Altered Pharmacology of Synaptic and Extrasynaptic GABAA Receptors

Home , Convulsant, Pentylenetetrazol

0022-3565/04/3103-1234–1245$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 310, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 67983/1164611 JPET 310:1234–1245, 2004 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, rents by diazepam and zolpidem was lost, whereas potentiation 2003) that chronic intermittent ethanol (CIE) treatment leads to by the ␣4 subunit-preferring benzodiazepine Ro15-4513 (ethyl signs of alcohol dependence, including anxiety and hyperac- 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzo- tivity, accompanied by reduced synaptic ␥-aminobutyric acid diazepine-3-carboxylate) and THIP was only partially reduced.

(A) receptor (GABAAR) function and altered sensitivity to its Potentiation of synaptic GABAAR currents by zolpidem was elim- allosteric modulators consistent with a measured switch in inated after CIE, whereas THIP slightly inhibited mIPSCs from subunit composition. In this study, we separated the synaptic control rats and greatly enhanced them after CIE treatment. These ␣ and extrasynaptic components of GABAAR activation in record- results are consistent with 1 subunit decreases at synaptic and ␣ ings from pyramidal CA1 cells of hippocampal slices and dem- extrasynaptic GABAARs, whereas 4 subunits are increased at onstrated marked differences in the responsiveness of synaptic synaptic and decreased at extrasynaptic GABAARs. Behaviorally, and extrasynaptic GABAARs to agonists and allosteric modu- THIP was active as a hypnotic and anxiolytic but not as an anti- lators in control rats, and in the way they are altered following convulsant against pentylenetetrazol seizures in control rats. Only CIE treatment. Notably, tonic inhibition mediated by extrasyn- slight tolerance was observed to the sleep time, but not to the aptic GABAARs was differentially sensitive to the partial agonist anxiolytic, effect of THIP after CIE. Thus, differential alterations in gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; THIP) synaptic and extrasynaptic GABAARs appear to play an important and the allosteric modulator zolpidem, compared with the minia- role in the brain plasticity of alcohol dependence, and withdrawal ture inhibitory synaptic currents (mIPSCs) in the same cells from signs may be profitably treated with GABAergic drugs such as saline-treated rats. After CIE treatment, potentiation of tonic cur- THIP, which does not show cross-tolerance with ethanol.

The molecular mechanisms of ethanol tolerance and de- (Mhatre et al., 1988, 1993; Buck et al., 1991; Devaud et al., pendence appear to involve changes in GABAAR function 1995; Mahmoudi et al., 1997; Matthews et al., 1998). (Buck and Harris, 1990; Morrow et al., 1990; Kang et al., The chronic intermittent ethanol (CIE) treatment of rats,

1996). GABAAR function, including responsiveness to many which includes multiple (Ն60) withdrawal episodes, has re- clinically important drugs, is highly dependent on the sub- cently been validated as a model of human alcohol with- unit composition of native receptors (Olsen et al., 1990; See- drawal and dependence (Cagetti et al., 2003). CIE treatment burg et al., 1990; Whiting et al., 2000). Region-specific alter- leads to a kindling-like state with a persistent decrease in ations in GABAAR subunit composition have been observed pentylenetetrazol seizure threshold (Kokka et al., 1993), per- in several animal models of chronic ethanol consumption sistently reduced hippocampal GABAA receptor (GABAAR)- mediated synaptic function measured 2 to 40 days after the This work was supported by National Institutes of Health Grant AA07680. ethanol treatment is ended (Kang et al., 1996, 1998), and Article, publication date, and citation information can be found at decreased sedative/hypnotic responses to positive allosteric http://jpet.aspetjournals.org. doi:10.1124/jpet.104.067983. 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-5- methyl-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 GABAAR Pharmacology in Alcoholism 1235

and, to a lesser extent, pentobarbital (Cagetti et al., 2003). withdrawal. Some of these results have been reported previ- Immunoblotting revealed a decrease in ␣1 and ␦ subunit ously in abstract form (Liang et al., 2003a,b). expression and an increase in ␥2 and ␣4 subunits in hippocampus of CIE rats, confirmed by an increase in diazepam- insensitive binding for Ro15-4513, a marker for ␣4␥2 recep- Materials and Methods tors. Analogous changes in ␣1 and ␣4 subunit peptide levels Production of CIE Rats. The Institutional Animal Care and Use were previously reported in the hippocampus of rats after 40 Committee approved all animal experiments. Male Sprague-Dawley days of continuous ethanol administration (Matthews et al., rats (170–190 g) were housed in the vivarium under a 12-h light/ 1998). Recordings in hippocampal slices from CIE rats re- dark cycle and had free access to food and water. Intoxicating doses

