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

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.106161v1 319/1/219    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Werner, D. F. Articles by Homanics, G. E. Search for Related Content PubMed PubMed Citation Articles by Werner, D. F. Articles by Homanics, G. E.

NEUROPHARMACOLOGY

Knockin Mice with Ethanol-Insensitive 1-Containing -Aminobutyric Acid Type A Receptors Display Selective Alterations in Behavioral Responses to Ethanol

Departments of Pharmacology (D.F.W., G.E.H.) and Anesthesiology (G.E.H.), University of Pittsburgh, Pittsburgh, Pennsylvania; Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, Texas (Y.A.B., C.M.B., R.A.H.); Departments of Physiology and Pharmacology (O.J.A., Y.S., E.L., J.L.W.) and Program in Neuroscience (J.L.W.), Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Psychology, University of Memphis, Memphis, Tennessee (R.B.B., D.B.M.); C.V. Starr Laboratory for Molecular Neuropharmacology, Departments of Anesthesiology and Pharmacology, Weill Medical College, Cornell University, New York, New York (N.L.H.)

Received April 13, 2006; accepted June 15, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Despite the pervasiveness of alcohol (ethanol) use, it is unclear how the multiple molecular targets for ethanol contribute to its many behavioral effects. The function of GABA type A receptors (GABA_A-Rs) is altered by ethanol, but there are multiple subtypes of these receptors, and thus far, individual subunits have not been definitively linked with specific behavioral actions. The 1 subunit of the GABA_A-R is the most abundant subunit in the brain, and the goal of this study was to determine the role of receptors containing this subunit in alcohol action. We designed an 1 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity and constructed knockin mice containing this mutant subunit. Hippocampal slice recordings from these mice indicated that the mutant receptors were less sensitive to ethanol's potentiating effects. Behaviorally, we observed that mutant mice recovered more quickly from the motor-impairing effects of ethanol and etomidate, but not pentobarbital, and showed increased anxiolytic effects of ethanol. No differences were observed in ethanol-induced hypnosis, locomotor stimulation, cognitive impairment, or in ethanol preference and consumption. Overall, these studies demonstrate that the postsynaptic effects of ethanol at GABAergic synapses containing the 1 subunit are important for specific ethanol-induced behavioral effects.

GABA_A-Rs are pentameric chloride channels composed of multiple subunits. Numerous subunits have been cloned and are categorized into different classes, with some containing several isoforms: six , three , three , one , one , one , one , and three (Sieghart and Sperk, 2002). However, the majority of GABA_A-Rs are thought to consist of two , two , and a or subunit (Tretter et al., 1997). Because of the numerous subunits, it is quite possible that receptors with a specific subunit composition can mediate specific ethanol-induced behavior as is the case for benzodiazepines (Atack, 2005) and i.v. anesthetics (Grasshoff et al., 2005).

We hypothesized that 1 subunit-containing GABA_A-Rs are important for ethanol action based on several lines of evidence. The 1 subunit is the most abundant subunit in adult brain and is present in 50% of all GABA_A-Rs (McKernan and Whiting, 1996; Sieghart and Sperk, 2002). 1-Containing GABA_A-Rs mediate benzodiazepine-induced sedation and memory impairment (Rudolph et al., 1999; McKernan et al., 2000) and pharmacologic studies using the 1-selective ligand zolpidem have suggested a role for this subunit in ethanol sensitivity (Criswell et al., 1995). In addition, previous work with 1 global knockout mice suggests this subunit is important for certain ethanol-related effects. Specifically, abolition of 1 increases ethanol-induced locomotor activity (Blednov et al., 2003b; Kralic et al., 2003), decreases ethanol-induced hypnosis (Blednov et al., 2003a), and nonsedative doses of ethanol eliminate essential tremor observed in knockouts (Kralic et al., 2005).

The compensatory changes and other limitations of null mutants can often be avoided by constructing knockin mice in which the wild-type gene is replaced by a mutant sequence possessing a drug-insensitive, but otherwise normally responsive, protein. This has proven very useful for defining the role of specific GABA_A-R subunits in behavioral actions of i.v. anesthetics and benzodiazepines. For example, construction of mice containing mutant GABA_A-Rs with normal responses to GABA, but lacking modulation by benzodiazepines, have shown that the 1, 2, and 3 subunits are each responsible for distinct actions (e.g., sedative, antianxiety, myorelaxation, respectively) of these drugs (e.g., Rudolph et al., 1999). Two previous attempts to construct knockin mice with glycine or GABA_A-Rs resistant to ethanol were not completely successful because the normal function of these receptors was impaired by the mutations (Findlay et al., 2003; Homanics et al., 2005).

Numerous studies have shown that mutation of amino acids in the transmembrane domains of the GABA_A-R subunits can eliminate alcohol modulation, suggesting that construction of a knockin mouse with alcohol-resistant GABA_A-Rs is feasible. Because the serine 270 to histidine (S270H) mutation in the TM2 region of the 1 subunit eliminated GABA_A-R enhancement by ethanol (Ueno et al., 2000), we initially used this mutation for knockin mice. However, this mutation by itself resulted in receptors that were hypersensitive to GABA (Nishikawa et al., 2002), and mice bearing this mutation were behaviorally impaired (Homanics et al., 2005). However, incorporating a second mutation, leucine 277 to alanine (L277A), resulted in receptors that displayed normal GABA sensitivity but no modulation by ethanol and a reduced potentiation by etomidate and pentobarbital. Importantly, these receptors showed normal modulation by flunitrazepam, and mice harboring this mutation appeared normal (Borghese et al., 2006b).

