against PTZ-induced seizures. First, the seizure threshold to PTZ was determined in saline-treated (n ⫽ 6) and CIE rats (n ⫽ 6). Rats were gently restrained and PTZ (25 mg/ml in 0.9% saline) was injected slowly, at a constant rate, into the tail vein via a 24 gauge shielded i.v. catheter (BD Medical, Franklin Lakes, NJ). The endpoint was taken as the time to the first myoclonic twitch of the head. Seizure thresholds were calculated from the volume injected for body weight and are presented as milligrams per kilogram of PTZ. Once the seizure threshold for each rat was established, 2 days later the rats were tested for the anticonvulsant effect of THIP

(they had no ethanol during the extra 2 days). Rats were injected with 8 mg/kg THIP (diluted in 0.9% saline, 2 ml/kg i.p.) 30 min before infusion of PTZ, and seizure threshold was measured again. Data were analyzed by ANOVA. Anxiolytic Effect of THIP on the Elevated Plus-Maze. Rats were tested for the anxiolytic effect of THIP (1.25 and 2.5 mg/kg) on the elevated plus-maze. Rats were brought to the procedure room 2 h before testing. The plus-maze was constructed as described previously (Pellow et al., 1985). Rats were randomly divided into the following groups: saline controls treated with vehicle (saline) or THIP, and CIE rats treated with vehicle or THIP. Rats were injected 1 h before testing (volume 2 ml/kg i.p.). Rats were placed on the central area of the maze, tested for 5 min, and videotaped in a room absent of any humans. The following measures were scored: number of entries into open arms, closed arms, or center platform, and time spent in each of these areas. An arm entry was defined as the entry of all four feet into one arm. Data are reported as mean ⫾ S.E.M. percentage of entries into the open arms and percentage of time spent in open arms. Statistical differences were determined using ANOVA.

Results CA1 Neurons Exhibit Synaptic (Phasic) and Extrasynaptic (Tonic) GABAAR-Mediated Inhibition. Initially, we sought to isolate the tonic component of inhibition in CA1 neurons from the synaptic component through the use of the GABAAR antagonist gabazine (McCabe et al., 1988; Bai et al., 2001). We also wanted to ascertain that glycine receptor activation does not contribute to the Cl⫺ currents attributed to GABAAR activation. Figure 1 illustrates that strychnine (1 ␮M) application had no effect on the kinetics of averaged GABAAR-mediated mIPSCs or the tonic current (Ih). However, subsequent application of gabazine (1 ␮M) selectively abolished the mIPSCs without affecting the tonic current. Loss of Diazepam and Decreased Ro15-4513 Effect on Tonic GABAAR-Mediated Current in CIE-Treated Rats. Application of diazepam enhanced the isolated tonic current (without any appreciable desensitization) in CA1 neurons from saline-treated rats, whereas subsequent addition of the GABAAR blocker (picrotoxin, 50 ␮M) blocked this response (Fig. 1; summarized in Fig. 3). By contrast, diazepam application in slices from CIE-treated rats had no effect

Fig. 1. Pharmacological separation of synaptic and extrasynaptic GABAAR-mediated currents in CA1 neurons from saline- and CIE-treated rats. In these and all subsequent experiments, membrane voltage was clamped at 0 mV, and the initial extracellular solution containing tetrodotoxin (TTX) and amino acid receptor blockers was applied for at least 10 min. Application of strychnine in the presence of glutamate and GABAB receptor blockade has no effect on the tonic holding current (Ih) or synaptic mIPSCs (top traces averaged over the indicated 100-s periods). Subsequent application of gabazine completely blocks mIPSCs without affecting the tonic current. Diazepam application enhances the tonic current, which is blocked by picrotoxin application. By contrast, diazepam has no effect on the tonic current in a CA1 neuron from a CIE-treated rat. Note the continued effectiveness of picrotoxin on Ih after CIE treatment. Dashed lines indicate the average Ih before strychnine application.