vealed that the decay time of GABAAR-mediated miniature of ethanol (Pharmco Products, Brookfield, CT) were administered by inhibitory postsynaptic currents (mIPSCs) in CA1 pyramidal oral intubation on a chronic regimen: for the first five doses, rats cells was decreased, and potentiation of mIPSCs by positive 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% modulators of GABAARs was also reduced, compared with controls. However, mIPSC potentiation by the ␣4-preferring (w/v) once every day. The control group received saline (20 ml/kg of body weight). This ethanol regimen led rats to experience multiple benzodiazepine ligands bretazenil and Ro15-4513 was main- cycles of intoxication and withdrawal phases. The blood ethanol tained, and increased, respectively (Cagetti et al., 2003). concentrations peaked (300–400 mg/dl) at 1 h after gavage and Spillover of GABA released in the synaptic cleft and the declined rapidly such that they were undetectable at 8 h after treat- presence of ambient GABA (Lerma et al., 1986; Tossman et ment (Kang et al., 1998). Although saline-treated rats initially al., 1986; Attwell et al., 1993) activate extrasynaptic tended to gain weight faster than ethanol-treated rats, the weights of the two groups after 60 doses were not significantly different. After GABAARs. Persistent activation of extrasynaptic GABAARs results in a tonic inhibitory influence on neurons. This small, the treatment and 2 days of withdrawal, rats were subjected to but significant, GABAergic current has been observed in behavioral experiments. Alternatively, rats were withdrawn for 2 to 5 days and euthanized, and tissues were prepared for experiments. various brain regions (Otis et al., 1991; Brickley et al., 1996; Since it was reported that certain effects of chronic alcohol admin- Salin and Prince, 1996; Bai et al., 2001; Nusser and Mody, istration revert to control levels within 24 h of alcohol withdrawal, 2002). Immunocytochemical studies in these brain regions whereas others persist for more than a week (Buck and Harris, 1990; have provided evidence that the relative densities and sub- Buck et al., 1991; Devaud et al., 1996), we compared the properties

unit composition of extrasynaptic GABAARs are quite differ- of GABAAR activation at 2 and at 5 days after the last CIE dose.

ent from those of synaptic GABAARs (Soltesz et al., 1990; There were no significant differences in the kinetics of mIPSCs or the ϭ Nusser et al., 1995, 1998; Devor et al., 2001; Scotti and tonic GABAAR-mediated currents at 2 days (n 10 cells, 4 rats) or ϭ Reuter, 2001; Brunig et al., 2002). Functional studies deter- at 5 days (n 5 cells, 2 rats) after alcohol withdrawal. Furthermore, ␮ mined that extrasynaptic GABA Rs activate at lower GABA the effects of THIP (0.3 and 3 M) application on synaptic and A extrasynaptic currents did not differ between the 2-day and 5-day concentrations and desensitize more slowly than do the syn- withdrawal groups. Therefore, results combine the electrophysiolog- aptic GABAARs (Brickley et al., 1999; Robello et al., 1999; ical data from CIE rats at 2 to 5 days of withdrawal. Banks and Pearce, 2000; Hutcheon et al., 2000; Nusser and Electrophysiology. Transverse slices (400 ␮m thick) of dorsal Mody, 2002). The differences in subunit composition also hippocampus were obtained using standard techniques (Spigelman confer considerable pharmacological differences between the et al., 1992; Kang et al., 1996). Whole-cell patch clamp recordings were obtained from cells located in the CA1 pyramidal layer at 34 Ϯ synaptic and extrasynaptic GABAARs (Robello et al., 1999; Hutcheon et al., 2000; Bai et al., 2001; Nusser and Mody, 0.5°C during perfusion with artificial cerebrospinal fluid (ACSF) composed of 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl , 2 mM MgCl , 2002). 2 2 26 mM NaHCO , and 10 mM D-glucose. The ACSF was continuously In the present study, we sought to determine the effect of 3 bubbled with a 95/5% mixture of O2/CO2 to ensure adequate oxygen- CIE treatment on different pools of receptors, the extrasyn- ation of slices and a pH of 7.4. Patch pipettes contained 135 mM aptic (tonic) and synaptic GABA R-mediated inhibition in A cesium gluconate, 2 mM MgCl2, 1 mM CaCl2, 11 mM ethylene CA1 neurons, and to obtain additional evidence supporting glycol-bis(␤-aminoethyl ether)-N,N,NЈ,NЈ-tetraacetic acid, 10 mM