In the present study, we used these knockin mice to test the hypothesis that 1-containing GABA_A-Rs mediate specific behavioral responses to ethanol. We demonstrate that some, but not all, ethanol-induced behavioral responses are altered in these knockin mice.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Mouse Production
Wild-type (homozygous for serine at 270 and leucine at 277; genotype referred to as SL/SL) and knockin (homozygous for histidine at 270 and alanine at 277; genotype referred to as HA/HA) mice used for these experiments were produced from heterozygous (SL/HA) breeding pairs at the University of Pittsburgh (Pittsburgh, PA), the University of Texas (Austin, TX), or the University of Memphis (Memphis, TN). All mice were genotyped by Southern blot or polymerase chain reaction analysis of tail DNA as described previously (Borghese et al., 2006b). All mice were of a mixed C57BL/6Jx129SvJ background of the F₃ to F₆ generations. After weaning, mice were housed under specific pathogen-free conditions with ad libitum access to rodent chow and water with 12-h light/dark cycles (lights on at 7:00 AM). All mice were between 8 and 12 weeks of age for behavioral studies and 3 to 5 months for electrophysiological experiments. For most experiments, only male mice were used; however, in cases where sufficient numbers of male mice were not available, both male and female mice were used, and this is noted in the figure legends. Each mouse was used for only one experiment, and all mice were ethanol naive at the start of each experiment. All experiments were approved by Institutional Animal Care and Use Committees and were conducted in accordance with National Institutes of Health guidelines with regard to the use of animals in research.

Electrophysiology in Hippocampal Slices
Brain slice electrophysiological experiments were carried out on 3- to 5-month old male and female mice. Mice were anesthetized with halothane and euthanized by decapitation. Experiments on SL/SL and HA/HA mice were performed on an alternating day schedule, and experimenters were blinded to genotype. Brain slice preparation and electrophysiological recordings were carried out as described previously (Ariwodola et al., 2003; Ariwodola and Weiner, 2004). In brief, transverse hippocampal slices (400 µm) were prepared using a vibrating tissue slicer (Leica VT 1000S; Leica Microsystems, Bannockburn, IL), and slices were maintained at ambient temperature for at least2hin oxygenated artificial cerebrospinal fluid containing: 124 mM NaCl, 3.3 mM KCl, 2.4 mM MgCl₂, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄,10mM D-glucose, and 25 mM NaHCO₃, saturated with 95% O₂-5% CO₂. Whole-cell patch-clamp recordings of hippocampal CA1 pyramidal neurons were performed at ambient temperature using a patch pipette filling solution containing: 130 mM potassium gluconate, 10 mM KCl, 1 mM EGTA, 100 µM CaCl₂, 2 mM Mg-ATP, 200 µM Tris-guanosine 5'-triphosphate, 5 mM QX-314, and 10 mM HEPES (pH adjusted with KOH; 275-280 mOsM). Neurons were voltage-clamped at -45 to -60 mV, and only cells with a stable access resistance of 5 to 20 M were included in the data analysis. GABA_A IPSCs were evoked every 20 s by electrical stimulation (0.2-ms duration, constant current) using a concentric bipolar stimulating electrode (FHC, Bowdoinham, ME) placed near the CA1 somatic layer ("proximal" stimulation) (Weiner et al., 1997). Stimulation intensity was adjusted to evoke responses that were 10 to 20% of maximal currents (typically 50-200 pA). GABA_A IPSCs were pharmacologically isolated using a cocktail of 50 µM 2-amino-5-phosphonovalerate and 20 µM 2,3-dihydroxy-6,7-dinitroquinoxaline to block N-methyl-D-aspartate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. Unless otherwise stated, all drugs used were purchased from Sigma-Aldrich (St. Louis, MO). Drugs were made up as 100- to 400-fold concentrates and applied to slices via calibrated syringe pumps (Razel Scientific Instruments, Stamford, CT). A 4 M ethanol solution was prepared immediately before each experiment from a 95% stock solution (Aaper Alcohol and Chemical, Shelbyville, KY) kept in a glass storage bottle. Drug effects were quantified as the percentage change in the area under the curve of synaptic currents relative to the mean of control and washout values. Statistical analyses of drug effects were carried out using the two-tailed Student's paired or unpaired t tests or analysis of variance followed by the Newman-Keuls post hoc test.

Behavioral and Pharmacokinetic Analyses
Acute Withdrawal. Mice were scored for handling-induced convulsion (HIC) severity 30 min and immediately before i.p. ethanol administration. These two predrug baseline scores were averaged. A dose of 4.0 g/kg ethanol in saline was injected i.p., and the mice were tested for HIC every hour until the HIC level reached baseline. Acute withdrawal was quantified as the area under the curve but above basal response (Crabbe et al., 1991). In brief, each mouse was picked up gently by the tail and, if necessary, gently rotated 180°, and the HIC was scored as follows: 5, tonic-clonic convulsion when lifted; 4, tonic convulsion when lifted; 3, tonic-clonic convulsion after a gentle spin; 2, no convulsion when lifted, but tonic convulsion elicited by a gentle spin; 1, facial grimace only after a gentle spin; and 0, no convulsion. HIC response curves were integrated to determine the area both under the curve and above basal response. Data were analyzed by Student's t test.

Rotorod. Mice were trained on a fixed speed (5.0 rpm) rotorod [Economex; Columbus Instruments (Columbus, OH) for ethanol and etomidate studies; Ugo Basile Rota-Rod, model 7650; Stoelting Co. (Wood Dale, IL) for pentobarbital studies], and training was considered complete when mice were able to remain on the rotorod for 60 s. After drug administration, each mouse was placed back on the rotorod, and time spent on the rotorod was measured for up to 60 s. Behavior of mice was measured at 30-, 5-, or 10-min intervals for ethanol (2.0 g/kg i.p.), etomidate (20 mg/kg i.p.), or pentobarbital (35 mg/kg i.p.), respectively. Data were analyzed using two-way repeated measures ANOVA. Time was the repeated measure factor and genotype was the between-group factor.

Loss of Righting Reflex. Mice were tested for the sedative/hypnotic effects of ethanol (3.8 g/kg i.p.) and etomidate (20 or 30 mg/kg i.p.). Mice were injected with drug and observed for loss of righting reflex (LORR). Once this occurred, mice were placed on their backs in v-shaped troughs and monitored until they were able to right themselves three times in a 30-s period. LORR was determined as the length of time from when the mouse was placed in a supine position until it was able to right itself. Data were analyzed using unpaired Student's t test.