on the tonic current. Picrotoxin was still able to reduce Ih, indicating that the GABAAR-mediated tonic current was still present in CIE-treated CA1 neurons. These data suggested that the extrasynaptic GABAARs in CIE rats lose the diazepam-sensitive ␣1, -2, and -5 subunits and/or gain the diazepam-insensitive ␣4 subunits. To test for this possibility, we next compared the effects of Ro15-4513 on the tonic current in saline- and CIE-treated rats. Ro15-4513 does not possess agonist activity at ␣1- and ␣2-containing GABAARs, but is a high-affinity agonist at ␣4-containing GABAARs (Wisden et al., 1991; Knoflach et al., 1996; Whiting et al., 2000). Application of Ro15-4513 in slices of saline-treated rats potentiated Ih from its baseline level, an effect that was blocked by subsequent picrotoxin application (Fig. 2; summarized in Fig. 3). This suggested that ␣4-containing GABAARs contribute to the tonic current in rat CA1 neurons. In slices from CIE-treated rats, Ro15-4513 still potentiated Ih, but this potentiation was smaller than that in slices from saline-treated rats. Quantification of Ih changes after application of various GABAAR modulators confirmed these findings (Fig. 3). Loss of Zolpidem Potentiation of Synaptic and Extrasynaptic GABAARs after CIE Treatment. The benzodiazepine, zolpidem, is an allosteric positive modulator of GABAAR function with selectivity for the ␣1-containing GABAARs, with intermediate potency on ␣2/3 receptors, and no effect on ␣5-containing GABAARs (Whiting et al., 2000). Application of zolpidem potentiated both the mIPSCs and the tonic current in a concentration-dependent manner in CA1 neurons from saline-treated rats (Figs. 4 and 5). By contrast, zolpidem (0.1 ␮M) application in slices from CIE-treated rats had no effect either on mIPSCs or the tonic current, whereas 0.3 ␮M zolpidem had a very small potentiating effect on the synaptic and extrasynaptic GABAAR-mediated currents. Examination of the mIPSC kinetics revealed that zolpidem increased the mIPSC area (total charge transfer) by prolonging the decay time constants without affecting mIPSC amplitude (Fig. 6). The mIPSC rise time was also increased by zolpidem, but only in saline-treated rats (Fig. 6). These data pointed to the loss of ␣1-containing extrasynaptic GABAARs, further extending our previous findings of an apparent decrease in synaptic ␣1-containing GABAARs (Cagetti et al., 2003), and also suggested that the zolpidem-insensitive ␣5-containing GABAARs do not solely contribute to the tonic current in CA1 neurons, since some zolpidem-enhanced receptors are present.

