Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABAA-R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABAA-Rs and individual GABAA-R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABAA-Rs that harbor the 1 subunit. To test this, we used global knockout mice that completely lack the 1 subunit and forebrain-specific, conditional knockout mice that lack the 1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that 1-containing GABAA-Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane. Inhaled anesthetics produce anesthesia by unknown mechanisms. The prevailing theory, initially proposed by Franks and Lieb (1984), posits that specific protein targets in the nervous system are the molecular sites of action of inhaled anesthetics. Numerous putative protein targets have been identified, including a wide variety of ion channels (Campagna et al., 2003). However, the contribution that each of these targets makes to whole-animal behavioral responses to inhaled anesthetics is not clear. A plausible target that has received considerable attention is the GABA A receptor (GABAA-R). The GABAA-R is a fivesubunit chloride channel activated by GABA and muscimol and is blocked competitively by bicuculline and noncompetitively by picrotoxin (Olsen, 1982). Eccles et al. (1963) noted that many general anesthetics prolong the inhibition of spinal motoneurons, an effect mediated by GABA. Nicoll (1972) suggested that GABA-mediated enhancement of synaptic inhibition might underlie anesthetic actions. Consistent with this suggestion, Pearce et al. (1989) reported that anesthetics greatly prolong the time course of recurrent inhibition in the rat hippocampus. However, our knowledge of the importance of GABAA-Rs and individual GABAA-R subunits to anesthetic-induced behavioral effects remains incomplete. Inhaled anesthetics produce two universal clinical effects: amnesia for events during surgery, and immobility in response to noxious stimulation (e.g., surgical incision). Although the primary neuroanatomic site at which inhaled anesthetics act to produce immobility is the spinal cord (Antognini and Schwartz, 1993; Rampil et al., 1993), supraspinal structures probably mediate amnestic effects. A plausible site of action by which inhaled anesthetics interfere with memory is the hippocampus, where GABAA-Rs participate in memory formation (Bailey et al., 2002; Collinson et al., 2002). This work was supported by National Institutes of Health grants AA10422, GM52035, and GM47818. E.I.E. is a paid consultant to Baxter Healthcare Corp. Baxter Healthcare Corp supplied the desflurane and isoflurane used in these studies. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.009936. ABBREVIATIONS: GABAA-R, GABA type A receptor; MAC, minimum alveolar concentration; LORR, loss of righting reflex; Ro 15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]benzodiazepine-3-carboxylate; TBPS, t-butylbicyclophosphorothionate. 0026-895X/05/6801-61–68$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 1 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 9936/3039620 Mol Pharmacol 68:61–68, 2005 Printed in U.S.A. 61 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from The 1 subunit seems to be of particular importance in this process. The 1 subunit of the GABAA-R is the most abundant subunit in the adult brain (McKernan and Whiting, 1996) and is expressed at high levels in many brain regions, including the hippocampus (Sperk et al., 1997). Benzodiazepines, which only act by allosterically enhancing the action of GABA, specifically cause amnesia by an action on the 1 GABAA-R (Rudolph et al., 1999). This study investigated the hypothesis that 1 subunitcontaining hippocampal GABAA-Rs partly mediate the amnesia caused by inhaled anesthetics. We used genetically engineered mice that completely lack the 1 subunit (Vicini et al., 2001) in all cells of the body (i.e., a global knockout) and mice that conditionally lack the 1 subunit in restricted neuronal populations (i.e., forebrain-specific knockout) to address this hypothesis. We assessed amnesia by Pavlovian fear conditioning. For comparison, we tested whether either knockout would influence the capacity of an inhaled anesthetic to produce loss of the righting reflex or suppression of nociceptive reflexes. We predicted that these effects would not be influenced by the 1 subunit. Materials and Methods Mouse Production. Global 