the interpretation that there is a subunit composition switch HEPES, 2 mM K2ATP, 0.2 mM Na2GTP; pH adjusted to 7.25 with in this model. Our electrophysiological data demonstrate a CsOH. GABAAR-mediated mIPSCs were pharmacologically isolated loss of the potentiation of tonic GABA current by positive by adding tetrodotoxin (0.5 ␮M), D-(Ϫ)-2-amino-5-phosphonopen- ␮ ␮ allosteric modulators (diazepam and zolpidem). Further- tanoate (40 M), 6-cyano-7-nitroquinoxaline-2,3-dione (10 M), and ␮ more, reduced tonic current potentiation by Ro15-4513 and CGP 54626 (1 M) to the ACSF from stock solutions. Stock solutions of 6-cyano-7-nitroquinoxaline-2,3-dione, bretazenil, diazepam, zolpi- the directly acting agonist 4,5,6,7-tetrahydroisoxazolo[5,4- dem, and Ro15-4513 were made with pure dimethyl sulfoxide. The c]pyridin-3-ol (THIP; also known as gaboxadol) suggests re- final concentration of dimethyl sulfoxide did not exceed 42 ␮Minthe ductions in extrasynaptic ␣1 and ␣4 subunit-containing recording chamber. Signals were recorded in voltage-clamp mode GABAARs. Interestingly, THIP-induced decreases in mIPSC with an amplifier (Axoclamp 2B, Axon Instruments Inc., Union City, charge transfer in saline-treated rats are “switched” to po- CA). Whole-cell access resistances were in the range of 2.5 to 15 M⍀ tentiation in CIE-treated rats, consistent with the evident before electrical compensation by about 90%. During voltage-clamp ␣ recordings, access resistance was monitored by measuring the size of increases in 4 subunits in synaptic GABAARs. In addition, we demonstrate that the soporific effects of THIP are only the capacitative transient in response to a 5-mV step command, and experiments were abandoned if changes Ͼ20% were encountered. At modestly reduced and its anxiolytic effects are maintained in least 10 min was allowed for equilibration of the pipette solution CIE rats. The novel enhancement by THIP of synaptic GABA with the intracellular milieu before commencing mIPSC recordings. receptors tempered by the reduced sensitivity of extrasynap- Data were acquired with pClamp 8 software (Axon Instruments tic receptors after CIE treatment suggests potential useful- Inc.), digitized at 20 kHz (Digidata 1200B; Axon Instruments Inc.), ness of THIP in ameliorating the symptoms of human alcohol and analyzed using the Clampfit software (Axon Instruments Inc.) 1236 Liang et al.