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Partial Agonist, THIP, Potently Activates Tonic GABAAR Currents in CA1 Neurons from Untreated Rats. To extend our observations on GABAAR subunit changes, we decided to use a directly acting agonist at GABAARs, THIP. THIP has been demonstrated to act as a high-efficacy and reasonably high-affinity agonist at recombinant ␣4␤3␦-containing GABAARs (Adkins et al., 2001; Brown et al., 2002) and is, therefore, a partial agonist at certain other GABAARs. Unlike GABA, THIP is not subject to rapid uptake and, thus, its application in brain slices would be somewhat analogous to increasing ambient [GABA] in the slice. Since the concentration-dependent actions of THIP on synaptic and extrasynaptic GABAAR-mediated currents in CA1 neurons have not been previously characterized in detail, we first studied the effects of THIP application in slices from control (untreated) rats at 34.5°C. Bath application of THIP produced a concentration-dependent increase in Ih, without appreciable response desensitization at THIP concentrations up to 10 ␮M (Figs. 7, A and B, and Fig. 8). Some desensitization of the response was observed at THIP concentrations of 30 and 100 ␮M. The EC50 for THIP was at approximately 1 ␮M (Fig. 7B). THIP Inhibits mIPSCs in CA1 Neurons from Untreated Rats. Synaptic GABAARs in CA1 neurons were recently demonstrated to be biophysically and pharmacologically distinct from extrasynaptic receptors (Banks and Pearce, 2000; Bai et al., 2001; Yeung et al., 2003). Thus, synaptic GABAARs possess higher unitary conductance and lower affinity for GABA than do their extrasynaptic counterparts (Yeung et al., 2003). They also desensitize more rapidly than do extrasynaptic GABAARs and are selectively blocked by gabazine and penicillin (Banks and Pearce, 2000; Yeung et al., 2003). In the continued presence of a partial agonist such as THIP, these receptors would be expected to exhibit decreased responses to the full agonist (GABA) released in the synaptic cleft (Kurata et al., 1999; Bianchi and Macdonald, 2003). For example, one way in which a partial agonist can reduce GABA currents is to accelerate the desensitization of GABA-induced currents (Nakahiro et al., 1991; Kurata et al., 1999). Indeed, examination of averaged mIPSC current kinetics revealed a concentration-dependent decrease in mIPSC area by THIP (Fig. 7C). This decrease was primarily due to the speeding up of the mIPSC decay, since the rise time and amplitude of mIPSCs were little affected by THIP at concentrations ⱕ10 ␮M. In addition, there was a substantial concentration-dependent decrease in mIPSC frequency

Fig. 2. Reduced effect of Ro15-4513 on the tonic current (Ih) in a CA1 neuron from a CIE-treated rat. Dashed lines indicate the average Ih before strychnine application. Note that picrotoxin application reduces Ih to a level below that seen before or after gabazine application. TTX, tetrodotoxin.

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Fig. 3. CIE treatment-induced changes in allosteric modulation of tonic GABAAR-mediated current. A, summary graphs of diazepam-induced changes in GABAAR-mediated tonic current. The average Ih values before strychnine application were 50 ⫾ 3.0 pA (saline, n ⫽ 6 cells, 3 rats) and 50 ⫾ 2.7 pA (CIE, n ⫽ 7 cells, 4 rats), respectively. Note the complete loss of diazepam effect on the tonic current in neurons from CIE-treated rats. B, summary graphs of Ro15-4513-induced changes in GABAAR-mediated tonic current. The mean Ih values before strychnine application were 47 ⫾ 3.5 pA (saline, n ⫽ 5 cells, 3 rats) and 49 ⫾ 3.4 pA (CIE, n ⫽ 6 cells, 4 rats), respectively. The enhancement of Ih by Ro15-4513 is significantly reduced in neurons from CIE-treated compared with saline-treated rats. The picrotoxin-induced decreases in Ih tend to be lower in CIE- than in saline-treated rats, but the difference does not reach statistical significance (p ⬎ 0.05).

Fig. 4. Decreased potentiation of synaptic and extrasynaptic GABAAR-mediated currents by zolpidem in CA1 neurons from CIE-treated rats. Numbered top traces represent mIPSCs averaged over the indicated 100-s periods during continuous recordings (lower traces). Note the increased mIPSC area after zolpidem application in saline-treated rats and an almost complete loss of this effect after similar application in CA1 neurons from CIE-treated rats. Also note the decreased effect of zolpidem on Ih after CIEtreatment.


Fig. 5. Summary graph of zolpidem effects on tonic GABAAR-mediated current (Ih) in CA1 neurons from saline- and CIE-treated rats. Asterisks denote significant difference between saline and CIE groups (two-way repeated measures ANOVA). Daggers represent significant difference from control (predrug) condition. Note the large decrease in the zolpidem effect in neurons from CIE rats.

by THIP, which suggested THIP-mediated decreases in presynaptic GABA release. This decrease in frequency was not likely to be due to failure to detect mIPSCs of reduced am-