Two-Bottle Choice. The two-bottle choice protocol was carried out as described previously (Blednov et al., 2003b). In brief, mice were allowed to acclimate for 1 week to individual housing. Experiments were carried out in standard 7.5- x 12.5-inch cages in sliding racks. Two drinking tubes were continuously available to each mouse, and tubes were weighed daily. One tube always contained water. Food was available ad libitum, and mice were weighed every 4 days. After 4 days of water available on both tubes, mice were offered 3% (v/v) ethanol versus water for 4 days. Tube positions were changed every day to control for position preferences. Quantity of ethanol consumed (grams per kilogram of body weight per 24 h) was calculated for each mouse, and these values were averaged for every concentration of ethanol. Immediately following 3% ethanol, a choice between 6% (v/v) ethanol and water was offered for 4 days, then 9% (v/v) ethanol and water for 4 days, and finally 12% (v/v) ethanol and water for 4 days. Throughout the experiment, evaporation/spillage estimates were calculated every day from two bottles placed on an empty cage; one contained water, and the other contained the appropriate ethanol solution. Wild-type and knockin mice were also tested for saccharin and quinine consumption. One tube always contained water, and the other contained the tastant solution. Mice were serially offered saccharin (0.033 and 0.066%) and quinine hemisulfate (0.03 and 0.06 mM), and intakes were calculated. Each concentration was offered for 4 days, with bottle position changed every day. Within each tastant, the low concentration was always presented first, followed by the higher concentrations. Between tastants, mice had two bottles with water for 2 weeks. Preference was defined as the ratio between the amount of consumed ethanol or tastant solution and total liquid intake. If the ratio was less than 0.5, the behavior was considered avoidance, and if it was more than 0.5, the behavior was considered preference. Data were analyzed using two-way repeated measures ANOVA with Bonferroni post hoc test when necessary. Ethanol, saccharin, or quinine concentration was the repeated measure factor, and genotype was the between-group factor.

Hypothermia. Ethanol-induced hypothermia was determined by measuring rectal body temperature via a digital thermometer (Thermalert Model TH-8 with probe RET-3; Physiotemp Instruments, Clifton, NJ). Mice were injected with either 2.0 or 3.0 g/kg ethanol. Body temperatures were measured at times of 15, 30, 45, 60, 90, and 120 min postinjection. Data were assessed using repeated measures ANOVA with time as the repeated measure factor and genotype or dose as the between-group factor.

Morris Water Maze. The water maze consisted of a galvanized steel, circular tub 3 feet in diameter and 24 inches high. Four points labeled along the rim corresponded to compass points and served as start points during training day sessions. Water level was raised 0.25 inches above the level of the clear, Plexiglas escape platform and made invisible by clouding the water with water-based paint. The water maze was located in a room that was rich with distal cues in fixed locales. Recording measurements were made using a video camera (video tracking system; HVS Image, Co., Buckingham, UK) mounted above the pool. Animals were trained using the standard spatial version of the Morris water maze task (Morris, 1981). The procedure occurred over a successive 10-day period. Training took place on days 1 through 9 with testing occurring on day 10. Escape platform location was held constant throughout the procedure. Mice were given four trials a day, one from each starting position, with each trial lasting 45 s. A Latin square design allowed for a unique starting position order each day. For each trial, the investigator lowered mice into the pool facing the wall while simultaneously activating the tracking system. Latency to reach the platform was assessed for each trial. On test day, mice received 2.25 g/kg ethanol i.p. 30 min prior to testing. Dose was based on previous work demonstrating reliably impaired spatial memory (Berry and Matthews, 2004). Animals were tested in the same manner as employed during training. Data were analyzed first by multivariate ANOVA [genotype by day (last day training, testing under ethanol)] followed by a direct comparison between conditions using a Student's t test.

Elevated Plus Maze. Mice were evaluated for basal anxiety as well as ethanol-induced anxiolysis using the elevated plus maze. Mice were transported to the testing room 1 day prior to testing. Animals were tested between 9:00 and 11:00 AM under ambient room light. Mice were weighed and injected with 0.75, 1.0, or 1.5 g/kg ethanol or saline 10 min prior to testing. Each mouse was placed on the central platform of the maze facing an open arm. Mice were allowed to freely explore the maze for 5 min during which the following measurements were manually recorded: number of open arm entries, number of closed arm entries, total number of entries, time spent in open arms, and time spent in closed arms. A mouse was considered to be on the central platform or any arm when all four paws were within its perimeter. Data were analyzed using ANOVA with genotype and dose as the between-subject factors. Data were further analyzed with Fischer's post hoc test, or pair-wise comparisons were made with Student's t test where appropriate.