1 knockout mice were produced as described previously (Vicini et al., 2001). Mice heterozygous for a floxed 1 allele (exon 8 flanked by loxP sites) and a cre-recombined, inactive 1 allele that lacks exon 8 were interbred to produce homozygous floxed control mice and heterozygous and homozygous global knockout mice. Expression of the unrecombined floxed allele does not differ from wild-type 1 expression; the recombined 1 allele is a true null allele (Vicini et al., 2001; Kralic et al., 2002). Global knockout mice and control mice were of a mixed C57BL/6J X strain 129S1/X1 FVB/N hybrid background (Vicini et al., 2001) of the F6 generation. CamKII-cre transgenic mice, line T29-1 (Tsien et al., 1996), were crossed with B6;129S4-Gt(ROSA)26Sor/J (Soriano, 1999) or B6.Cg-Tg(xstpx-lacZ)32/J (Zinyk et al., 1998) reporter mice obtained from The Jackson Laboratory (Bar Harbor, ME). Adult (56 days of age) F1 generation mice from these crosses were analyzed for functional -galactosidase activity to reveal tissue-specific patterns of cre-mediated recombination, as described below. Crossing the CamKII-cre transgene (Tsien et al., 1996) onto the 1 floxed background (Vicini et al., 2001) produced forebrain-specific 1 knockout mice. Breeding pairs were used in which the male lacked the CamKII-cre transgene (Cre-) but was homozygous floxed 1, and the female was hemizygous for the CamKII-cre transgene (Cre ) and homozygous for the floxed 1 gene. Conditional knockout mice and control mice were of a mixed C57BL/6J X strain 129Sv/SvJ hybrid background of the F6–9 generation. All mice were maintained under a 12-h light/dark schedule with lights on at 7:00 AM. Mice were group housed, provided ad libitum access to food and water, and genotyped by Southern blot analysis. All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the Institutional Animal Care and Use Committees at the University of California at San Francisco and the University of Pittsburgh. Histology and Immunohistochemistry. Tissue sections derived from mice at 8 weeks of age were analyzed for cre-activated -galactosidase staining to determine the extent of recombination throughout the brain using standard techniques (Tsien et al., 1996). Slides were counterstained with eosin and examined by light microscopy. For GABAA-R 1 immunostaining, animals (56 days of age) were deeply anesthetized with pentobarbital (Nembutal 40 mg/kg; Ovation Pharmaceuticals Inc., Deerfield, IL) and perfused transcardially (Fritschy and Mohler, 1995). The GABAA-R 1 subunit was visualized in 40m sections processed for immunoperoxidase staining with subunit-specific antisera raised against amino acids 1 to 16 of the 1 subunit (Gao et al., 1993). Free-floating sections were washed three times for 10 min each in Tris buffer (Tris saline, pH 7.4, and 0.05% Triton X-100) and incubated at 4°C overnight in primary antibody solution (1:20,000) diluted in Tris buffer containing 2% normal goat serum. Sections were then washed three times for 10 min each in Tris buffer and incubated in biotinylated secondary antibody solution (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) diluted 1:300 in Tris buffer containing 2% normal goat serum for 30 min at room temperature. After additional washing, sections were transferred to the avidin-peroxidase solution (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA) for 20 min, washed, and processed using diaminobenzidine hydrochloride (Sigma-Aldrich, St. Louis MO) as the chromogen. Slides were airdried, dehydrated with ascending series of ethanol, cleared with xylene, and coverslipped with Eukitt (Electron Microscopy Services, Hatfield, PA). Changes in regional distribution of GABAA-R 1 subunits were analyzed by light microscopy (Zeiss Axioplan microscope; Carl Zeiss Inc., Thornwood, NY). Ligand Autoradiography. Whole brains from 5 adult (11.6–12.7 weeks of age) male mice of each genotype were used. The autoradiographic procedures for regional localization of the benzodiazepine (labeled with [H]Ro 15-4513; PerkinElmer Life and Analytical Sciences, Boston, MA), GABA ([H]muscimol; PerkinElmer Life and Analytical Sciences) and convulsant ionophoric (t-butylbicyclophosphoro[S]thionate, [S]TBPS; PerkinElmer Life and Analytical Sciences) binding sites were as described previously (Mäkelä et al., 1997). Nonspecific binding was determined with 10 M flumazenil (donated by F. Hoffmann-La