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plitude since 1) THIP concentrations ⱕ10 ␮M had little affect on the amplitude of averaged mIPSCs, and 2) the current noise increases after THIP application were small. The root mean square of current noise increased from 4.5 ⫾ 0.14 pA in the absence of THIP to a maximum of 6.3 ⫾ 0.33 pA at 1 ␮M THIP, other concentrations producing smaller root mean square increases in current noise. At THIP concentrations of 30 and 100 ␮M, the average mIPSC amplitude also decreased (not shown), but the frequency of single analyzable events was too low to permit robust analysis of mIPSC kinetics. Decreased Tonic Current Activation by THIP in CIETreated Rats. The concentration-response for THIP was very similar between slices from untreated and salinetreated rats (compare Fig. 7B and Fig. 9). However, application of THIP in slices from CIE rats was less effective in evoking the tonic GABAAR-mediated current than in slices from saline-treated rats (Figs. 8 and 9). These data suggested an overall decrease in extrasynaptic GABAARs after CIE treatment. Also, given the high efficacy and reasonably high affinity of THIP at ␣4␤3␦-containing GABAARs (Adkins et al., 2001; Brown et al., 2002), such GABAARs may be preferentially decreased at extrasynaptic locations after CIE treatment. THIP Potentiates GABAAR-Mediated Synaptic Inhibition in CIE-Treated Rats. The concentration-dependent effects of THIP on mIPSC kinetics in saline-treated rats were almost indistinguishable from the effects in untreated (con-

Fig. 6. Summary graphs of zolpidem (0.1 and 0.3 ␮M) effects on kinetics of averaged mIPSCs in CA1 neurons after saline (n ⫽ 5 cells, 2 rats) and CIE treatment (n ⫽ 6 cells, 2 rats). Asterisks denote significant difference between saline and CIE groups (two-way repeated measures ANOVA). Daggers represent significant difference from control (predrug) condition. Note the large decrease in the zolpidem effect in neurons from CIE rats. Note also that the zolpidem-induced increases in mIPSC area are primarily due to prolongation of the decay time constants (␶1 and ␶2).

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Fig. 7. Opposite THIP actions on synaptic and extrasynaptic GABAAR-mediated currents in CA1 neurons from control (untreated rats). A, bath application of THIP activates the tonic current without any appreciable desensitization of the response during prolonged application. B, concentrationresponse relationship of THIP and Ih. Each point represents the mean ⫾ S.E.M. of responses to THIP in 4 to 14 CA1 neurons. A sigmoid concentration-response function, y ⫽ min ⫹ (max ⫺ min)/(1 ⫹ 10(log EC50 ⫺ log[x])) was fitted to the data points by nonlinear regression. The peak response appears to plateau between 10 and 30 ␮M and gives a half-maximal response at 1 ␮M THIP. C, summary graphs of THIP (0.1–10 ␮M) effects on kinetics of averaged mIPSCs. Note that unlike THIP effects on the tonic current, mIPSCs are inhibited by THIP in a concentration-dependent manner. The greatest effect of THIP on total charge transfer (area) of averaged mIPSCs is through speeding up their decay. Each point represents the mean ⫾ S.E.M. of responses to THIP in 4 –13 CA1 neurons from 10 untreated rats.

trol) rats (compare Figs. 7C and 10). However, comparison of THIP effects on mIPSC kinetics between saline- and CIEtreated rats revealed a dramatic shift from a depressant action on mIPSCs in slices from saline-treated rats to a potentiating effect in CIE rats (Fig. 10). The predominant effect was a prolongation of both decay time constants, resulting in increased area of mIPSCs. Interestingly, the greatest potentiation was observed at the lowest concentration of THIP tested. Nevertheless, even at the highest concentration (10 ␮M), there was a significant increase in the mIPSC area. This behavior is consistent with a partial agonist model for THIP in the presence of the agonist GABA, with differential effects in CIE- versus saline-treated rats due to differential efficacy and kinetics of GABAARs with an altered subunit composition. There was also a concentration-dependent increase in mIPSC rise time in CIE- but not in saline-treated rats. The amplitude and frequency of mIPSCs were affected similarly by THIP in both saline- and CIE-treated rats. Reduced Hypnotic and Maintained Anxiolytic Effects of THIP in CIE-Treated Rats. The dramatic shift from a depressant to a potentiating effect of THIP on mIPSCs