and the Mini Analysis Program (versions 5.2.2 and 5.4.8; Synap- (they had no ethanol during the extra 2 days). Rats were injected tosoft, Decatur, GA). with 8 mg/kg THIP (diluted in 0.9% saline, 2 ml/kg i.p.) 30 min before Detection and Analysis of mIPSCs and Tonic Currents. The infusion of PTZ, and seizure threshold was measured again. Data recordings were low-pass filtered off-line (Clampfit software) at 2 were analyzed by ANOVA. kHz. The mIPSCs were detected (Mini Analysis Program) with Anxiolytic Effect of THIP on the Elevated Plus-Maze. Rats threshold criteria of 5 pA amplitude and 20 pA ⅐ ms area. Frequency were tested for the anxiolytic effect of THIP (1.25 and 2.5 mg/kg) on of mIPSCs was determined from all automatically detected events in the elevated plus-maze. Rats were brought to the procedure room 2 h a given 100-s recording period. For kinetic analysis, only single-event before testing. The plus-maze was constructed as described previ- mIPSCs were chosen during visual inspection of the recording trace. ously (Pellow et al., 1985). Rats were randomly divided into the This included mIPSCs with a stable baseline, sharp rising phase, following groups: saline controls treated with vehicle (saline) or and exponential decay. Double- and multiple-peak mIPSCs were THIP, and CIE rats treated with vehicle or THIP. Rats were injected excluded. The mIPSC kinetics were obtained from analysis of the 1 h before testing (volume 2 ml/kg i.p.). Rats were placed on the averaged chosen single events (Ͼ120 events/100-s recording period) central area of the maze, tested for 5 min, and videotaped in a room aligned with half-rise time in each cell. Decay time constants were absent of any humans. The following measures were scored: number obtained by fitting a double exponential to the falling phase of the of entries into open arms, closed arms, or center platform, and time averaged mIPSC in each neuron. The tonic current magnitudes were spent in each of these areas. An arm entry was defined as the entry obtained from the mean baseline current during the 100-s recording of all four feet into one arm. Data are reported as mean Ϯ S.E.M. periods. The investigator performing the recordings and mIPSC percentage of entries into the open arms and percentage of time analysis was blind to the treatment (saline or CIE) that the rats spent in open arms. Statistical differences were determined using received. All comparisons of group differences in mIPSC kinetics and ANOVA. drug effects were made with ANOVA (Sigmastat; SPSS Inc., Chi- cago, IL). Results Sleep Time Assay. The hypnotic effect of THIP (30 mg/kg i.p.; H. Lundbeck A/S, Copenhagen, Denmark) was tested on CIE- and sa- CA1 Neurons Exhibit Synaptic (Phasic) and Extra- ϭ line-treated rats (n 11). THIP was dissolved in 0.9% saline. Injec- synaptic (Tonic) GABAAR-Mediated Inhibition. Ini- tion volume was 2 ml/kg. The latency time (the time required after tially, we sought to isolate the tonic component of inhibition drug injection to lose the righting reflex) and sleep time were mea- in CA1 neurons from the synaptic component through the use sured. Sleep time was determined as follows. After drug injection of the GABA R antagonist gabazine (McCabe et al., 1988; and loss of righting reflex, rats were placed on their backs in a A Bai et al., 2001). We also wanted to ascertain that glycine V-shaped trough and a timer was started. The sleep time period Ϫ ended when the animal was able to flip over three times in 30 s. receptor activation does not contribute to the Cl currents Latency and sleep time are reported as mean (minutes) Ϯ S.E.M. attributed to GABAAR activation. Figure 1 illustrates that Statistical significance was calculated by ANOVA. strychnine (1 ␮M) application had no effect on the kinetics of Pentylenetetrazol (PTZ) Seizure Threshold and Anticon- averaged GABAAR-mediated mIPSCs or the tonic current ␮ vulsant Effect of THIP. After 2 days of treatment withdrawal, a (Ih). However, subsequent application of gabazine (1 M) group of CIE- and saline-treated rats were tested for the anticonvul- selectively abolished the mIPSCs without affecting the tonic sant effect of THIP against PTZ-induced seizures. First, the seizure current. threshold to PTZ was determined in saline-treated (n ϭ 6) and CIE Loss of Diazepam and Decreased Ro15-4513 Effect on ϭ rats (n 6). Rats were gently restrained and PTZ (25 mg/ml in 0.9% Tonic GABA R-Mediated Current in CIE-Treated saline) was injected slowly, at a constant rate, into the tail vein via A Rats. Application of diazepam enhanced the isolated tonic 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 current (without any appreciable desensitization) in CA1 the head. Seizure thresholds were calculated from the volume in- neurons from saline-treated rats, whereas subsequent addi- ␮ jected for body weight and are presented as milligrams per kilogram tion of the GABAAR blocker (picrotoxin, 50 M) blocked this of PTZ. Once the seizure threshold for each rat was established, 2 response (Fig. 1; summarized in Fig. 3). By contrast, diaze- days later the rats were tested for the anticonvulsant effect of THIP pam 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 effective-

ness of picrotoxin on Ih after CIE treatment. Dashed lines indicate the average Ih before strychnine application. GABAAR Pharmacology in Alcoholism 1237

on the tonic current. Picrotoxin was still able to reduce Ih, Partial Agonist, THIP, Potently Activates Tonic indicating that the GABAAR-mediated tonic current was still GABAAR Currents in CA1 Neurons from Untreated present in CIE-treated CA1 neurons. These data suggested Rats. To extend our observations on GABAAR subunit that the extrasynaptic GABAARs in CIE rats lose the diaze- changes, we decided to use a directly acting agonist at ␣ pam-sensitive 1, -2, and -5 subunits and/or gain the diaze- GABAARs, THIP. THIP has been demonstrated to act as a pam-insensitive ␣4 subunits. high-efficacy and reasonably high-affinity agonist at recom- ␣ ␤ ␦ To test for this possibility, we next compared the effects of binant 4 3 -containing GABAARs (Adkins et al., 2001; Ro15-4513 on the tonic current in saline- and CIE-treated Brown et al., 2002) and is, therefore, a partial agonist at ␣ rats. Ro15-4513 does not possess agonist activity at 1- and certain other GABAARs. Unlike GABA, THIP is not subject ␣ 2-containing GABAARs, but is a high-affinity agonist at to rapid uptake and, thus, its application in brain slices ␣ 4-containing GABAARs (Wisden et al., 1991; Knoflach et al., would be somewhat analogous to increasing ambient [GABA] 1996; Whiting et al., 2000). Application of Ro15-4513 in slices in the slice. Since the concentration-dependent actions of

of saline-treated rats potentiated Ih from its baseline level, THIP on synaptic and extrasynaptic GABAAR-mediated cur- an effect that was blocked by subsequent picrotoxin applica- rents in CA1 neurons have not been previously characterized tion (Fig. 2; summarized in Fig. 3). This suggested that in detail, we first studied the effects of THIP application in ␣ 4-containing GABAARs contribute to the tonic current in rat slices from control (untreated) rats at 34.5°C. Bath applica- CA1 neurons. In slices from CIE-treated rats, Ro15-4513 still tion of THIP produced a concentration-dependent increase in

potentiated Ih, but this potentiation was smaller than that in Ih, without appreciable response desensitization at THIP ␮ slices from saline-treated rats. Quantification of Ih changes concentrations up to 10 M (Figs. 7, A and B, and Fig. 8). after application of various GABAAR modulators confirmed Some desensitization of the response was observed at THIP ␮ these findings (Fig. 3). concentrations of 30 and 100 M. The EC50 for THIP was at Loss of Zolpidem Potentiation of Synaptic and Ex- approximately 1 ␮M (Fig. 7B).

