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Perioperative Medicine  |   March 2013
Bidirectional Regulation of Intravenous General Anesthetic Actions by &agr;3-containing &ggr;-aminobutyric AcidAReceptors
Author Affiliations & Notes
  • Carolin J. Straub, Ph.D.
    *
  • Hew Mun Lau, B.Sc.
    †
  • Rosanna Parlato, Ph.D.
    ‡
  • Guenther Schuetz, M.D.
    §
  • Jean-Marc Fritschy, Ph.D.
    ║
  • Uwe Rudolph, M.D.
    #
  • *Predoctoral Research Fellow, Laboratory of Genetic Neuropharmacology, McLean Hospital and Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts, and Institute of Pharmacology and Toxicology, University of Zürich, Zürich, Switzerland. †Associate in Psychiatry, Laboratory of Genetic Neuropharmacology, McLean Hospital and Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts. ‡Postdoctoral Research Fellow, §Helmholtz Professor, Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany. ║Professor, Institute of Pharmacology and Toxicology, University of Zürich, Zürich, Switzerland. #Associate Professor, Laboratory of Genetic Neuropharmacology, McLean Hospital and Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts.
Article Information
Perioperative Medicine / Pharmacology
Perioperative Medicine   |   March 2013
Bidirectional Regulation of Intravenous General Anesthetic Actions by &agr;3-containing &ggr;-aminobutyric AcidAReceptors
Anesthesiology 3 2013, Vol.118, 562–576. doi:10.1097/ALN.0b013e3182800d76
Anesthesiology 3 2013, Vol.118, 562–576. doi:10.1097/ALN.0b013e3182800d76
What We Already Know about This Topic
  • The &ggr;-aminobutyric acidA (GABAA) receptor system mediates actions of many general anesthetics in the central nervous system. However, the contributions of the different GABAAreceptor subtypes and the role of noradrenergic neurons require further clarification

What This Article Tells Us That Is New
  • GABAAreceptors containing the &agr;3 subunit mediate anesthetic responses to etomidate and midazolam. Lack of the &agr;3 subunit results in enhanced responses to the combination ketamine/xylazine, an effect that is rescued in part by expression of the &agr;3 subunit selectively in noradrenergic neurons, indicating an involvement of noradrenergic neurons in modulating hypnosis

&ggr;-AMINOBUTYRIC acidA(GABAA) receptors are chloride channels, which mediate fast synaptic inhibition and tonic inhibition in the central nervous system. They are composed of five subunits drawn from a repertoire of eight different subunit families comprising at least 19 different genes (&agr;1–6, &bgr;1–3, &ggr;1–3, &dgr;, &egr;, &thgr;, &pgr;, &rgr;1–3).1–4 Depending on the exact subunit composition GABAAreceptors possess different physiological and pharmacological properties.1,2 Many general anesthetics potentiate the activity of GABAAreceptors, and the role of several GABAAreceptors subtypes in mediating anesthetic effects has been described.3,4 We have previously shown that &bgr;3(N265M) knock-in mice display dramatically reduced hypnotic responses to etomidate and pentobarbital and cannot be immobilized by these compounds, demonstrating that a subpopulation of GABAAreceptors is essential for the anesthetic actions of these anesthetic agents.5 Most frequently, the GABAAreceptors are classified by the &agr; subunit that is present. On the basis of studies in global knockout (KO) mice, the &agr;1 subunit,6 but not the &agr;57 and &agr;6 subunits,8 contribute to the hypnotic actions of intravenous general anesthetics. However, the significance of &agr;3 subunit–containing GABAAreceptors for anesthetic effects remained to be clarified.
Performing stereotaxic surgeries we made the unexpected and surprising observation that a combination of ketamine, an N  -methyl-D-aspartate receptor antagonist and xylazine, an &agr;2-adrenergic agonist, induces remarkably prolonged anesthesia in &agr;3KO mice compared with wild-type (WT) mice. The &agr;3 subunit of the GABAAreceptor is present in 10–15% of all GABAAreceptors in the central nervous system and GABAAreceptors containing the &agr;3 subunit are expressed widely in the brain.1,2 However, in monoaminergic neurons the &agr;3 subunit seems to be the major &agr; subunit expressed. High expression levels in noradrenergic neurons9–11 suggest that &agr;3 subunit–containing GABAAreceptors might be important modulators of the noradrenergic system.
The noradrenergic system (cell groups A1–A10), which includes the locus coeruleus (A6) whose neurons project widely throughout the neuraxis and other noradrenergic nuclei (e.g.  , A1 and A2, which project mainly to the hypothalamus) plays an important role in the regulation of sleep and arousal as well as thermoregulation.12–14 Activity of the noradrenaline-containing locus coeruleus neurons depends on the behavioral state (e.g.  , sleeping or waking) of the organism and this system is likely involved in switching between these different states.15 Moreover, &agr;2-adrenoceptor agonists such as dexmedetomidine have been shown to exert their sedative/hypnotic effects through endogenous sleep pathways,16 at least in part through inhibition of the locus coeruleus.17 
In order to obtain insights into the role of &agr;3-containing GABAAreceptors in general anesthetic actions, we investigated (1) in &agr;3KO mice whether &agr;3-containing GABAAreceptors mediate the action of intravenous general anesthetics known to act at least in part via  GABAAreceptors such as etomidate, midazolam, and pentobarbital; (2) whether &agr;3-containing GABAAreceptors play a role in the actions of the N  -methyl-D-aspartate receptor antagonist ketamine and of the &agr;2-adrenergic agonist medetomidine, either alone or in combination, using &agr;3KO mice; and (3) whether &agr;3-containing GABAAreceptors on noradrenergic neurons are responsible for the increased sensitivity to ketamine/xylazine. To this end, using conditional cre-lox  P-mediated recombination limited to noradrenergic neurons, we generated &agr;3KO mice in which the &agr;3 subunit was reintroduced exclusively in noradrenergic neurons.
Materials and Methods
Animals
Male mice on the C57BL/6J background between 8 and 18 weeks of age at beginning of experiments were used. Biochemical and morphological studies were performed in Zurich (Switzerland) with approval of the Cantonal Veterinary Office of Zurich and performed in accordance with international guidelines on animal use and care (European Community Council Directive 86/609/EEC). All behavioral experiments were conducted in Belmont in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals, and were approved by the McLean Hospital Institutional Animal Care and Use Committee, Belmont, Massachusetts. Mice lacking the &agr;3 subunit of the GABAAreceptor (&agr;3KO mice) have been described previously.18 For behavioral testing mice were group-housed (3–4 per cage) and kept under a reversed 12/12 h light–dark cycle (lights on from 9:00 PM to 9:00 AM) for at least 2 weeks before beginning of experimental testing and throughout the study.