Ethanol Metabolism and Clearance. Following injection of ethanol (3.5 g/kg i.p.), blood was collected from the retro-orbital sinus at 30, 60, 90, and 120 min postinjection. Blood ethanol concentrations (BECs) were determined as described previously (Harris et al., 1995). In brief, blood samples were collected in heparinized capillary tubes, mixed with 3% perchloric acid, and centrifuged for 10 min at 1500g at 4°C. Supernatants were subsequently used to assess ethanol concentration via an alcohol dehydrogenase enzymatic spectrophotometric method. The rate of clearance was determined by linear regression analysis. Data were analyzed by Student's t test.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Hippocampal Electrophysiology. Whole-cell recordings were made from CA1 pyramidal neurons, and pharmacologically isolated GABA_A IPSCs were evoked as described in the methods. Experiments were carried out on male and female mice, and since no sex differences in the acute effects of ethanol on IPSCs were noted, data from males and females were pooled for the genotype comparisons. Bath application of ethanol significantly potentiated the area of GABA_A IPSCs in both groups of mice in a concentration-dependent manner (Fig. 1A). The potentiation was significant at all three concentrations tested, and there were no genotype differences in the magnitude of the enhancement at 20 and 40 mM ethanol (e.g., 40 mM: SL/SL = 23.0 ± 7.6%; HA/HA = 24.7 ± 4.6%; p > 0.05). In contrast, at 80 mM ethanol, IPSCs recorded from HA/HA slices were markedly less sensitive to ethanol (SL/SL = 59.1 ± 6.3%; HA/HA = 27.7 ± 3.5%, p < 0.01). As illustrated in the summary time course graphs (Fig. 1B), the ethanol enhancement was stable throughout the duration of an 8-min application in both groups and reversed completely upon washout.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Pharmacological characterization of GABAA mIPSCs recorded from SL/SL and HA/HA CA1 pyramidal neurons. A, bar graph summarizing the effect of 20, 40, and 80 mM ethanol on the area of pharmacologically isolated GABAA IPSCs recorded from SL/SL and HA/HA CA1 pyramidal neurons. n = 12-38 neurons/group. *, p < 0.05, significant difference relative to control; #, p < 0.01, significant difference between SL/SL and HA/HA; NS, no significant difference. B, summary time courses of the effect of an 8-min bath application of 80 mM ethanol on the area of evoked GABAA IPSCs recorded from SL/SL (n = 38) and HA/HA (n = 34) CA1 pyramidal neurons. Traces above the graph are averages of 8 to 10 IPSCs recorded from a representative SL/SL and HA/HA neuron prior to, during, and 10 min after 80 mM ethanol treatment. C, bar graph summarizing the effect of 1 µM FLU and 50 µM PB on the area of GABAA IPSCs recorded from SL/SL and HA/HA CA1 pyramidal neurons. n = 5-7 neurons/group. *, p < 0.05, significant difference relative to control; NS, no significant difference between SL/SL and HA/HA.

Acute Withdrawal. HICs were scored before and after injection of ethanol. The initial effect of ethanol is to eliminate the HIC, which returns to preinjection baseline after approximately 6 h, and then increases above baseline during h 6 to 10. The increase above baseline is a measure of acute withdrawal and was not different for the SL/SL and HA/HA mice (areas under the curve but above basal response 3.5 ± 0.8 and 4.3 ± 1.2, respectively) (Fig. 2). However, the recovery of HIC appeared to be somewhat more rapid for the HA/HA mice (Fig. 2), although the area below baseline was not significantly different for the two genotypes (6.3 ± 0.6 and 4.5 ± 0.8, respectively, p = 0.08).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Severity of acute ethanol withdrawal using HICs. Mice were injected with 4 g/kg ethanol at time 0, and handling-induced convulsions were scored hourly for 13 h. The increase above basal levels is a measure of acute alcohol withdrawal and showed a similar withdrawal severity for SL/SL and HA/HA mice. Values are mean ± S.E.M., n = 8 per genotype.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effects of ethanol, etomidate, and pentobarbital on motor coordination using rotorod. Mice were injected with 2 g/kg ethanol (A), 20 mg/kg etomidate (B), or 35 mg/kg sodium pentobarbital (C), and the ability to balance on a rotorod was measured at 30-, 5-, and 10-min intervals, respectively. HA/HA mice recovered motor coordination more rapidly than SL/SL mice after ethanol (p < 0.0001) and etomidate (p < 0.01) administration, but the lines did not differ for pentobarbital. Values are mean ± S.E.M., n = 7-11 per genotype. Male and female mice were used for pentobarbital experiments, but no gender differences were observed.

Loss of Righting Reflex. The LORR assay was used to examine the hypnotic effects of ethanol and etomidate. For ethanol, there were no significant differences between genotypes (Fig. 4A), but for etomidate, the knockin mice displayed a shorter duration of LORR than the wild type (Fig. 4, B and C). It should be noted that etomidate gave somewhat variable LORR values, and these experiments were independently conducted in two different laboratories using a large number of mice (n = 17-31) to assure the validity of this conclusion.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Loss of righting reflex produced by ethanol or etomidate. Mice were injected i.p. with 3.8 g/kg ethanol (A) or with 20 (B) or 30 (C) mg/kg etomidate, and the duration of LORR was measured. The HA/HA mice did not differ from SL/SL mice for ethanol-induced LORR (n = 6-7), but the etomidate LORR was significantly shorter for HA/HA than for SL/SL mice (p < 0.005, n = 24-31 for 20 mg/kg; p < 0.01, n = 17-18 for 30 mg/kg). Values are mean ± S.E.M. Male and female mice were used for etomidate experiments, but no gender differences were observed.

For ethanol consumption, there was a significant main effect of concentration (F_3,51 = 15.96, p < 0.0001) but no effect of genotype or the interaction of genotype x concentration (Fig. 5A). Analysis of total fluid intake (ethanol + water) indicated a main effect of concentration (F_3,51 = 4.55, p < 0.01) and genotype (F_1,51 = 18.47, p < 0.001) but no interaction of genotype x concentration (Fig. 5B). Subsequent pairwise comparisons indicate HA/HA mice had an increased total fluid intake at 6% (v/v) ethanol (p < 0.05). Analysis of ethanol preference revealed no effect of concentration, genotype, or interaction of concentration x genotype (Fig. 5C). Overall, these results indicate that although genotypes do not differ in preference for ethanol, there is an increase in total fluid intake in HA/HA mice.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Voluntary consumption of ethanol and water. The two bottle choice test was used to measure voluntary consumption of ethanol solutions and water. HA/HA mice showed increased total fluid (alcohol + water) consumption at 6% (v/v) ethanol (B) compared with SL/SL mice, but alcohol consumption (A) and preference (C) were not different between HA/HA and SL/SL mice. Values are mean ± S.E.M., n = 9-10. *, p < 0.05.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Voluntary consumption of saccharin and quinine. The two-bottle choice test was used to measure voluntary consumption of two different concentrations of saccharin or quinine solutions versus water. There were no differences between genotypes in preference for saccharin (A) or avoidance of quinine (C). Main effects of genotype were observed for both saccharin (B, p < 0.05) and quinine (D, p < 0.005) in total fluid intake. Pair-wise comparisons indicate no difference in genotype at individual concentrations of saccharin (B), but HA/HA mice had increased total fluid intake (quinine + water) at 0.06 mM quinine compared with SL/SL mice (D). Values are mean ± S.E.M., n = 9-10. *, p < 0.05.