trasynaptic GABAARs after CIE Treatment. The benzo- THIP Inhibits mIPSCs in CA1 Neurons from Un- diazepine, zolpidem, is an allosteric positive modulator of treated Rats. Synaptic GABAARs in CA1 neurons were ␣ GABAAR function with selectivity for the 1-containing recently demonstrated to be biophysically and pharmacolog- ␣ GABAARs, with intermediate potency on 2/3 receptors, and ically distinct from extrasynaptic receptors (Banks and ␣ no effect on 5-containing GABAARs (Whiting et al., 2000). Pearce, 2000; Bai et al., 2001; Yeung et al., 2003). Thus, Application of zolpidem potentiated both the mIPSCs and the synaptic GABAARs possess higher unitary conductance and tonic current in a concentration-dependent manner in CA1 lower affinity for GABA than do their extrasynaptic counter- neurons from saline-treated rats (Figs. 4 and 5). By contrast, parts (Yeung et al., 2003). They also desensitize more rapidly ␮ zolpidem (0.1 M) application in slices from CIE-treated rats than do extrasynaptic GABAARs and are selectively blocked had no effect either on mIPSCs or the tonic current, whereas by gabazine and penicillin (Banks and Pearce, 2000; Yeung et 0.3 ␮M zolpidem had a very small potentiating effect on the al., 2003). In the continued presence of a partial agonist such

synaptic and extrasynaptic GABAAR-mediated currents. Ex- as THIP, these receptors would be expected to exhibit de- amination of the mIPSC kinetics revealed that zolpidem increased responses to the full agonist (GABA) released in the creased the mIPSC area (total charge transfer) by prolonging synaptic cleft (Kurata et al., 1999; Bianchi and Macdonald, the decay time constants without affecting mIPSC amplitude 2003). For example, one way in which a partial agonist can (Fig. 6). The mIPSC rise time was also increased by zolpidem, reduce GABA currents is to accelerate the desensitization of but only in saline-treated rats (Fig. 6). These data pointed to GABA-induced currents (Nakahiro et al., 1991; Kurata et al., ␣ the loss of 1-containing extrasynaptic GABAARs, further 1999). Indeed, examination of averaged mIPSC current ki- extending our previous findings of an apparent decrease in netics revealed a concentration-dependent decrease in ␣ synaptic 1-containing GABAARs (Cagetti et al., 2003), and mIPSC area by THIP (Fig. 7C). This decrease was primarily also suggested that the zolpidem-insensitive ␣5-containing due to the speeding up of the mIPSC decay, since the rise

GABAARs do not solely contribute to the tonic current in CA1 time and amplitude of mIPSCs were little affected by THIP neurons, since some zolpidem-enhanced receptors are at concentrations Յ10 ␮M. In addition, there was a substan- present. tial 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 ap-

plication reduces Ih to a level below that seen before or after gabazine application. TTX, tetrodotoxin. 1238 Liang et al.

Fig. 3. CIE treatment-induced changes in allosteric modulation of tonic GABAAR-mediated current. A, summary graphs of diazepam-induced changes Ϯ ϭ Ϯ 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 ϭ 7 cells, 4 rats), respectively. Note the complete loss of diazepam effect on the tonic current in neurons from CIE-treated rats. B, summary Ϯ graphs of Ro15-4513-induced changes in GABAAR-mediated tonic current. The mean Ih values before strychnine application were 47 3.5 pA (saline, ϭ Ϯ ϭ 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 Ͼ 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 CIE- treatment. GABAAR Pharmacology in Alcoholism 1239

plitude since 1) THIP concentrations Յ10 ␮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 Ϯ 0.14 pA in the absence of THIP to a maximum of 6.3 Ϯ 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 CIE- Treated Rats. The concentration-response for THIP was very similar between slices from untreated and saline- treated 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

Fig. 5. Summary graph of zolpidem effects on tonic GABAAR-mediated treatment. Also, given the high efficacy and reasonably high current (I ) in CA1 neurons from saline- and CIE-treated rats. Asterisks ␣ ␤ ␦ h affinity of THIP at 4 3 -containing GABAARs (Adkins et denote significant difference between saline and CIE groups (two-way repeated measures ANOVA). Daggers represent significant difference al., 2001; Brown et al., 2002), such GABAARs may be prefer- from control (predrug) condition. Note the large decrease in the zolpidem entially decreased at extrasynaptic locations after CIE treat- effect in neurons from CIE rats. ment.