Generation of Global Rescue Mice
The generation of mice lacking the &agr;3 subunit of the GABAAreceptor (&agr;3KO mice) has been described in detail.18 The absence of &agr;3 messenger RNA was demonstrated by reverse transcription polymerase chain reaction, and the absence of the &agr;3 protein by Western blot and immunohistochemistry.18 As the nomenclature of exons in the Gabra  3 gene has changed over time, exon 4 in the study by Yee et al.  18 is identical to exon 5 as described here. These mice carry an artificial exon flanked by lox  P sites in the Gabra3  gene, which is located on the X-chromosome. In mice that carry both the mutant gene and express the Cre recombinase this artificial exon is removed, thereby restoring &agr;3 subunit expression. Mice in which the &agr;3 subunit is rescued globally have been generated by crossing &agr;3KO mice with EIIa-Cre mice, which express the Cre recombinase in oocytes and preimplantation stages of the embryo.19 Mice carrying both the mutant gene and the Cre recombinase gene have been selected by polymerase chain reaction genotyping. These mice were crossed with C57BL/6J WT mice. Hemizygote male mice carrying only the mutant gene were then selected for experiments to ensure that the rescue of the &agr;3 subunit had been present in the germline.
Generation of Neuron-specific Rescue Mice
Mice expressing the &agr;3 subunit of the GABAAreceptor exclusively in noradrenergic (dopamine &bgr; hydroxylase [DBH]-rescue mice) and dopaminergic (dopamine transporter [DAT]-rescue mice) neurons, respectively, have been generated by crossing female &agr;3KO mice with male mice expressing the improved Cre (iCre) recombinase specifically in (nor)adrenergic (DBH-iCre mice) or dopaminergic neurons (DAT-iCre mice). DBH-iCre mice express the iCre recombinase under the control of the promoter of the DBH gene and have been described previously.20,21 DAT-iCre mice express the iCre recombinase under the control of the promoter of the DAT gene and have been described previously.22 Male mice carrying both the &agr;3KO gene and the respective iCre transgene have been selected by polymerase chain reaction genotyping.
Immunoperoxidase Staining
Four WT and four global rescue mice (9 weeks of age) were deeply anaesthetized with pentobarbital (Nembutal®, Abbott, Chicago, IL; 50 mg/kg, intraperitoneally) and perfused through the ascending aorta with fixative solution (4% paraformaldehyde in 0.15 M phosphate buffer and 15% saturated picric acid solution; pH 7.4). Brains were postfixed in fixative solution for 3 h and incubated in citrate buffer (0.2 M Na2HPO4dihydrate, 0.1 M citric acid; pH 4.5) overnight. The next day brains were cut parasagittally into approximately 8-mm thick blocks and irradiated in citrate buffer at 650 watt in the microwave for 90 s for antigen retrieval.23 Brains were cryoprotected in 10% dimethyl sulfoxide in phosphate buffered saline (PBS) for at least 2 h before sectioning them transversally at 40 µm with a sliding microtome. The sections were collected in PBS. Free-floating sections were incubated overnight at 4°C with a primary antibody directed against the &agr;3 subunit of the GABAAreceptor (1:5,000; antibody was developed in-house and is described in detail)9 in TrisTriton buffer (0.05 M Tris, 0.15 M NaCl, 0.2% Triton X-100; pH 7.4) containing 2% normal goat serum. The next day sections were washed three times in Tris-Triton buffer and incubated for 30 min at room temperature in biotinylated secondary antibody (1:300; Jackson Immunoresearch, West Grove, PA) in Tris-Triton buffer containing 2% normal goat serum. After three washes in Tris-Triton buffer, sections were incubated in the Avidin-Peroxidase (ABC)-Solution (1:100 in TrisTriton buffer) for 30 min (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA) and after another wash finally reacted with diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO) in Tris-Triton buffer (pH 7.7) containing 0.015% hydrogen peroxide. After 15 min the color reaction was stopped with ice-cold PBS. After two washes in PBS at room temperature, sections were mounted on gelatin-coated slides and air-dried. Finally, they were dehydrated with an ascending series of ethanol, cleared in xylene, and coverslipped with Eukitt (Erne Chemie, Dällikon, Switzerland). Brains and sections from WT and global rescue mice were processed in parallel under identical conditions to minimize variability in staining intensity. Images were taken with a stereo microscope (Zeiss, Jena, Germany).
Immunofluorescence Staining
DAT-Rescue (n = 4), DBH-Rescue (n = 3), WT (n = 3), and &agr;3KO (n =2) mice (8–14 weeks of age) were examined to confirm selective expression of the &agr;3 subunit in dopaminergic and noradrenergic neurons, respectively. For optimal and simultaneous detection of tyrosine hydroxylase, a neuronal marker for dopaminergic and noradrenergic neurons, and the &agr;3 subunit of the GABAAreceptor a novel protocol was used, which has been described in detail previously.24,25 In brief, mice were anesthetized with isoflurane and decapitated. The brain was quickly removed and placed in ice-cold artificial cerebrospinal fluid (composition in mM: NaCl, 125; NaHCO3, 26; NaH2PO4, 1.25; KCl, 2.5; MgCl2, 1; CaCl2, 2.5; and glucose, 11; oxygenated with 95% O2–5% CO2). The brain was affixed to a vibratome stage with cyanoacrylate and kept in the ice-cold artificial cerebrospinal fluid for slicing. Parasagittal 300 µm-thick slices were prepared and incubated at 33°C for 20 min in oxygenated artificial cerebrospinal fluid. They were then fixed by immersion for 10 min in 4% paraformaldehyde in 0.15 M phosphate buffer, washed with PBS, cryoprotected overnight in 30% sucrose in PBS, and frozen on a flat surface in the cryostat. Parasagittal sections were cut at a thickness of 16 µm, mounted onto gelatin-coated glass slides, air-dried at room temperature for 30 s, and stored at 20°C for at least 1 h. Sections were preincubated for 1 h at room temperature in PBS containing 10% normal goat serum and 0.2% Triton X-100, followed by overnight incubation at 4°C with primary antibodies against tyrosine hydroxylase raised in rabbit (1:5,000; Merck Millipore, Billerica, MA) and against the &agr;3 subunit of the GABAAreceptor raised in guinea pig (1:3,000)9 diluted in the same solution. Sections were then washed extensively in PBS and incubated for 1 h at room temperature in corresponding affinity-purified goat immunoglobulin G coupled to Cy3 (1:500) or Alexa 488 (1:1000; Jackson Immunoresearch). Sections were washed again, and coverslipped with aqueous mounting medium (Dako, Carpinteria, CA). Immunofluorescence staining was visualized by confocal laser scanning microscopy (LSM-510 Meta; Zeiss) with a 40× objective (N.A. 1.3) and sequential acquisition of separate color channels. Image-acquisition settings were adjusted to cover the entire dynamic range of the photomultipliers. The pinhole size was set to 1.0 Airy units for each channel, and stacks of 12 sections (512 × 512) spaced by 0.4 µm were acquired. For display, images were processed with the image-analysis program Imaris (Bitplane, Zurich, Switzerland). Images from both channels were overlaid, background was subtracted, and a low-pass filter (“edge preserving” filter) was applied. Images are displayed as extended projection of the entire stack.