coupled with a moderate reduction in its effect on the tonic current in CIE-treated rats was in marked contrast to the almost complete loss of potentiation by benzodiazepines such as diazepam and zolpidem of both synaptic and extrasynaptic GABAARs. Previously, we showed that the hypnotic effects of the benzodiazepine flurazepam were greatly attenuated after CIE treatment (Cagetti et al., 2003). Based on the current in vitro studies, we suspected that the in vivo actions of THIP may be affected differently from those of benzodiazepines after CIE treatment. To this end, we compared THIP for its anticonvulsant, soporific, and anxiolytic effects in saline- and CIE-treated rats. CIE rats showed partial tolerance to the hypnotic effect of THIP (Table 1). The mean sleep time of 92 ⫾ 8 min for saline-treated rats was significantly reduced to 59 ⫾ 6 min for CIE rats at 30 mg/kg (ⴱ, p ⫽ 0.003). The latency time of 20 ⫾ 2 min was significantly longer for CIE rats compared with 13 ⫾ 1 min for controls (ⴱ, p ⫽ 0.003). The anticonvulsant effect of THIP (8 mg/kg) against PTZ seizure threshold was measured in CIE and control rats. As demonstrated previously (Kokka et al., 1993), the PTZ seizure threshold was significantly reduced in CIE rats com-


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Fig. 8. Changes in THIP effects on GABAARmediated currents in CA1 neurons after CIE treatment. Tonic current activation by THIP is reduced in a CA1 neuron after CIE treatment compared with a saline-treated control. However, inhibition of mIPSCs by THIP in slices from saline-treated rats is changed to potentiation of mIPSCs in slices from CIE-treated rats. Numbered top traces represent mIPSCs averaged over the indicated 100-s periods during continuous recordings (lower traces). Note the decreased mIPSC area after THIP application in a slice from a saline-treated rat and increased mIPSC area after similar application in a CA1 neuron from a CIE-treated rat.

saline controls (p ⫽ 0.02) and CIE rats (p ⫽ 0.04) and time spent in them (p ⬍ 0.05) (Fig. 12, A and B).

Discussion

Fig. 9. Summary graph of THIP effects on tonic GABAAR-mediated current (Ih) in CA1 neurons after saline (six rats) and CIE treatment (six rats). Each point is a mean ⫾ S.E.M. of responses to THIP in five or six CA1 neurons.

pared with saline controls (Fig. 11): the seizure threshold was 28 ⫾ 2 mg/kg PTZ in CIE (n ⫽ 6) and 48 ⫾ 6 mg/kg PTZ in saline groups, respectively (p ⫽ 0.01). At the concentration tested, THIP failed to exhibit anticonvulsant activity in either group. CIE- and saline-treated rats were tested on the elevated plus-maze with two doses of THIP (1.25 and 2.5 mg/kg). At both doses, THIP had no effect on the total number of entries in either saline or CIE rats. Total entries for saline rats treated with vehicle, 1.25 and 2.5 mg/kg THIP, were (mean ⫾ S.E.M.): 13 ⫾ 1, 10.5 ⫾ 0.9, and 10.2 ⫾ 0.9. For CIE rats, they were: 8.1 ⫾ 0.7, 8.7 ⫾ 0.9, and 8 ⫾ 1.0. Total entries were significantly lower in CIE- compared with saline-treated rats (two-way ANOVA; p ⬍ 0.05). The lower dose of THIP did not have an anxiolytic effect in either group (Fig. 12, A and B). However, when tested at the concentration of 2.5 mg/kg, THIP had an anxiolytic effect in both groups. THIP increased significantly the number of entries into the open arms by