Roche, Basel, Switzerland), 100 M picrotoxinin (Sigma-Aldrich), and 100 M GABA (Sigma-Aldrich) in [H]Ro 15-4513, [S]TBPS, and [H]muscimol assays, respectively. Autoradiography films were quantified using AIS image analysis system (Imaging Research, St. Catherines, ON, Canada) as described previously (Mäkelä et al., 1997). Binding densities for each brain area were averaged from measurements of one to three sections per brain. The standards exposed simultaneously with brain sections were used as reference with the resulting binding values given as radioactivity levels estimated for gray matter areas (in nanocuries per milligram for H and nanocuries per gram for C). The significance of the differences between the mouse lines in each brain region was assessed with analysis of variance followed by Tukey-Kramer post-test. Fear Conditioning and Minimum Alveolar Concentration Determinations. At 8 to 12 weeks of age, 99 control, 154 heterozygous, and 100 homozygous GABAA-R 1 global knockout mice, and 40 control and 37 conditional GABAA-R 1 knockout mice were studied. Fear conditioning was performed as described previously (Eger et al., 2003). In brief, animals were exposed to target concentrations of isoflurane in oxygen or oxygen alone (control) for 30 min and then were placed in a gas-tight training chamber containing the same concentration of isoflurane. After allowing 3 min for exploration of the training chamber, a 90-dB, 2-kHz tone sounded, coterminating with a 2-s foot shock. This was repeated twice, with 1 min between tones. Foot shock intensity varied between 1 and 3 mA as a function of anesthetic concentration to equalize the response of mice to the foot shock. Animals were observed by closed-circuit television. The following day, fear to tone was tested by placing animals in a different context. After allowing 3 min for exploration, the animals were exposed to a 90-dB, 2-kHz tone for 8 min and then were immediately returned to the animal’s home container. Fear to context was tested later that day by placing the mice in the original training chamber for 8 min with no tone imposed. Animals were observed by closed-circuit television. Fear was assessed by behav62 Sonner et al. at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from ioral freezing (i.e., immobility except for respiration) and was measured every 8 s for 8 min per mouse by a blinded observer. The number of freezes of the 60 measurements gave the probability of freezing (“freeze score”) for each animal. The minimum alveolar concentration (MAC) of anesthetic preventing movement in 50% of animals in response to a noxious stimulus (a tail clamp) is a standard EC50 measure of anesthetic potency. MAC values for desflurane, halothane, and isoflurane were measured as described previously (Sonner et al., 2000). For each anesthetic, MAC values and regression parameters estimated in calculating amnestic EC50 values for different genotypes were compared using either an analysis of variance with a StudentNewman-Keuls test for post hoc multiple comparison testing or a Student’s t test. Nonlinear regression was performed to calculate an EC50 value and the maximum value of the dose-response curve (A) for fear conditioning according to the following equation: Freeze Score A 1 isoflurane isoflurane ED50 Means S.E. are reported except where otherwise noted. P 0.05 was considered statistically significant. Loss of Righting Reflex. Groups of six to eight mice (8–19 weeks old; 15.9–33.1 g) were tested for loss of righting reflex (LORR) in individual wire-mesh cages in a rotating carrousel enclosed in a sealed acrylic chamber as described previously (Homanics et al., 1997; Quinlan et al., 1998). Halothane and isoflurane (both from Halocarbon Laboratories, River Edge, NJ) mixed with oxygen were monitored with an infrared anesthesia analyzer (Datex-Ohmeda Inc., Andover, MA). Constant anesthetic concentrations were supplied for 15 min before testing. A blinded observer scored the mice as positive for LORR if they passively rolled twice in a 75-s time period while the carousel rotated at 4 rpm. Mice tested with both volatile drugs were given at least 7 days to recover between anesthetics. Sleep Time. The sleep time (duration of the loss of the righting reflex) was used to assess the sedative/hypnotic effects of pentobarbital (45 mg/kg, i.p.) and zolpidem (60 mg/kg, i.p.). Normothermia was maintained with a heat lamp.