THIP Potentiates GABAAR-Mediated Synaptic Inhi- by THIP, which suggested THIP-mediated decreases in pre- bition in CIE-Treated Rats. The concentration-dependent synaptic GABA release. This decrease in frequency was not effects of THIP on mIPSC kinetics in saline-treated rats were likely to be due to failure to detect mIPSCs of reduced am- 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 ϭ 5 cells, 2 rats) and CIE treatment (n ϭ 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). 1240 Liang et al.

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, concentration- Ϯ response 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 ϭ min ϩ (max Ϫ min)/(1 ϩ 10(log EC50 Ϫ 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 Ϯ 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 coupled with a moderate reduction in its effect on the tonic THIP effects on mIPSC kinetics between saline- and CIE- current in CIE-treated rats was in marked contrast to the treated rats revealed a dramatic shift from a depressant almost complete loss of potentiation by benzodiazepines such action on mIPSCs in slices from saline-treated rats to a as diazepam and zolpidem of both synaptic and extrasynaptic potentiating effect in CIE rats (Fig. 10). The predominant GABAARs. Previously, we showed that the hypnotic effects of effect was a prolongation of both decay time constants, re- the benzodiazepine flurazepam were greatly attenuated after sulting in increased area of mIPSCs. Interestingly, the great- CIE treatment (Cagetti et al., 2003). Based on the current in est potentiation was observed at the lowest concentration of vitro studies, we suspected that the in vivo actions of THIP THIP tested. Nevertheless, even at the highest concentration may be affected differently from those of benzodiazepines (10 ␮M), there was a significant increase in the mIPSC area. after CIE treatment. To this end, we compared THIP for its This behavior is consistent with a partial agonist model for anticonvulsant, soporific, and anxiolytic effects in saline- and THIP in the presence of the agonist GABA, with differential CIE-treated rats. CIE rats showed partial tolerance to the effects in CIE- versus saline-treated rats due to differential hypnotic effect of THIP (Table 1). The mean sleep time of Ϯ efficacy and kinetics of GABAARs with an altered subunit 92 8 min for saline-treated rats was significantly reduced p ϭ 0.003). The ,ء) composition. There was also a concentration-dependent in- to 59 Ϯ 6 min for CIE rats at 30 mg/kg crease in mIPSC rise time in CIE- but not in saline-treated latency time of 20 Ϯ 2 min was significantly longer for CIE .(p ϭ 0.003 ,ء) rats. The amplitude and frequency of mIPSCs were affected rats compared with 13 Ϯ 1 min for controls similarly by THIP in both saline- and CIE-treated rats. The anticonvulsant effect of THIP (8 mg/kg) against PTZ Reduced Hypnotic and Maintained Anxiolytic Ef- seizure threshold was measured in CIE and control rats. As fects of THIP in CIE-Treated Rats. The dramatic shift demonstrated previously (Kokka et al., 1993), the PTZ sei- from a depressant to a potentiating effect of THIP on mIPSCs zure threshold was significantly reduced in CIE rats com- GABAAR Pharmacology in Alcoholism 1241

Fig. 8. Changes in THIP effects on GABAAR- mediated 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. How- ever, 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 ϭ 0.02) and CIE rats (p ϭ 0.04) and time spent in them (p Ͻ 0.05) (Fig. 12, A and B).