Western Blotting
Membrane preparation and Western blotting have been conducted as described previously.26 Fresh frozen brains from five WT and five global rescue mice (10 weeks of age) were homogenized in 5 ml sucrose buffer (0.32 M sucrose, 5 mM ethylenediaminetetraacetic acid, 10 mM Tris pH 7.5, 0.02% NaN3, phenylmethanesulfonyl fluoride 1:500, complete, mini protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany]) and centrifuged for 10 min at 1,000g  . The supernatant was collected and the step repeated. Both supernatants were combined and centrifuged for 20 min at 40,000g  to obtain the crude membrane fraction. The pellet was resuspended in 3 ml sucrose buffer. Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL). Samples were incubated for 5 min at 95°C with an equal volume of 125 mM Tris-HCl pH 6.8, 20% glycerol, 0.002% bromphenol blue, 10% &bgr;-mercaptoethanol, 4% sodium dodecyl sulfate and stored at −20°C. Before use samples were incubated for 10 min at 60°C and diluted to a concentration of 1 mg/ml. Aliquots with increasing protein content (2.5, 5, 7.5, and 10 &mgr;g) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using 10% minigels (Mini protean II; Bio-Rad, Hercules, CA). Proteins were transferred onto nitrocellulose membranes using a Trans Blot Mini Cell (Bio-Rad). The blots were blocked for 1–2 h in 10 mM Tris-HCl pH 8, 0.15 M NaCl, 0.05% Tween 20 containing 5% nonfat dry milk at room temperature, followed by incubation with affinity-purified antiserum against the &agr;3 subunit of the GABAAreceptor (1:500)9 together with a monoclonal antibody directed against &bgr;-actin (1:20,000, Merck Millipore) overnight at 4°C in 10 mM Tris-HCl pH 8, 0.15 M NaCl, 0.05% Tween 20/5% nonfat dry milk. The blots were washed once with 20 mM Tris pH 7.5, 60 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 0.4% SDS, 0.4% Triton-X 100, and 0.4% deoxycholate and four times with 10 mM Tris-HCl pH 8, 0.15 M NaCl, 0.05% Tween 20. Incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies was carried out for 1 h at room temperature. After extensive washing immunoreactivity was detected by the enhanced chemoluminescence method (Super Signal West Pico chemoluminescence, Pierce), images were captured using a Fujifilm (Tokyo, Japan) LAS-1000 Plus Gel Documentation System, and immunoreactive bands were quantified with the AIDA software (Version 3.25, Raytest, Pforzheim, Germany). Actin immunoreactivity was used to monitor equal sample loading. The specificity of the antibody against the &agr;3 subunit has been confirmed previously using &agr;3KO mice.18 
Drugs
Ketamine-HCl/Xylazine-HCl solution (Sigma-Aldrich) was administered at a volume of 20 ml/kg. Ketamine-HCl (Sigma-Aldrich), medetomidine-HCl solution (Domitor®, Pfizer Animal Health, New York, NY) and pentobarbital-Na (Sigma-Aldrich) were administered at a volume of 10 ml/kg. Etomidate (Amidate®, Hospira, Lake Forest, IL) was administered at a volume of 15 ml/kg. Midazolam-HCl solution (Bedford Laboratories, Bedford, OH) was administered at a volume of 16.5 ml/kg. All drugs were dissolved or diluted in physiological (0.9%) saline where necessary and drug doses were calculated as mg/kg base. All drugs were administered by intraperitoneal injection. The doses administered are reported in the Results section.
Behavioral Testing
Tests were conducted during the dark phase (9:00 AM–9:00 PM) and each test was conducted at approximately the same time of day. The tester was blind to the genotype of the animals. Animals were tested in three cohorts. Cohort 1 containing WT, &agr;3KO, DBH-Rescue, and DBH-iCre mice received a single injection of ketamine/xylazine and anesthetic properties were observed. Cohort 2 containing WT and &agr;3KO mice were first tested for medetomidine-induced sedation in the locomotor activity test, 3 days later for medetomidine-induced hypothermia, and 4–6 days later for anesthetic properties of ketamine, medetomidine, or pentobarbital. Animals received increasing doses of ketamine, medetomidine, or pentobarbital every 7 days. Mice were counterbalanced for drug experience. Cohort 3 containing WT and &agr;3KO mice were first tested for anesthetic properties of midazolam and 7 days later for anesthetic properties of etomidate.
Locomotor Activity
Animals were moved into the testing room at least 1 h before testing. Animals were injected with medetomidine (30 µg/kg or 60 µg/kg) or vehicle immediately before testing. The testing apparatus consisted of four evenly illuminated (approximately 130 lux) clear plexiglas (J. Freeman, Inc., Dorchester, MA) boxes (41 × 41 × 31 cm) separated by white cardboard. Boxes were cleaned with 70% ethanol, between animals. Locomotor activity was recorded for 60 min using the video-tracking system EthoVisionXT (Noldus Information Technology, Wageningen, The Netherlands).
Medetomidine-induced Hypothermia
Animals were moved into the testing room directly before testing. Body temperature was determined using a TH-5 digital thermometer (Physitemp Instruments, Clifton, NJ) with a RET3-Iso thermal probe for mice (inserted 2 cm deep) immediately before drug injection (vehicle, 60 µg/kg, 120 µg/kg medetomidine) and 30, 60, 90, and 120 min thereafter. Mice were kept together in their home cage throughout the experiment.
Anesthetic Endpoints
The assessment of anesthetic end points was adapted from published protocols.27–29 Animals were moved into the testing room directly before testing. Each animal was placed in a clean holding cage without bedding material immediately after drug injection. Body temperature was determined using a TH-5 digital thermometer (Physitemp Instruments) with an RET3-Iso thermal probe for mice (inserted 2 cm deep) immediately before drug injection and every 30 min thereafter. Once the animal stopped moving it was turned on its back. Beginning of loss of righting reflex (LORR) was defined as the time-point when the animal was placed on its back. End of LORR was defined as the time-point when the animal righted itself spontaneously from dorsal to sternal recumbency with all four extremities touching the ground. The time between beginning and end of LORR was defined as duration of LORR. After regaining the righting reflex animals were turned on their back every minute and time to righting was scored: less than 2 s (score = 1), between 2 and 10 s (score = 2), more than 10 s (score = 3). Animals were defined as having fully recovered when they were able to right themselves three times consecutively within 2 s. Time to recovery was defined as the time between beginning of LORR and full recovery. A total LORR score was calculated by adding the score values for every minute from beginning of LORR until full recovery. The loss of hind limb withdrawal reflex (LHWR) was determined every 5 min by pinching the paw firmly with an atraumatic forceps alternating between the left and right hind paw until animals regained the righting reflex. LHWR was defined as present if no signs of reaction to the pinch were observed.
Anesthetic properties of medetomidine were assessed using a different protocol. Animals were moved into the testing room directly before testing and each animal was placed in a clean holding cage without bedding material immediately after drug injection. Animals were gently rolled on their side 30, 60, 90, and 120 min after injection and time to righting defined as completely returning to the prone position with all four extremities touching the ground was noted. LORR was defined as present if animals were not able to roll back to the prone position within 10 s.