The present study provides an electrophysiological and pharmacological analysis of synaptic and extrasynaptic GABAARs in a rat model of alcohol intoxication and withdrawal. Our data specifically point to subunit-selective alterations in CA1 hippocampal neuron GABAARs after CIE treatment. In addition, our data suggest that in contrast to classical benzodiazepines, certain GABAergic drugs that are active at ␣4-containing GABAARs may be more efficacious as sedative-hypnotic and anxiolytic agents in chronic alcoholism, as a result of up-regulation of such GABAAR isoforms in this condition. The differences in subunit composition confer considerable pharmacological differences between the synaptic and extrasynaptic GABAARs (Robello et al., 1999; Hutcheon et al., 2000; Bai et al., 2001; Nusser and Mody, 2002). Although precise physiological roles have not been identified for the tonic inhibitory currents mediated by extrasynaptic GABAARs, they are clearly subject to modulation by clinically important drugs. Our data confirm the presence of a tonic GABAAR-mediated current in rat CA1 neurons (Bai et al., 2001; Yeung et al., 2003) and demonstrate that the potentiation of this current by several allosteric positive modulators is either reduced or eliminated after CIE treatment. Specifically, diazepam (0.3 ␮M) potentiation of the isolated tonic current is eliminated (Figs. 1 and 3), similar to the elimination of mIPSC potentiation we reported previously in CIE rats (Cagetti et al., 2003). Here we showed that zolpidem (0.1 and 0.3 ␮M) effects are similarly reduced, consistent with a reduction in ␣1-containing and/or increases in ␣4containing GABAARs at both synaptic and extrasynaptic locations. However, whereas the potentiation of mIPSCs by Ro15-4513 is increased (Cagetti et al., 2003), the potentiation of the isolated tonic current by Ro15-4513 is reduced in CIE rats (Figs. 2 and 3). Since Ro15-4513 does not possess agonist activity at ␣1-containing GABAARs, but is a high-affinity

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Fig. 10. Summary graphs of THIP (0.1–10 ␮M) effects on kinetics of averaged mIPSCs in CA1 neurons after saline (six rats) and CIE treatment (six rats). Asterisks denote significant difference between saline and CIE groups (two-way repeated measures ANOVA). Daggers represent significant difference from control (predrug) condition. Note that the THIP-induced decrease in area of averaged mIPSCs in saline-treated rats is replaced by a potentiating effect in CIE rats. Note also that the THIP-induced changes in mIPSC area are primarily due to changes in the decay time constants (␶1 and ␶2). TABLE 1 Sleep time assay for THIP (30 mg/kg i.p).

Sleep time (min) Latency (min)

Saline (n ⫽ 11)

CIE (n ⫽ 12)

92 ⫾ 8 13 ⫾ 2

59 ⫾ 6* 20 ⫾ 1*

* The reduction in mean sleep time and the increase in latency for CIE rats compared with controls were significant (p ⫽ 0.003 and p ⫽ 0.003, respectively).

agonist at ␣4-containing GABAARs (Wisden et al., 1991; Knoflach et al., 1996; Whiting et al., 2000), these data collectively suggest that a reduction in ␣1 subunits is compensated by the introduction of ␣4 subunits into synaptic GABAARs after CIE treatment. However, decreases in the ␣1 subunits at extrasynaptic GABAARs do not appear to be fully compensated by increases in ␣4 subunits because of the reduced effectiveness of Ro15-4513 (␣4 subunit-selective) in potentiating the tonic current in CIE rats. Further evidence for decreases in extrasynaptic GABAARs was obtained by demonstrating that tonic current potentiation by THIP, a partial agonist with reported high efficacy at certain ␣4-containing GABAARs, was also reduced in CIE rats (Figs. 8 and 9). The large potentiation of synaptic currents by THIP in CIE rats is further evidence for the insertion of ␣4 subunits into synaptic GABAARs. Previously, increased expression of ␣4␤3␦ subunits in a rat model of progesterone withdrawal was shown to double the affinity and increase the efficacy of CA1 neuron GABAARs for THIP (Sundstrom-Poromaa et al., 2002). However, this subunit combination is unlikely to be inserted into