Discussion 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 alter-

ations 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 alcohol-

ism, 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 extra- Fig. 9. Summary graph of THIP effects on tonic GABAAR-mediated synaptic GABAARs (Robello et al., 1999; Hutcheon et al., current (Ih) in CA1 neurons after saline (six rats) and CIE treatment (six rats). Each point is a mean Ϯ S.E.M. of responses to THIP in five or six 2000; Bai et al., 2001; Nusser and Mody, 2002). Although CA1 neurons. precise physiological roles have not been identified for the tonic inhibitory currents mediated by extrasynaptic pared with saline controls (Fig. 11): the seizure threshold GABAARs, they are clearly subject to modulation by clini- was 28 Ϯ 2 mg/kg PTZ in CIE (n ϭ 6) and 48 Ϯ 6 mg/kg PTZ cally important drugs. Our data confirm the presence of a ϭ in saline groups, respectively (p 0.01). At the concentration tonic GABAAR-mediated current in rat CA1 neurons (Bai et tested, THIP failed to exhibit anticonvulsant activity in ei- al., 2001; Yeung et al., 2003) and demonstrate that the po- ther group. tentiation of this current by several allosteric positive mod- CIE- and saline-treated rats were tested on the elevated ulators is either reduced or eliminated after CIE treatment. plus-maze with two doses of THIP (1.25 and 2.5 mg/kg). At Specifically, diazepam (0.3 ␮M) potentiation of the isolated both doses, THIP had no effect on the total number of entries tonic current is eliminated (Figs. 1 and 3), similar to the in either saline or CIE rats. Total entries for saline rats elimination of mIPSC potentiation we reported previously in treated with vehicle, 1.25 and 2.5 mg/kg THIP, were (mean Ϯ CIE rats (Cagetti et al., 2003). Here we showed that zolpidem S.E.M.): 13 Ϯ 1, 10.5 Ϯ 0.9, and 10.2 Ϯ 0.9. For CIE rats, they (0.1 and 0.3 ␮M) effects are similarly reduced, consistent were: 8.1 Ϯ 0.7, 8.7 Ϯ 0.9, and 8 Ϯ 1.0. Total entries were with a reduction in ␣1-containing and/or increases in ␣4- significantly lower in CIE- compared with saline-treated rats containing GABAARs at both synaptic and extrasynaptic lo- (two-way ANOVA; p Ͻ 0.05). The lower dose of THIP did not cations. However, whereas the potentiation of mIPSCs by have an anxiolytic effect in either group (Fig. 12, A and B). Ro15-4513 is increased (Cagetti et al., 2003), the potentiation However, when tested at the concentration of 2.5 mg/kg, of the isolated tonic current by Ro15-4513 is reduced in CIE THIP had an anxiolytic effect in both groups. THIP increased rats (Figs. 2 and 3). Since Ro15-4513 does not possess agonist ␣ significantly the number of entries into the open arms by activity at 1-containing GABAARs, but is a high-affinity 1242 Liang et al.

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).

Saline (n ϭ 11) CIE (n ϭ 12) Sleep time (min) 92 Ϯ 859Ϯ 6* Latency (min) 13 Ϯ 220Ϯ 1* * The reduction in mean sleep time and the increase in latency for CIE rats compared with controls were significant (p ϭ 0.003 and p ϭ 0.003, respectively). ␣ agonist at 4-containing GABAARs (Wisden et al., 1991; Knoflach et al., 1996; Whiting et al., 2000), these data collec- tively 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 potenti- Fig. 11. Anticonvulsant effect of THIP against PTZ-induced seizures in ating the tonic current in CIE rats. Further evidence for saline (n ϭ 6) and CIE rats (n ϭ 6). Rats were tested first for the PTZ (25 decreases in extrasynaptic GABAARs was obtained by dem- mg/ml i.v.) seizure threshold. CIE rats exhibited a reduced PTZ seizure onstrating that tonic current potentiation by THIP, a partial threshold compared with saline controls (white bars; ૺ, p ϭ 0.01). The ␣ same rats were tested again after 2 days for the anticonvulsant effect of agonist with reported high efficacy at certain 4-containing THIP. Pretreatment with THIP (8 mg/kg i.p.) failed to protect either GABAARs, was also reduced in CIE rats (Figs. 8 and 9). The group against PTZ-induced seizures. large potentiation of synaptic currents by THIP in CIE rats is further evidence for the insertion of ␣4 subunits into synaptic CA1 synapses in CIE rats because 1) ␦ subunit has very low ␣ ␤ ␦ GABAARs. Previously, increased expression of 4 3 sub- expression levels in the hippocampal CA1 region (Persohn et units in a rat model of progesterone withdrawal was shown to al., 1992), and 2) ␦ subunit protein levels are decreased by double the affinity and increase the efficacy of CA1 neuron ϳ50% in the hippocampus of CIE rats (Cagetti et al., 2003).

GABAARs for THIP (Sundstrom-Poromaa et al., 2002). How- Also, it should be noted that THIP was demonstrated to ever, this subunit combination is unlikely to be inserted into activate channels in rat CA1 neurons that are modulated by GABAAR Pharmacology in Alcoholism 1243