Statistical Analysis
Data were analyzed using the appropriate statistical test (Student t  test, ANOVA followed by post hoc  Dunnett t  test, ANOVA with repeated measures followed by post hoc t  tests with Bonferroni correction) or Sheffé post hoc  test. Hypothesis testing was two-tailed. Data for medetomidine-induced LORR were analyzed by fitting standard least-squares mixed models with subject as a random effect. Drug dose and genotype were included as fixed effects. All effects were assessed by F tests based on the fitted regression models. This analysis was followed by post hoc  Mann–Whitney U tests. Data are expressed as the mean ± SEM. P  value less than 0.05 was considered to be statistically significant. Statistical analysis was performed using the SPSS (SPSS, Inc., Chicago, IL) and GraphPad (GraphPad Software, Inc., La Jolla, CA) software packages.
Results
When performing stereotaxic surgeries for implantation of electrodes into the medial forebrain bundle under ketamine/xylazine (139/21 mg/kg intraperitoneally) anesthesia, we unexpectedly observed that using the same doses of anesthetics &agr;3KO mice were anesthetized longer than WT mice (approximately 130 min compared with 80 min), prompting us to investigate the role of &agr;3-containing GABAAreceptors in the action of intravenous general anesthetics.
Anesthetic Endpoints with Etomidate, Midazolam, and Pentobarbital
The anesthetic effects of etomidate, midazolam and pentobarbital, three compounds known to act at least in part via  GABAAreceptors, were assessed by determining the following endpoints: duration of LORR, time to recovery and total LORR score as a measure of hypnosis, LHWR as a measure of immobility, and body temperature as a measure of hypothermic effects of the drug (fig. 1and table 1).
Table 1. Anesthetic Properties (Ketamine, Pentobarbital, Midazolam, Etomidate)
Image not available
Table 1. Anesthetic Properties (Ketamine, Pentobarbital, Midazolam, Etomidate)
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Fig. 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t  tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t  test). (B  ) Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250 mg/kg ketamine, 75 mg/kg midazolam, and 30 mg/kg etomidate. t  Tests with Bonferroni correction revealed that the temperature decrease was more pronounced in &agr;3KO mice compared with WT mice 60 min and 120 min after administration of ketamine and pentobarbital, respectively, and that the temperature decrease was less pronounced in &agr;3KO mice compared with WT mice 90 min after administration of midazolam or etomidate. Data represent the mean ± SEM; *P  < 0.05, **P  < 0.01 and ***P  < 0.001, n = 9 WT and n = 7 &agr;3KO for ketamine; n = 9 for pentobarbital; n = 11 for etomidate and midazolam. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on (A 
	) duration of LORR and (B 
	) core body temperature. (A 
	) Duration of LORR was significantly increased in &agr;3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t 
	tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t 
	test). (B 
	) Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250 mg/kg ketamine, 75 mg/kg midazolam, and 30 mg/kg etomidate. t 
	Tests with Bonferroni correction revealed that the temperature decrease was more pronounced in &agr;3KO mice compared with WT mice 60 min and 120 min after administration of ketamine and pentobarbital, respectively, and that the temperature decrease was less pronounced in &agr;3KO mice compared with WT mice 90 min after administration of midazolam or etomidate. Data represent the mean ± SEM; *P 
	< 0.05, **P 
	< 0.01 and ***P 
	< 0.001, n = 9 WT and n = 7 &agr;3KO for ketamine; n = 9 for pentobarbital; n = 11 for etomidate and midazolam. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t  tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t  test). (B  ) Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250 mg/kg ketamine, 75 mg/kg midazolam, and 30 mg/kg etomidate. t  Tests with Bonferroni correction revealed that the temperature decrease was more pronounced in &agr;3KO mice compared with WT mice 60 min and 120 min after administration of ketamine and pentobarbital, respectively, and that the temperature decrease was less pronounced in &agr;3KO mice compared with WT mice 90 min after administration of midazolam or etomidate. Data represent the mean ± SEM; *P  < 0.05, **P  < 0.01 and ***P  < 0.001, n = 9 WT and n = 7 &agr;3KO for ketamine; n = 9 for pentobarbital; n = 11 for etomidate and midazolam. KO = knockout; LORR = loss of righting reflex; WT = wild type.
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Etomidate
Etomidate (30 mg/kg) was less effective in &agr;3KO mice compared with WT mice. Duration of LORR (WT: 88.3 ± 4.3 min, &agr;3KO: 72.7 ± 5.8 min) and total LORR score (WT: 273.7 ± 10.3, &agr;3KO: 224.9 ± 17.5) were significantly decreased in &agr;3KO mice (unpaired t  test, P  = 0.0439 and P  = 0.0263, respectively), whereas time to recovery was not different (WT: 91.1 ± 4.5 min, &agr;3KO: 77.2 ± 5.8 min, unpaired t  test, P  = 0.0710). Anesthetic-induced decrease in body temperature was smaller in &agr;3KO mice compared with WT controls (two-way ANOVA with repeated measures of time: significant effect of time F(2,40), P  < 0.0001; significant effect of genotype F(1,20) = 4.90, P  = 0.0386; significant time × genotype interaction F(2,40) = 8.12, P  = 0.0011). Post hoc t  tests with Bonferroni correction showed that the temperature differed significantly 90 min after injection (P  < 0.01).
Midazolam
The hypnotic action of midazolam (75 mg/kg) was decreased in &agr;3KO mice compared with WT mice. Although the difference in the duration of LORR failed to reach significance (unpaired t  test, P  = 0.0691), the time to recovery (WT: 101.1 ± 7.5 min, &agr;3KO: 53.8 ± 9.9 min) and the total LORR score (WT: 298.8 ± 23.6, &agr;3KO: 159.4 ± 27.3) were significantly lower in &agr;3KO mice (unpaired t  test, P  = 0.0011 and P  = 0.001, respectively). Similarly, the reduction in body temperature was smaller in &agr;3KO mice (two-way ANOVA with repeated measures of time: significant effect of time F(2,40) = 17.65, P  < 0.0001; significant effect of genotype F(1,20), P  = 0.0395 and significant time × genotype interaction F(2,40), P  = 0.0008). Post hoc t  tests with Bonferroni correction showed that the temperature difference was significant 90 min after injection (P  < 0.01).
Pentobarbital
For pentobarbital two-way ANOVAs using drug dose and genotype as factors revealed main effects of drug and genotype on LORR (drug F(2,32) = 160.92, P  < 0.0001; genotype F(1,16) = 5.64, P  = 0.304), on time to recovery (drug F(2,32) = 163.15, P  < 0.0001; genotype F(1,16) = 5.51, P  = 0.0321) and on total LORR score (drug F(2,32) = 158.06, P  < 0.0001, genotype F(1,16) = 5.63, P  = 0.305). Furthermore, four out of nine &agr;3KO mice lost the hind limb withdrawal reflex at the highest dose of pentobarbital (60 mg/kg), whereas none of the WT mice lost the reflex. This difference was not statistically significant (Fisher’s exact probability test: P  = 0.0824). At the highest dose of pentobarbital (60 mg/kg), two-way ANOVA with repeated measures of time showed a significant effect of time F(3,48) = 27.19, P  < 0.0001 but no effect of genotype F(1,16) = 3.60, P  = 0.0761). However, with respect to body temperature, there was a significant time x genotype interaction (F(3,48) = 5.56, P  = 0.0023) and t  tests with Bonferroni correction showed that body temperature was significantly lower in &agr;3KO mice 120 min after injection (P  < 0.001).