Hypothermia. We next asked whether genotypes differed in ethanol-induced decreases in body temperature. Treatment with ethanol (2 or 3 g/kg) reduced body temperature (Fig. 7) at all of the time points measured compared with preinjection baseline. Analysis indicated a significant main effect of dose (F_1,40 = 29, p < 0.0001), time (F_6,40 = 211, p < 0.0001), and interaction of dose x time (F_6,40 = 9, p < 0.0001). No difference was observed with respect to genotype.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Ethanol-induced hypothermia. Ethanol (2 or 3 g/kg) caused a dose-dependent decrease in body temperature (p < 0.0001) that was similar for both genotypes. Values are mean ± S.E.M., n = 10-11 per genotype per dose. Male and female mice were used, but no gender differences were observed.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Effect of ethanol on spatial memory. Latency (seconds) for mice to escape the water on the last training day and the effects of ethanol impairment. Acute ethanol administration significantly impaired spatial memory (p < 0.001), but the impairment was not dependent upon genotype. Data represent mean ± S.E.M., n = 26 SL/SL mice and 12 HA/HA mice. Male and female mice were used, but no gender differences were observed.

Elevated Plus Maze. Locomotor activity was assessed by total number of entries, whereas anxiety was measured by percentage of time spent in open arm entries and percentage of open arm entries. Basal performance on the elevated plus maze (following saline injection) did not differ with respect to genotype for these measures (Fig. 9, A-C). Thus, locomotor activity and indicators of anxiety-like behavior were not altered by the mutations in the GABA_A-R 1 subunit.

View larger version (27K): [in this window] [in a new window]   Fig. 9. Evaluation of anxiety and activity using the elevated plus maze. Total arm entries (A), percentage of open arm entries (B), and percent total time spent in open arms (C) are shown. Saline or ethanol was administered 10 min prior to testing. The locomotor stimulant effect was dependent on ethanol dose and genotype. The anxiolytic effect of ethanol was detected in both genotypes; ethanol increased the percentage of open arm entries relative to total number of entries and percentage of time spent in open arms relative to the total time. HA/HA mice had increased responses compared with SL/SL mice at specific doses (0.75 for anxiolytic effect; 1.0 for locomotor-stimulant effect). Data represent mean ± S.E.M., n = 10-11 per group per genotype. Male and female mice were used for experiments. No effect of gender was observed. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent comparison with the saline groups (within genotype). #, p < 0.05; and ##, p < 0.01 represent differences between genotypes at each ethanol dose.

With respect to the anxiolytic effects of ethanol, ethanol increased the percentage of open arm entries relative to total number of entries (Fig. 9B) and percentage of time spent in open arms relative to the total time (Fig. 9C). Two-way ANOVA indicated a significant main effect of dose (F_3,73 = 7.0; p < 0.001) and interaction of genotype x dose (F_3,73 = 4.3, p < 0.01) but no main effect of genotype in the percentage of open arm entries. For percent time spent in open arms, the statistical analysis indicated a significant effect of dose (F_3,73 = 6.0, p < 0.01), genotype (F_3,73 = 4.0, p < 0.05), and interaction of genotype x dose (F_3,73 = 2.9, p < 0.05).

Because the anxiolytic effects observed at 1.0 and 1.5 g/kg may be potentially complicated by the genotypic differences observed in total arm entries, we limited our analysis and interpretation to the 0.75 g/kg dose. At this dose, ethanol decreased anxiety-like behavior in knockin mice but not in controls. This is evidenced by the increase in percent open arm entries in HA/HA mice (Fig. 9B, p < 0.05) and percent time in open arms (Fig. 9C, p < 0.01). Therefore, HA/HA mice appear to be more sensitive to the anxiolytic effects of ethanol.

Metabolism and Clearance. To determine whether ethanol pharmacokinetics differed between wild-type and knockin mice, BECs were measured every 30 min for 2 h following injection of 3.5 g/kg i.p. ethanol BECs (e.g., at 30 min postinjection, 415 ± 17 mg/dl for SL/SL; 414 ± 13 mg/dl for HA/HA) and clearance (SL/SL, 1.13 ± 0.22 mg/dl/min, n = 5; HA/HA, 1.22 ± 0.19 mg/dl/min, n = 6) did not differ with respect to genotype, thus allowing valid comparisons between genotypes for ethanol-related behaviors. Clearance rates are similar to previously published rates for mice with a similar genetic background (Mihalek et al., 2001).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we used mice in which double-mutant 1 subunits of GABA_A-Rs replaced the wild-type subunit (knockin mice). We chose mutations that render the receptor resistant to ethanol (and also to isoflurane) but provide a near normal response to GABA and normal modulation by flunitrazepam (Borghese et al., 2006b). This allowed us to test the importance of 1-containing GABA_A-Rs to specific cellular and behavioral responses to ethanol.

We first determined the effects of the knockin mutations on synaptic sensitivity to ethanol using hippocampal electrophysiological analysis. We recorded from proximal GABAergic synapses onto CA1 pyramidal neurons because these synapses are thought to express high levels of the 1 subunit (Nusser et al., 1996), and the ethanol sensitivity of these synapses has been extensively characterized (for review, see Siggins et al., 2005; Weiner and Valenzuela, 2006). Interestingly, the knockin mutation did not alter the potentiating effect of ethanol on evoked IPSCs at low concentrations (20 or 40 mM) but markedly attenuated the effect at a relatively high ethanol concentration (80 mM). These data suggest that, at least in the hippocampal CA1 region, ethanol facilitation of GABAergic synapses at low concentrations is mediated primarily by mechanisms that do not require the 1 subunit (i.e., facilitation of GABA release, for review, see Siggins et al., 2005; Weiner and Valenzuela, 2006) but that a direct interaction with postsynaptic GABA_A-Rs contributes to the potentiation observed at higher ethanol concentrations.