Fig. 11. Anticonvulsant effect of THIP against PTZ-induced seizures in saline (n ⫽ 6) and CIE rats (n ⫽ 6). Rats were tested first for the PTZ (25 mg/ml i.v.) seizure threshold. CIE rats exhibited a reduced PTZ seizure threshold compared with saline controls (white bars; 多, p ⫽ 0.01). The same rats were tested again after 2 days for the anticonvulsant effect of THIP. Pretreatment with THIP (8 mg/kg i.p.) failed to protect either group against PTZ-induced seizures.

CA1 synapses in CIE rats because 1) ␦ subunit has very low expression levels in the hippocampal CA1 region (Persohn et al., 1992), and 2) ␦ subunit protein levels are decreased by ⬃50% in the hippocampus of CIE rats (Cagetti et al., 2003). Also, it should be noted that THIP was demonstrated to activate channels in rat CA1 neurons that are modulated by


Fig. 12. Anxiolytic effect of THIP (1.25 and 2.5 mg/kg) in saline and CIE rats in the elevated plus-maze. Rats were divided into six groups: vehicle–treated saline controls (n ⫽ 11) and vehicle-treated CIE rats (n ⫽ 10), THIP (1.25 mg/kg)-treated saline controls (n ⫽ 11) and CIE rats (n ⫽ 10), THIP (2.5 mg/kg)-treated saline controls (n ⫽ 5) and CIE rats (n ⫽ 5). Data are reported as mean ⫾ S.E.M value for percentage of open arms entries (A) and percentage of time spent in open arms (B). THIP (1.25 mg/kg) failed to induce anxiolysis in both saline controls and CIE rats compared with their respective vehicle-treated group. At a higher dose, THIP (2.5 mg/kg) significantly increased in both groups the number of entries into the open arms and time spent in them compared with their respective vehicle-treated groups; 多, p ⬍ 0.05.

diazepam (Lindquist et al., 2003), and therefore, its actions are not limited to ␣4-containing GABAARs. Similarly, it should be noted that the allosteric actions of Ro15-4513 may not be entirely selective for ␣4-containing GABAARs, and therefore, the decreased potentiation of the tonic current after CIE treatment by Ro15-4513 could also be attributed to decreases in other extrasynaptic GABAAR subunit combinations. In saline-treated rats, THIP exhibits both anxiolytic and sedative-hypnotic properties (Fig. 12; Table 1). In hippocampal slices from these rats, THIP application activates the extrasynaptic GABAAR-mediated tonic current but occludes mIPSCs generated by GABA release in the synaptic cleft (Figs. 7 and 8). This suggests that potentiation of extrasynaptic GABAARs alone may be sufficient to reduce anxiety or, at higher concentrations of this agonist, to induce sleep. We estimated the half-maximal concentration for THIP activation of the tonic current in control rats to be approximately 1 ␮M (Fig. 7B), which is substantially lower than the 80 ␮M value reported previously (Sundstrom-Poromaa et al., 2002). Interestingly, the plasma concentration of THIP that improves sleep quality in humans was also estimated to be about 1 ␮M (Schultz et al., 1981), and brain concentrations are unlikely to be much higher (Madsen et al., 1983; Faulhaber et al., 1997). THIP was demonstrated to promote deep, slow-wave sleep without affecting rapid eye movement sleep in both rodents and humans (Faulhaber et al., 1997; Lancel and Langebartels, 2000). Recent studies in normal