Anesthetic Endpoints with Ketamine and Medetomidine
Ketamine
There were no differences in duration of LORR, time to recovery, total LORR score or LHWR between WT and &agr;3KO mice at any dose of ketamine, although two &agr;3KO mice died after administration of 150 and 250 mg/kg, respectively. However, the reduction in body temperature was significantly larger in &agr;3KO mice compared with WT mice by 250 mg/kg ketamine (ANOVA with repeated measures of time: significant effect of time F(1,14) = 7.99 P  = 0.0134; significant effect of genotype F(1,14) = 7.99 P  = 0.025). Post hoc t  tests with Bonferroni correction showed that body temperature was significantly different 60 min after injection (P  < 0.05).
Medetomidine
Sedative Effects of Medetomidine
The sedative effects of the &agr;2-adrenergic agonist medetomidine in &agr;3KO and WT mice were assessed by measuring the locomotor activity in a novel open field over 60 min following drug injection. The total distance traveled was decreased by medetomidine (30 or 60 µg/kg) (fig. 2A) but there were no differences between &agr;3KO and WT mice (two-way ANOVA: significant effect of drug (F(2,48) = 32.25, P  < 0.0001) (insert in fig. 2A). Analysis with two-way ANOVA with repeated measures of 5-min time bins also did not reveal any difference between genotypes, although a general reduction in activity over time was observed in both genotypes (Vehicle: significant effect of time F(11,176) = 32.41, P  < 0.0001; 30 µg/kg: significant effect of time F(11,176) = 41.85, P  < 0.0001; 60 µg/kg: significant effect of time F(11,176) = 47.54, P  < 0.0001) (fig. 2A).
Fig. 2. Effects of medetomidine on locomotor activity (A  ), duration of LORR (B  ) and body temperature (C  ) in &agr;3KO and wt mice. (A  ) Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 min time period (insert) and looking at 5-min time bins. (B  ) Duration of LORR was significantly increased in &agr;3KO mice by 250 µg/kg medetomidine compared with WT mice (Sheffé post hoc  tests). (C  ) Body temperature was comparable between &agr;3KO mice and WT mice. Data represent the mean ± SEM; n = 9 per genotype. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 2. Effects of medetomidine on locomotor activity (A 
	), duration of LORR (B 
	) and body temperature (C 
	) in &agr;3KO and wt mice. (A 
	) Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 min time period (insert) and looking at 5-min time bins. (B 
	) Duration of LORR was significantly increased in &agr;3KO mice by 250 µg/kg medetomidine compared with WT mice (Sheffé post hoc 
	tests). (C 
	) Body temperature was comparable between &agr;3KO mice and WT mice. Data represent the mean ± SEM; n = 9 per genotype. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 2. Effects of medetomidine on locomotor activity (A  ), duration of LORR (B  ) and body temperature (C  ) in &agr;3KO and wt mice. (A  ) Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 min time period (insert) and looking at 5-min time bins. (B  ) Duration of LORR was significantly increased in &agr;3KO mice by 250 µg/kg medetomidine compared with WT mice (Sheffé post hoc  tests). (C  ) Body temperature was comparable between &agr;3KO mice and WT mice. Data represent the mean ± SEM; n = 9 per genotype. KO = knockout; LORR = loss of righting reflex; WT = wild type.
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Hypnotic Effects of Medetomidine
The hypnotic effects of medetomidine in &agr;3KO and WT mice were assessed 60 min after injection by rolling the mice on their side and noting the time to righting (fig. 2B). When righting did not occur within 30 min the observation was stopped. Data were analyzed by fitting standard least-squares mixed models with subject as a random effect. Drug dose and genotype were included as fixed effects. Effects were assessed by F tests based on the fitted regression models. This revealed a significant overall effect of genotype (F[1,16] = 8.90, P  = 0.0088) and dose (F[1,34] = 36.96, P  < 0.0001). Post hoc  Sheffé tests showed that the LORR was significantly increased in &agr;3KO mice (298 ± 192 s) compared with WT mice (6 ± 4 s) by 250 µg/kg medetomidine (P  = 0.0098). At this dose, two of nine WT mice lost the righting reflex (loss of righting reflex defined as inability to right within 10 s) in contrast to seven of nine &agr;3KO mice.
Hypothermic Effects of Medetomidine
The hypothermic effects of medetomidine in &agr;3KO and WT mice were assessed by determining the rectal body temperature immediately before drug or vehicle injection and every 30 min thereafter over a period of 120 min (fig. 2C).
The baseline body temperature before injection was increased in &agr;3KO mice compared with WT mice in the groups that received vehicle (WT = 38.29 ± 0.11°C, &agr;3KO = 39.01 ± 0.18°C, unpaired t  test, P  < 0.01) and 60 µg/kg medetomidine (WT = 38.297 ± 0.15°C, &agr;3KO = 39.13 ± 0.17°C, unpaired t  test, P  < 0.01) in &agr;3KO mice (39.0 ± 0.2°C), but not in the group that received 120 µg/kg medetomidine (WT = 38.82 ± 0.14°C, &agr;3KO = 38.97 ± 0.09°C, independent samples t  test, P  = 0.398). This precluded direct statistical analysis of the hypothermic effects of 60 µg/kg medetomidine. For the 120 µg/kg medetomidine dose, a genotype × time two-way ANOVA did not reveal a significant genotype main effect or genotype × time interaction. In the entire study, a difference in baseline temperature was only present in 2 of the 11 experiments performed in total with midazolam, pentobarbital, and ketamine. We therefore assume that extraneous undefined factors specific to the experiments with medetomidine are responsible for this baseline difference, and that overall WT mice and &agr;3KO mice do not display a difference in baseline body temperature. When we analyzed the change in body temperature from baseline, we still did not obtain any significant differences between genotypes both for 60 µg/kg and for 120 µg/kg medetomidine (not shown).