We also carried out a limited characterization of some of the other pharmacological properties of CA1 GABA_A IPSCs from wild-type and knockin mice. Importantly, flunitrazepam and pentobarbital potentiation were not affected by the double mutation in the 1 subunit, suggesting that the mutations did not alter other pharmacological properties of 1-containing GABA_A-Rs in hippocampal synapses. These findings are not entirely consistent with the results of similar experiments in recombinant receptors (Borghese et al., 2006b): in 12/32s GABA_A-Rs expressed in Xenopus oocytes, flunitrazepam potentiation was not modified in mutated 1-containing GABA_A-Rs, but pentobarbital potentiation was decreased by approximately 30%. However, it should be emphasized that only a single concentration of each drug was tested in the initial studies reported here. A more thorough analysis of the concentration dependence of the effects of benzodiazepines, barbiturates, and other subunit-selective modulators will be needed to confirm that the mutations only disrupted the pharmacological effects of ethanol on synaptic GABA_A-Rs. Moreover, even though 1 may be present in high levels in these hippocampal synapses, other subunits present could participate and result in a normal response to pentobarbital. In addition, the mutation did not change paired-pulse facilitation or paired-pulse depression (data not shown), suggesting that the 1 HA/HA knockin did not result in dramatic compensatory alterations in the presynaptic properties of these GABAergic synapses.

Behaviorally, knockin mice displayed a faster recovery from the ataxic effects of ethanol or etomidate than wild-type mice, but the recovery from pentobarbital ataxia was not affected by the mutation. Ataxia is due, at least in part, to drug effects on cerebellar function, and it is important to note that normal 1 protein levels were observed in the cerebellum of mutant animals (Borghese et al., 2006b). Knockin mice also displayed decreased behavioral responses to the hypnotic actions of etomidate, but hypnotic effects of ethanol were normal. The decreased behavioral sensitivity to etomidate is consistent with previously observed electrophysiological recordings (Borghese et al., 2006b). The discrepancy in the responses to etomidate and ethanol may be due to the fact that etomidate has a more selective mechanism of action than ethanol. A single mutation in the 3 subunit of the GABA_A-R markedly reduced the LORR duration after etomidate administration (Jurd et al., 2003), whereas the ethanol effects are likely determined by its action on multiple targets, including non-GABA_A-R targets (Markel et al., 1996; Crabbe et al., 2006).

There were no differences between genotypes in preference toward ethanol, saccharine, and quinine. However, knockin mice showed an increased total consumption of fluids in each of these tests. The GABA_A-R has been strongly implicated in appetitive behaviors: administration of positive modulators such as benzodiazepines induces hyperdipsia and hyperphagia, whereas inverse agonists of the benzodiazepine site induce hypophagia, possibly through the modulation of palatability (Cooper, 2005). This would suggest that GABAergic transmission in the knockin mice is enhanced in the centers that control appetitive behaviors, probably due to compensatory changes, since the 1-containing GABA_A-Rs do not seem to mediate the hyperphagic effect of benzodiazepines (Cooper, 2005).

In contrast, knockin mice were observed to be more sensitive to the anxiolytic effects of ethanol as tested on the elevated plus maze assay. This effect was dependent on dose and was detected in both measurements of anxiety-like behavior at an ethanol dose of 0.75 g/kg. If 1-containing receptors were responsible for this effect of ethanol, then the knockin mice would be predicted to be less sensitive, not more sensitive. However, the anxiolytic effects of benzodiazepines have been linked to GABA_A-Rs containing the 2 and 3 subunits, not the 1 subunit (Atack, 2005). We found that the 3 and possibly 2 subunits are increased in the forebrain of these knockin mice (Borghese et al., 2006b). Therefore, we posit that increased expression of other ethanol-sensitive subunits may contribute to the observed phenotype and may represent molecular targets for the anxiolytic effects of ethanol.

Comparing these results with studies of the 1 null mutant (knockout) mice (Blednov et al., 2003a,b; Kralic et al., 2003, 2005), the knockout mice showed differences in more behavioral effects of ethanol, including consumption, tremor suppression, acute withdrawal, and hypnotic effects. However, the knockout mice displayed substantial and widespread compensatory changes in synaptic physiology, gene expression, and GABAergic circuitry (Kralic et al., 2006; Ponomarev et al., 2006), and it is likely that these secondary changes, in response to deletion of a large fraction of brain GABA_A-Rs, contribute to phenotypes seen in the knockout, but not the knockin mice.

It is somewhat surprising that the behavioral actions of ethanol that are changed by the knockin mutation are observed with moderate doses (e.g., 1 g/kg) of ethanol, whereas the electrophysiological differences were only seen with high concentrations of ethanol. It is possible that the electrophysiological methods used in this study were not sensitive enough to detect differences in the effects at low concentrations of ethanol. For example, analysis of ethanol effects on mIPSC frequency and kinetics in SL/SL and HA/HA mice may help to clarify the relative contribution of pre- and postsynaptic mechanisms to ethanol's overall facilitatory effects on GABAergic synapses in these mice. Alternatively, GABA_A-Rs that do not contain the 1 subunit may mediate the effects of low concentrations of ethanol via extrasynaptic GABA_A-Rs (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003; Hanchar et al., 2005), although these low-dose effects have not been observed in all studies (Borghese et al., 2006a). Lastly, because ethanol is known to affect multiple systems in the brain, it is possible that non-GABAergic systems may be responsible for certain behavioral effects or the combined effect of ethanol on multiple systems. Multiple genes mediate each of ethanol's effects (Crabbe et al., 2006), and modifying one of them may not be sufficient to alter the behavioral endpoint.

In summary, we demonstrated that incorporation of two specific mutations into the 1 subunit that had been shown to abolish GABA_A-R sensitivity to ethanol in vitro significantly decreased ethanol-induced potentiation of synaptic responses at high ethanol concentrations in vitro. Moreover, using these knockin animals, we also demonstrated that 1-containing GABA_A-Rs may play a role in recovery from ethanol's ataxic effects, indicating that 1-containing GABA_A-Rs are important for the mediation of specific ethanol-induced behavioral responses.