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 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: ve- potentiation of 2-containing GABAARs (reviewed in Mohler hicle–treated saline controls (n ϭ 11) and vehicle-treated CIE rats (n ϭ et al., 2002). In hippocampal pyramidal cells, ␣2-containing 10), THIP (1.25 mg/kg)-treated saline controls (n ϭ 11) and CIE rats (n ϭ ϭ ϭ GABAARs are concentrated in synapses at the axon initial 10), THIP (2.5 mg/kg)-treated saline controls (n 5) and CIE rats (n 5). ␣ Data are reported as mean Ϯ S.E.M value for percentage of open arms segment (Nusser et al., 1996; Fritschy et al., 1998), and 2 entries (A) and percentage of time spent in open arms (B). THIP (1.25 mRNA does not appear to be reduced in CIE rats (Cagetti et mg/kg) failed to induce anxiolysis in both saline controls and CIE rats al., 2003), whereas the potentiating effects of diazepam on compared with their respective vehicle-treated group. At a higher dose, THIP (2.5 mg/kg) significantly increased in both groups the number of both synaptic and extrasynaptic currents in CA1 neurons entries into the open arms and time spent in them compared with their from CIE rats are lost. Thus, anxiolytic properties of diaze- respective vehicle-treated groups; ૺ, p Ͻ 0.05. pam in CIE rats are likely due to effects at GABAARs located elsewhere in the central nervous system. diazepam (Lindquist et al., 2003), and therefore, its actions Chronic ethanol treatment leads to increased GABAAR ␣ ␣ ␣ are not limited to 4-containing GABAARs. Similarly, it internalization and changes in 1/ 4 ratios, concurrently should be noted that the allosteric actions of Ro15-4513 may with changes in the way the protein kinase C (PKC) ␥ isoform ␣ not be entirely selective for 4-containing GABAARs, and couples to these subunits (Kumar et al., 2002, 2003). Protein therefore, the decreased potentiation of the tonic current phosphorylation plays an important role in regulating after CIE treatment by Ro15-4513 could also be attributed to GABAAR, including both GABAAR channel activity and traf- decreases in other extrasynaptic GABAAR subunit combina- ficking (Barnes, 1996, 2001; Kittler et al., 2002; Kittler and tions. Moss, 2003). PKC also has important effects on allosteric

In saline-treated rats, THIP exhibits both anxiolytic and modulation of GABAAR by ethanol and neurosteroids sedative-hypnotic properties (Fig. 12; Table 1). In hippocam- (Weiner et al., 1997; Brussaard and Koksma, 2003; Harney et pal slices from these rats, THIP application activates the al., 2003; Koksma et al., 2003). For example, PKC activation extrasynaptic GABAAR-mediated tonic current but occludes in young rat dentate granule cells makes them sensitive to a mIPSCs generated by GABA release in the synaptic cleft previously ineffective concentration of an endogenous neuro- (Figs. 7 and 8). This suggests that potentiation of extrasyn- steroid (Harney et al., 2003). Also, mutant mice lacking dif- aptic GABAARs alone may be sufficient to reduce anxiety or, ferent PKC isoforms exhibit altered sensitivity to ethanol in at higher concentrations of this agonist, to induce sleep. We vivo and altered GABAAR responsiveness in vitro, such that estimated the half-maximal concentration for THIP activa- PKC␥ null mice have reduced ethanol modulation (Harris et tion of the tonic current in control rats to be approximately 1 al., 1995; Proctor et al., 2003) and PKC⑀ null mice have ␮M (Fig. 7B), which is substantially lower than the 80 ␮M increased ethanol modulation (Hodge et al., 1999; Proctor et value reported previously (Sundstrom-Poromaa et al., 2002). al., 2003). To date, the possibility that changes in the

Interestingly, the plasma concentration of THIP that im- GABAAR phosphorylation state and trafficking may account proves sleep quality in humans was also estimated to be for the altered responsiveness of GABAAR to various alloste- about 1 ␮M (Schultz et al., 1981), and brain concentrations ric modulators after CIE treatment has not been examined, are unlikely to be much higher (Madsen et al., 1983; and therefore, these changes represent alternative explana- Faulhaber et al., 1997). THIP was demonstrated to promote tions to the subunit change hypothesis. deep, slow-wave sleep without affecting rapid eye movement The current study extends our knowledge of GABAAR sleep in both rodents and humans (Faulhaber et al., 1997; pharmacology in the hippocampus of CIE rats and is consis- Lancel and Langebartels, 2000). Recent studies in normal tent with plastic changes in subunit composition of 1244 Liang et al.

and subcellular targeting of GABA -receptor subtypes demonstrated in mouse GABAARs. In particular, the changes occur in both synapti- A hippocampal and olfactory neurons in vivo. Neurosci Lett 249:99–102. cally and extrasynaptically localized receptors which give Harney SC, Frenguelli BG, and Lambert JJ (2003) Phosphorylation influences rise to phasic and tonic inhibition in CA1 neurons, respec- neurosteroid modulation of synaptic GABAA receptors in rat CA1 and dentate gyrus neurones. Neuropharmacology 45:873–883. tively. Differential sensitivity to benzodiazepine modulators Harris RA, McQuilkin SJ, Paylor R, Abeliovich A, Tonegawa S, and Wehner JM and the partial agonist THIP suggests different subunit com- (1995) Mutant mice lacking the ␥ isoform of protein kinase C show decreased positions before and after CIE treatment, possibly including behavioral actions of ethanol and altered function of ␥-aminobutyrate type A receptors. 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