Generation of Mice Expressing the GABAAReceptor &agr;3 Subunit Exclusively in Noradrenergic Neurons
&agr;3KO mice used in this study have been generated using an insertion targeting strategy (fig. 1in reference 18) with an insertion-type targeting vector containing exon 5 of the Gabra  3 gene flanked by two lox  P sites. This vector was inserted into the target locus in its entirety by single reciprocal recombination, resulting in a duplication of exon 5 (i.e.  , exon 4 in fig. 1in the study by Yee et al.  ).18 The resulting &agr;3KO mice carry both an endogenous exon 5 and an artificial exon 5* (5* in fig. 3A). This artificial exon 5* provides a “STOP” signal functionally disrupting the Gabra  3 gene (fig. 3, Aand B). We predicted that a cre-lox  P-mediated excision of this lox  P-flanked artificial exon 5* would functionally restore the Gabra  3 gene. In a proof-of-principle experiment, &agr;3KO mice were crossed with EIIa-cre mice, which express the cre transgene at early embryonic stages19 (fig. 4A). As predicted, cre-lox  P-mediated recombination resulted in the elimination of the lox  P-flanked artificial exon 5*. Subsequently, the Ella-Cre transgene was bred out to ensure that the rescue of the &agr;3 subunit had been present in the germline. These global rescue mice were examined for &agr;3 subunit expression. Immunohistochemical analysis revealed that &agr;3 subunit expression is restored in these global rescue mice and the distribution pattern of &agr;3 subunit expression is indistinguishable from WT mice (fig. 4B). Western blot analysis showed that &agr;3 subunit expression levels in global rescue mice are 70 ± 7% compared with WT mice (fig. 4C). Thus, the genomic rearrangement in the rescue allele, which includes the presence of a plasmid backbone, reduces the expression of the &agr;3 subunit by approximately 30% without affecting its regional distribution. The precise mechanisms of this reduction are unknown.
Fig. 3. Schematic of genetic rescue of the &agr;3KO in noradrenergic neurons. (A  ) &agr;3KO and rescue alleles. The &agr;3KO allele (top  ) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety.18 It carries an artificial exon (light green  , 5*) flanked by lox  P sites (red  ), targeting vector-derived homologous genomic sequences (light brown  ), and the plasmid backbone of the targeting vector (blue  ). Genomic sequences that were not included in the targeting vector are shown as dark brown  . The most relevant feature is that the &agr;3KO allele contains the endogenous exon 5 (dark green  , 5) and an artificial exon 5 (light green  , 5*), which results in mRNA degradation, which is the likely mechanism of the &agr;3KO.18 In the rescue allele, elimination of the artificial exon by Cre-lox  P-mediated recombination results in &agr;3 mRNA (orange  ) being made and thus the expression of the &agr;3 subunit being restored. (B  ) Exonic structure of predicted gene products from WT, &agr;3KO, and rescue allele. Presence of the artificial exon 5, 5*, in the &agr;3KO results in mRNA breakdown and thus a functional KO. The exonic structure of WT and rescue alleles is identical. KO = knockout; mRNA = messenger RNA; WT = wild type.
Fig. 3. Schematic of genetic rescue of the &agr;3KO in noradrenergic neurons. (A 
	) &agr;3KO and rescue alleles. The &agr;3KO allele (top 
	) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety.18It carries an artificial exon (light green 
	, 5*) flanked by lox 
	P sites (red 
	), targeting vector-derived homologous genomic sequences (light brown 
	), and the plasmid backbone of the targeting vector (blue 
	). Genomic sequences that were not included in the targeting vector are shown as dark brown 
	. The most relevant feature is that the &agr;3KO allele contains the endogenous exon 5 (dark green 
	, 5) and an artificial exon 5 (light green 
	, 5*), which results in mRNA degradation, which is the likely mechanism of the &agr;3KO.18In the rescue allele, elimination of the artificial exon by Cre-lox 
	P-mediated recombination results in &agr;3 mRNA (orange 
	) being made and thus the expression of the &agr;3 subunit being restored. (B 
	) Exonic structure of predicted gene products from WT, &agr;3KO, and rescue allele. Presence of the artificial exon 5, 5*, in the &agr;3KO results in mRNA breakdown and thus a functional KO. The exonic structure of WT and rescue alleles is identical. KO = knockout; mRNA = messenger RNA; WT = wild type.
Fig. 3. Schematic of genetic rescue of the &agr;3KO in noradrenergic neurons. (A  ) &agr;3KO and rescue alleles. The &agr;3KO allele (top  ) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety.18 It carries an artificial exon (light green  , 5*) flanked by lox  P sites (red  ), targeting vector-derived homologous genomic sequences (light brown  ), and the plasmid backbone of the targeting vector (blue  ). Genomic sequences that were not included in the targeting vector are shown as dark brown  . The most relevant feature is that the &agr;3KO allele contains the endogenous exon 5 (dark green  , 5) and an artificial exon 5 (light green  , 5*), which results in mRNA degradation, which is the likely mechanism of the &agr;3KO.18 In the rescue allele, elimination of the artificial exon by Cre-lox  P-mediated recombination results in &agr;3 mRNA (orange  ) being made and thus the expression of the &agr;3 subunit being restored. (B  ) Exonic structure of predicted gene products from WT, &agr;3KO, and rescue allele. Presence of the artificial exon 5, 5*, in the &agr;3KO results in mRNA breakdown and thus a functional KO. The exonic structure of WT and rescue alleles is identical. KO = knockout; mRNA = messenger RNA; WT = wild type.
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Fig. 4. Localization and quantification of the &agr;3KO in global rescue mice. (A  ) Generation of global rescue mice. An &agr;3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse.19 Some of the offspring will carry both the &agr;3KO allele and the EIIa-Cre transgene. This mouse is bred with a WT mouse to breed out the EIIa-Cre transgene (depicted by 2 sequential horizontal arrows  ) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the &agr;3 subunit is expected to be expressed in all cells in which it is naturally expressed. (B  ) Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: &agr;3 subunit distribution pattern is equivalent in WT and global rescue mice. Representative sections from two WT and two global rescue mice are shown. (C  ) Left panel: Representative Western Blot: &agr;3 subunit expression level is decreased in global rescue mice compared with WT mice. Right panel: Quantification of Western blot signal: signals were normalized to the &agr;3 subunit signal at 10 &mgr;g protein in WT mice (100%). &agr;3 subunit expression level in global rescue mice is 70 ± 7% of WT expression. Data represent the mean ± SEM of five experiments. KO = knockout; WT = wild type.
Fig. 4. Localization and quantification of the &agr;3KO in global rescue mice. (A 
	) Generation of global rescue mice. An &agr;3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse.19Some of the offspring will carry both the &agr;3KO allele and the EIIa-Cre transgene. This mouse is bred with a WT mouse to breed out the EIIa-Cre transgene (depicted by 2 sequential horizontal arrows 
	) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the &agr;3 subunit is expected to be expressed in all cells in which it is naturally expressed. (B 
	) Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: &agr;3 subunit distribution pattern is equivalent in WT and global rescue mice. Representative sections from two WT and two global rescue mice are shown. (C 
	) Left panel: Representative Western Blot: &agr;3 subunit expression level is decreased in global rescue mice compared with WT mice. Right panel: Quantification of Western blot signal: signals were normalized to the &agr;3 subunit signal at 10 &mgr;g protein in WT mice (100%). &agr;3 subunit expression level in global rescue mice is 70 ± 7% of WT expression. Data represent the mean ± SEM of five experiments. KO = knockout; WT = wild type.