	Acknowledgements

 
We thank Carolyn Ferguson, Brian Sloat, and Robert J. Tomko, Jr. for expert technical assistance.

	Footnotes

 
This work was supported by National Institutes of Health Grants AA06399, U01 AA13520, GM47818, AA10422, AA014588, U01 AA013509, AA13960, AA11997, MH018273, and AA016046.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.106161.

ABBREVIATIONS: GABA_A-R, GABA type A receptor; QX-314, N-(2,6-dimethyl-phenylcarbamoylmethyl)-triethylammonium bromide; IPSC, inhibitory postsynaptic current; HIC, handling-induced convulsion; ANOVA, analysis of variance; LORR, loss of righting reflex; BEC, blood ethanol concentration; FLU, flunitrazepam; PB, pentobarbital.

¹ Both authors contributed equally to this work.

Address correspondence to: Dr. Gregg E. Homanics, University of Pittsburgh, Department of Anesthesiology, W1356 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: homanicsge{at}anes.upmc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Ariwodola OJ, Crowder TL, Grant KA, Daunais JB, Friedman DP, and Weiner JL (2003) Ethanol modulation of excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons. Alcohol Clin Exp Res 27: 1632-1639.[Medline]

Ariwodola OJ and Weiner JL (2004) Ethanol potentiation of GABAergic synaptic transmission may be self-limiting: role of presynaptic GABA(B) receptors. J Neurosci 24: 10679-10686.[Abstract/Free Full Text]

Atack JR (2005) The benzodiazepine binding site of GABA(A) receptors as a target for the development of novel anxiolytics. Exp Opin Investig Drugs 14: 601-618.[CrossRef]

Berry RB and Matthews DB (2004) Acute ethanol administration selectively impairs spatial memory in C57BL/6J mice. Alcohol 32: 9-18.[CrossRef][Medline]

Blednov YA, Jung S, Alva H, Wallace D, Rosahl T, Whiting PJ, and Harris RA (2003a) Deletion of the alpha1 or beta2 subunit of GABAA receptors reduces actions of alcohol and other drugs. J Pharmacol Exp Ther 304: 30-36.[Abstract/Free Full Text]

Blednov YA, Walker D, Alva H, Creech K, Findlay G, and Harris RA (2003b) GABAA receptor alpha 1 and beta 2 subunit null mutant mice: behavioral responses to ethanol. J Pharmacol Exp Ther 305: 854-863.[Abstract/Free Full Text]

Borghese CM, Storustovu S, Ebert B, Herd MB, Belelli D, Lambert JJ, Marshall G, Wafford KA, and Harris RA (2006a) The delta subunit of gamma-aminobutyric acid type A receptors does not confer sensitivity to low concentrations of ethanol. J Pharmacol Exp Ther 316: 1360-1368.[Abstract/Free Full Text]

Borghese CM, Werner DF, Topf N, Baron NV, Henderson LA, Boehm II S, Blednov YA, Saad A, Dai S, Pearce RA, et al. (2006b) An isoflurane- and alcohol-insensitive mutant GABA_A receptor alpha1 subunit with near normal affinity for GABA: characterization in heterologous systems and production of knock-in mice. J Pharmacol Exp Ther 319: 208-218.[Abstract/Free Full Text]

Cooper SJ (2005) Palatability-dependent appetite and benzodiazepines: new directions from the pharmacology of GABA(A) receptor subtypes. Appetite 44: 133-150.[CrossRef][Medline]

Crabbe JC, Merrill C, and Belknap JK (1991) Acute dependence on depressant drugs is determined by common genes in mice. J Pharmacol Exp Ther 257: 663-667.[Abstract/Free Full Text]

Crabbe JC, Phillips TJ, Harris RA, Arends MA, and Koob GF (2006) Alcohol-related genes: contributions from studies with genetically engineered mice. Addict Biol, in press.

Criswell HE, Simson PE, Knapp DJ, Devaud LL, McCown TJ, Duncan GE, Morrow AL, and Breese GR (1995) Effect of zolpidem on gamma-aminobutyric acid (GABA)-induced inhibition predicts the interaction of ethanol with GABA on individual neurons in several rat brain regions. J Pharmacol Exp Ther 273: 526-536.[Abstract/Free Full Text]

Findlay GS, Phelan R, Roberts MT, Homanics GE, Bergeson SE, Lopreato GF, Mihic SJ, Blednov YA, and Harris RA (2003) Glycine receptor knock-in mice and hyperekplexia-like phenotypes: comparisons with the null mutant. J Neurosci 23: 8051-8059.[Abstract/Free Full Text]

Grasshoff C, Rudolph U, and Antkowiak B (2005) Molecular and systemic mechanisms of general anaesthesia: the "multi-site and multiple mechanisms" concept. Curr Opin Anaesthesiol 18: 386-391.[Medline]

Grobin AC, Matthews DB, Devaud LL, and Morrow AL (1998) The role of GABA(A) receptors in the acute and chronic effects of ethanol. Psychopharmacology (Berl) 139: 2-19.[CrossRef][Medline]

Hanchar HJ, Dodson PD, Olsen RW, Otis TS, and Wallner M (2005) Alcohol-induced motor impairment caused by increased extrasynaptic GABA(A) receptor activity. Nat Neurosci 8: 339-345.[CrossRef][Medline]

Harris RA (1999) Ethanol actions on multiple ion channels: which are important? Alcohol Clin Exp Res 23: 1563-1570.[CrossRef][Medline]

Harris RA, McQuilkin SJ, Paylor R, Abeliovich A, Tonegawa S, and Wehner JM (1995) Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors. Proc Natl Acad Sci USA 92: 3658-3662.[Abstract/Free Full Text]

Homanics GE, Elsen FP, Ying SW, Jenkins A, Ferguson C, Sloat B, Yuditskaya S, Goldstein PA, Kralic JE, Morrow AL, et al. (2005) A gain-of-function mutation in the GABA receptor produces synaptic and behavioral abnormalities in the mouse. Genes Brain Behav 4: 10-19.[CrossRef][Medline]

Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, et al. (2003) General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 17: 250-252.[Abstract/Free Full Text]

Kralic JE, Criswell HE, Osterman JL, O'Buckley TK, Wilkie ME, Matthews DB, Hamre K, Breese GR, Homanics GE, and Morrow AL (2005) Genetic essential tremor in gamma-aminobutyric acidA receptor alpha1 subunit knockout mice. J Clin Investig 115: 774-779.[CrossRef][Medline]

Kralic JE, Sidler C, Parpan F, Homanics GE, Morrow AL, and Fritschy JM (2006) Compensatory alteration of inhibitory synaptic circuits in cerebellum and thalamus of gamma-aminobutyric acid type A receptor alpha1 subunit knockout mice. J Comp Neurol 495: 408-421.[CrossRef][Medline]

Kralic JE, Wheeler M, Renzi K, Ferguson C, O'Buckley TK, Grobin AC, Morrow AL, and Homanics GE (2003) Deletion of GABAA receptor alpha 1 subunit-containing receptors alters responses to ethanol and other anesthetics. J Pharmacol Exp Ther 305: 600-607.[Abstract/Free Full Text]

Markel PD, Fulker DW, Bennett B, Corley RP, DeFries JC, Erwin VG, and Johnson TE (1996) Quantitative trait loci for ethanol sensitivity in the LS x SS recombinant inbred strains: interval mapping. Behav Genet 26: 447-458.[CrossRef][Medline]

McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S, Myers J, Cook G, Ferris P, et al. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype. Nat Neurosci 3: 587-592.[CrossRef][Medline]

McKernan RM and Whiting PJ (1996) Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19: 139-143.[CrossRef][Medline]

Mihalek RM, Bowers BJ, Wehner JM, Kralic JE, VanDoren MJ, Morrow AL, and Homanics GE (2001) GABA(A)-receptor delta subunit knockout mice have multiple defects in behavioral responses to ethanol. Alcohol Clin Exp Res 25: 1708-1718.[CrossRef][Medline]

Morris RGM (1981) Spatial localization does not require the presence of local cues. Learn Motiv 12: 239-260.[CrossRef]

Nishikawa K, Jenkins A, Paraskevakis I, and Harrison NL (2002) Volatile anesthetic actions on the GABAA receptors: contrasting effects of alpha 1(S270) and beta 2(N265) point mutations. Neuropharmacology 42: 337-345.[CrossRef][Medline]

Nusser Z, Sieghart W, Benke D, Fritschy JM, and Somogyi P (1996) Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells. Proc Natl Acad Sci USA 93: 11939-11944.[Abstract/Free Full Text]

Ponomarev I, Maiya R, Harnett MT, Schafer GL, Ryabinin AE, Blednov YA, Morikawa H, Boehm SL 2nd, Homanics GE, Berman A, et al. (2006) Transcriptional signatures of cellular plasticity in mice lacking the alpha1 subunit of GABA_A receptors. J Neurosci 26: 5673-5683.[Abstract/Free Full Text]

Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, and Mohler H (1999) Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature (Lond) 401: 796-800.[CrossRef][Medline]

Sieghart W and Sperk G (2002) Subunit composition, distribution and function of GABA(A) receptor subtypes. Curr Top Med Chem 2: 795-816.[CrossRef][Medline]

Siggins GR, Roberto M, and Nie Z (2005) The tipsy terminal: presynaptic effects of ethanol. Pharmacol Ther 107: 80-98.[CrossRef][Medline]

Sundstrom-Poromaa I, Smith DH, Gong QH, Sabado TN, Li X, Light A, Wiedmann M, Williams K, and Smith SS (2002) Hormonally regulated alpha(4)beta(2)delta GABA(A) receptors are a target for alcohol. Nat Neurosci 5: 721-722.[Medline]

Tretter V, Ehya N, Fuchs K, and Sieghart W (1997) Stoichiometry and assembly of a recombinant GABAA receptor subtype. J Neurosci 17: 2728-2737.[Abstract/Free Full Text]

Ueno S, Lin A, Nikolaeva N, Trudell JR, Mihic SJ, Harris RA, and Harrison NL (2000) Tryptophan scanning mutagenesis in TM2 of the GABA(A) receptor alpha subunit: effects on channel gating and regulation by ethanol. Br J Pharmacol 131: 296-302.[CrossRef][Medline]

Wallner M, Hanchar HJ, and Olsen RW (2003) Ethanol enhances alpha 4 beta 3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low concentrations known to affect humans. Proc Natl Acad Sci USA 100: 15218-15223.[Abstract/Free Full Text]

Weiner JL, Gu C, and Dunwiddie TV (1997) Differential ethanol sensitivity of subpopulations of GABAA synapses onto rat hippocampal CA1 pyramidal neurons. J Neurophysiol 77: 1306-1312.[Abstract/Free Full Text]

Weiner JL and Valenzuela CF (2006) Ethanol modulation of GABAergic transmission: the view from the slice. Pharmacol Ther 111: 533-554.[CrossRef][Medline]

This article has been cited by other articles:

				G. Akk, P. Li, B. D. Manion, A. S. Evers, and J. H. Steinbach Ethanol Modulates the Interaction of the Endogenous Neurosteroid Allopregnanolone with the {alpha}1beta2{gamma}2L GABAA Receptor Mol. Pharmacol., February 1, 2007; 71(2): 461 - 472. [Abstract] [Full Text] [PDF]

				C. M. Borghese, D. F. Werner, N. Topf, N. V. Baron, L. A. Henderson, S. L. Boehm II, Y. A. Blednov, A. Saad, S. Dai, R. A. Pearce, et al. An Isoflurane- and Alcohol-Insensitive Mutant GABAA Receptor {alpha}1 Subunit with Near-Normal Apparent Affinity for GABA: Characterization in Heterologous Systems and Production of Knockin Mice J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 208 - 218. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.106161v1 319/1/219    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Werner, D. F. Articles by Homanics, G. E. Search for Related Content PubMed PubMed Citation Articles by Werner, D. F. Articles by Homanics, G. E.