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human volunteers demonstrated a lack of tolerance to the soporific effects of THIP during long-term (3-week) daily treatment, this in contrast to zolpidem, to which patients exhibited marked tolerance (Vogel et al., 2003). Therefore, THIP holds promise as a sedative-hypnotic that lacks the major side effects of classical benzodiazepine drugs. In CIE rats that exhibit large decreases in the hypnotic response to the benzodiazepine flurazepam and the steroid anesthetic alphaxalone (Cagetti et al., 2003), the anxiolytic effects of THIP are maintained (Fig. 12), and its sedative effects are only modestly reduced (Table 1), consistent with the decreased THIP actions at extrasynaptic receptors and a “switch” to potentiation of synaptic currents. By analogy with the rat CIE model, we expect that THIP would remain an active soporific agent in treating the insomnia symptoms of human alcohol withdrawal syndrome. However, it should be underscored that these conclusions are based on studies of only one cell type in the central nervous system and, therefore, must be treated as tentative. For example, as is the case with THIP, the anxiolytic actions of diazepam (Cagetti et al., 2003) and alphaxalone (Cagetti et al., 2004) are maintained in CIE rats. Recent point mutation studies in mice have suggested that the anxiolytic properties of benzodiazepines such as diazepam are mediated through potentiation of ␣2-containing GABAARs (reviewed in Mohler et al., 2002). In hippocampal pyramidal cells, ␣2-containing GABAARs are concentrated in synapses at the axon initial segment (Nusser et al., 1996; Fritschy et al., 1998), and ␣2 mRNA does not appear to be reduced in CIE rats (Cagetti et al., 2003), whereas the potentiating effects of diazepam on both synaptic and extrasynaptic currents in CA1 neurons from CIE rats are lost. Thus, anxiolytic properties of diazepam in CIE rats are likely due to effects at GABAARs located elsewhere in the central nervous system. Chronic ethanol treatment leads to increased GABAAR internalization and changes in ␣1/␣4 ratios, concurrently with changes in the way the protein kinase C (PKC) ␥ isoform couples to these subunits (Kumar et al., 2002, 2003). Protein phosphorylation plays an important role in regulating GABAAR, including both GABAAR channel activity and trafficking (Barnes, 1996, 2001; Kittler et al., 2002; Kittler and Moss, 2003). PKC also has important effects on allosteric modulation of GABAAR by ethanol and neurosteroids (Weiner et al., 1997; Brussaard and Koksma, 2003; Harney et al., 2003; Koksma et al., 2003). For example, PKC activation in young rat dentate granule cells makes them sensitive to a previously ineffective concentration of an endogenous neurosteroid (Harney et al., 2003). Also, mutant mice lacking different PKC isoforms exhibit altered sensitivity to ethanol in vivo and altered GABAAR responsiveness in vitro, such that PKC␥ null mice have reduced ethanol modulation (Harris et al., 1995; Proctor et al., 2003) and PKC⑀ null mice have increased ethanol modulation (Hodge et al., 1999; Proctor et al., 2003). To date, the possibility that changes in the GABAAR phosphorylation state and trafficking may account for the altered responsiveness of GABAAR to various allosteric modulators after CIE treatment has not been examined, and therefore, these changes represent alternative explanations to the subunit change hypothesis. The current study extends our knowledge of GABAAR pharmacology in the hippocampus of CIE rats and is consistent with plastic changes in subunit composition of

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GABAARs. In particular, the changes occur in both synaptically and extrasynaptically localized receptors which give rise to phasic and tonic inhibition in CA1 neurons, respectively. Differential sensitivity to benzodiazepine modulators and the partial agonist THIP suggests different subunit compositions before and after CIE treatment, possibly including decreases in ␣4 subunit-containing receptors at extrasynaptic locations and increases at synaptic locations, where they are coupled to ␥2 subunits. These changes in CA1 GABAARs might explain some but not all of the behavioral plasticity observed in the CIE model of alcohol withdrawal and dependence. These changes are complex, and pathway-, cell-, and subcellular location-dependent. Acknowledgments

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Address correspondence to: Dr. Igor Spigelman, UCLA School of Dentistry, 10833 Le Conte Avenue, 63-078 (CHS), Los Angeles, CA 90095-1668. E-mail: [email protected]