Fig. 4. Localization and quantification of the &agr;3KO in global rescue mice. (A  ) Generation of global rescue mice. An &agr;3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse.19 Some of the offspring will carry both the &agr;3KO allele and the EIIa-Cre transgene. This mouse is bred with a WT mouse to breed out the EIIa-Cre transgene (depicted by 2 sequential horizontal arrows  ) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the &agr;3 subunit is expected to be expressed in all cells in which it is naturally expressed. (B  ) Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: &agr;3 subunit distribution pattern is equivalent in WT and global rescue mice. Representative sections from two WT and two global rescue mice are shown. (C  ) Left panel: Representative Western Blot: &agr;3 subunit expression level is decreased in global rescue mice compared with WT mice. Right panel: Quantification of Western blot signal: signals were normalized to the &agr;3 subunit signal at 10 &mgr;g protein in WT mice (100%). &agr;3 subunit expression level in global rescue mice is 70 ± 7% of WT expression. Data represent the mean ± SEM of five experiments. KO = knockout; WT = wild type.
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In order to generate mice expressing the &agr;3 subunit exclusively in noradrenergic neurons, mice carrying the &agr;3 knockout allele and a DBH-iCre transgene (DBH-iCre) were generated (fig. 5A). In addition, mice carrying the &agr;3 KO allele and a DAT-iCre transgene (DAT-iCre) were generated to demonstrate the specificity of the rescue approach.
Fig. 5. Recue of &agr;3 expression in defined neuronal cell types. (A  ) An &agr;3KO mouse is crossed with a DBH-iCre mouse to obtain DBH-iCre Rescue mice (in our studies hemizygous males) containing both the &agr;3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the &agr;3 KO allele functions as a “STOP” signal, resulting in messenger RNA degradation.18 In noradrenergic neurons of the Rescue mouse, this “STOP” signal is removed by cre-lox  P-mediated recombination. Thus, in the Rescue mice, the &agr;3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the DAT-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. (B  ) Neuron-specific rescue of &agr;3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that the &agr;3 subunit is highly expressed in the LC in WT mice. In &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) &agr;3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of &agr;3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. (C  ) Neuron-specific rescue of &agr;3 subunit expression in the SNpc: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that &agr;3 subunit expression is found in dopaminergic neurons of the SNpc of WT mice and in &agr;3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the SNpc of &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. DBH-iCre = dopamine &bgr; hydroxylase impoved Cre; DAT = dopamine transporter; WT = wild type; LC, locus coeruleus; SNpc = substantia nigra pars compacta.
Fig. 5. Recue of &agr;3 expression in defined neuronal cell types. (A 
	) An &agr;3KO mouse is crossed with a DBH-iCre mouse to obtain DBH-iCre Rescue mice (in our studies hemizygous males) containing both the &agr;3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the &agr;3 KO allele functions as a “STOP” signal, resulting in messenger RNA degradation.18In noradrenergic neurons of the Rescue mouse, this “STOP” signal is removed by cre-lox 
	P-mediated recombination. Thus, in the Rescue mice, the &agr;3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the DAT-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. (B 
	) Neuron-specific rescue of &agr;3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the &agr;3 subunit (red 
	) and tyrosine hydroxylase (green 
	) shows that the &agr;3 subunit is highly expressed in the LC in WT mice. In &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) &agr;3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of &agr;3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. (C 
	) Neuron-specific rescue of &agr;3 subunit expression in the SNpc: Immunofluorescence double-labeling of the &agr;3 subunit (red 
	) and tyrosine hydroxylase (green 
	) shows that &agr;3 subunit expression is found in dopaminergic neurons of the SNpc of WT mice and in &agr;3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the SNpc of &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. DBH-iCre = dopamine &bgr; hydroxylase impoved Cre; DAT = dopamine transporter; WT = wild type; LC, locus coeruleus; SNpc = substantia nigra pars compacta.
Fig. 5. Recue of &agr;3 expression in defined neuronal cell types. (A  ) An &agr;3KO mouse is crossed with a DBH-iCre mouse to obtain DBH-iCre Rescue mice (in our studies hemizygous males) containing both the &agr;3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the &agr;3 KO allele functions as a “STOP” signal, resulting in messenger RNA degradation.18 In noradrenergic neurons of the Rescue mouse, this “STOP” signal is removed by cre-lox  P-mediated recombination. Thus, in the Rescue mice, the &agr;3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the DAT-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. (B  ) Neuron-specific rescue of &agr;3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that the &agr;3 subunit is highly expressed in the LC in WT mice. In &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) &agr;3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of &agr;3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. (C  ) Neuron-specific rescue of &agr;3 subunit expression in the SNpc: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that &agr;3 subunit expression is found in dopaminergic neurons of the SNpc of WT mice and in &agr;3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the SNpc of &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. DBH-iCre = dopamine &bgr; hydroxylase impoved Cre; DAT = dopamine transporter; WT = wild type; LC, locus coeruleus; SNpc = substantia nigra pars compacta.
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Immunofluorescence analysis confirmed that the &agr;3 subunit is exclusively expressed in noradrenergic neurons in &agr;3KO/DBH-iCre (DBH-Rescue) mice (fig. 5B), whereas it is exclusively expressed in dopaminergic neurons in &agr;3KO/DAT-iCre (DAT-Rescue) mice (fig. 5C). WT mice show intense expression of the &agr;3 subunit on noradrenergic neurons of the locus coeruleus and on unidentified neurons in the proximity, whereas DBH-Rescue mice express the &agr;3 subunit only on noradrenergic neurons (fig. 5B). &agr;3 subunit expression is not detectable in &agr;3KO mice and DAT-Rescue mice in this brain region (fig. 5B). To confirm the specificity of the rescue approach &agr;3 subunit expression in DAT-Rescue mice was examined further. In the dopaminergic neurons of the substantia nigra pars compacta the &agr;3 subunit protein is absent in &agr;3KO and DBH-Rescue mice, whereas high levels of &agr;3 subunit expression are found in WT mice, and expression is specifically restored in dopaminergic neurons in DAT-Rescue mice (fig. 5C). In other brain regions where the &agr;3 subunit is normally expressed, such as the cerebral cortex or the hippocampus but where no noradrenergic or dopaminergic neurons are present, no &agr;3 subunit protein was detectable in DBH- or DAT-rescue mice (not shown). Overall, these data demonstrate that the &agr;3 subunit can be rescued specifically and to a large extent in noradrenergic neurons.
Anesthetic Endpoints with Ketamine/Xylazine and Rescue of the Phenotype in Mice Expressing the GABAAReceptor &agr;3 Subunit Exclusively in Noradrenergic Neurons
In order to confirm the unexpected observation that ketamine/xylazine-induced anesthesia is significantly prolonged in &agr;3KO mice and to address the question whether &agr;3 subunit–containing GABAAreceptors on noradrenergic neurons modulate these anesthetic effects, we studied several anesthetic endpoints and the body temperature decrease during anesthesia in &agr;3KO mice, WT mice, and DBH-Rescue mice (fig. 6and table 2). To exclude the possibility that the iCre transgene, which is also present in the DBH-Rescue mice, causes any effects by itself (also referred to as potential “Cre toxicity”), control mice expressing the iCre transgene in noradrenergic neurons (DBH-iCre) were included.
Table 2. Anesthetic Endpoints with Ketamine (139 mg/kg)/ Xylazine (21 mg/kg)
Image not available
Table 2. Anesthetic Endpoints with Ketamine (139 mg/kg)/ Xylazine (21 mg/kg)
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Fig. 6. Effect of ketamine (139 mg/kg)/xylazine (21 mg/kg) on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice compared with WT mice, whereas mice expressing the &agr;3 subunit exclusively in noradrenergic neurons (DBH-Rescue) and mice carrying the iCre transgene (DBH-iCre) did not differ from WT mice (one-way ANOVA followed by post hoc  Dunnett t  tests). (B  ) Body temperature was significantly lower in &agr;3KO mice compared with WT mice 150 min and 180 min after injection. Body temperature in DBH-Rescue mice was significantly lower 180 min after injection compared with WT mice (two-way repeated measures ANOVA followed by post hoc t  tests with Bonferroni correction). Data represent the mean ± SEM; ***P  < 0.001 (&agr;3KO compared with WT), *P  < 0.05 (&agr;3KO compared with WT), #P  < 0.05 (DBH-Rescue compared with WT); n = 15 WT and DBH-iCre, n = 14 &agr;3KO and DBH-Rescue. LORR = loss of righting reflex; WT = wild type; DBH = dopamine &bgr; hydroxylase; iCre = improved cre.
Fig. 6. Effect of ketamine (139 mg/kg)/xylazine (21 mg/kg) on (A 
	) duration of LORR and (B 
	) core body temperature. (A 
	) Duration of LORR was significantly increased in &agr;3KO mice compared with WT mice, whereas mice expressing the &agr;3 subunit exclusively in noradrenergic neurons (DBH-Rescue) and mice carrying the iCre transgene (DBH-iCre) did not differ from WT mice (one-way ANOVA followed by post hoc 
	Dunnett t 
	tests). (B 
	) Body temperature was significantly lower in &agr;3KO mice compared with WT mice 150 min and 180 min after injection. Body temperature in DBH-Rescue mice was significantly lower 180 min after injection compared with WT mice (two-way repeated measures ANOVA followed by post hoc t 
	tests with Bonferroni correction). Data represent the mean ± SEM; ***P 
	< 0.001 (&agr;3KO compared with WT), *P 
	< 0.05 (&agr;3KO compared with WT), #P 
	< 0.05 (DBH-Rescue compared with WT); n = 15 WT and DBH-iCre, n = 14 &agr;3KO and DBH-Rescue. LORR = loss of righting reflex; WT = wild type; DBH = dopamine &bgr; hydroxylase; iCre = improved cre.
Fig. 6. Effect of ketamine (139 mg/kg)/xylazine (21 mg/kg) on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice compared with WT mice, whereas mice expressing the &agr;3 subunit exclusively in noradrenergic neurons (DBH-Rescue) and mice carrying the iCre transgene (DBH-iCre) did not differ from WT mice (one-way ANOVA followed by post hoc  Dunnett t  tests). (B  ) Body temperature was significantly lower in &agr;3KO mice compared with WT mice 150 min and 180 min after injection. Body temperature in DBH-Rescue mice was significantly lower 180 min after injection compared with WT mice (two-way repeated measures ANOVA followed by post hoc t  tests with Bonferroni correction). Data represent the mean ± SEM; ***P  < 0.001 (&agr;3KO compared with WT), *P  < 0.05 (&agr;3KO compared with WT), #P  < 0.05 (DBH-Rescue compared with WT); n = 15 WT and DBH-iCre, n = 14 &agr;3KO and DBH-Rescue. LORR = loss of righting reflex; WT = wild type; DBH = dopamine &bgr; hydroxylase; iCre = improved cre.
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One-way ANOVA showed a significant effect of genotype for all anesthetic parameters: duration of LORR (F[3,54] = 8.81, P  < 0.0001), time to recovery (F[3,54] = 9.52, P  < 0.0001), total LORR score (F[3,54] = 9.51, P  < 0.0001) and LHWR (F[3,54] = 10.76, P  < 0.0001) (table 2). Post hoc  Dunnett tests revealed that &agr;3KO mice show significantly increased measures on all anesthetic parameters (LORR, total LORR score, LHWR, P  < 0.001 and time to recovery P  < 0.01) compared with WT mice. In contrast, DBH-Rescue mice showed an increase only in the time to recovery and the total LORR score (P  < 0.05) compared with WT mice; they were not different from WTs in terms of LORR or LHWR times. In fact, the rescue mice were not different from either WT or &agr;3KO mice on these parameters, suggesting that they rank in between the two genotypes, leading to a “partial rescue” of the &agr;3KO phenotype. The body temperature decrease caused by ketamine/xylazine was analyzed by a two-way repeated measures ANOVA. A significant effect of genotype (F[3,54] = 2.85, P  = 0.0456), significant effect of time (F[5,270] = 475.04, P  < 0.0001) and a significant time × genotype interaction (F[15,270] = 5.69, P  < 0.0001) were observed. Post hoc t  tests with Bonferroni correction showed that the temperature was significantly lower in &agr;3KO mice 150 min (P  < 0.05) and 180 min (P  < 0.001) after injection compared with WT mice. DBH-Rescue mice showed a significantly lower temperature only 180 min (P  < 0.05) after injection compared with WT mice. DBH-iCre mice did not differ from WT mice in any anesthetic endpoint or in the body temperature, indicating that the iCre transgene itself has no effect on the parameters measured. Overall, our results indicate that the hypnotic phenotype present in &agr;3KO mice can be partially rescued by expression of the &agr;3 subunit in noradrenergic neurons in DBH-Rescue mice, whereas the hypothermic phenotype seems to be independent of the &agr;3 subunit in noradrenergic neurons.
Discussion
Clinically used intravenous general anesthetics are a structurally highly diverse class of drugs mediating their anesthetic effects through a variety of molecular targets.12 In this study, we evaluated the role of &agr;3-containing GABAAreceptors in the actions of intravenous anesthetics and found that although actions of GABAergic drugs like etomidate and midazolam are in part mediated by this receptor subtype, &agr;3-containing GABAAreceptors also constrain the actions of anesthetics that have partially (e.g.  , pentobarbital) or exclusively (ketamine/xylazine, medetomidine) non-GABAergic targets. We also demonstrate that &agr;3-containing GABAAreceptors on noradrenergic neurons may constrain the hypnotic but not the hypothermic action of ketamine/xylazine action.
Studies using mice carrying &bgr;2(N265S) or &bgr;3(N265M) point mutations rendering the mutated subunit insensitive to etomidate or etomidate and propofol, respectively, have revealed that GABAAreceptors containing the &bgr;2 or &bgr;3 subunits mediate the immobilizing (&bgr;3), hypnotic (&bgr;2 and &bgr;3), and hypothermic actions (mainly &bgr;2, minor role of &bgr;3 shown for etomidate and pentobarbital, but not for propofol) of etomidate, propofol, and pentobarbital.30–32 In line with previous reports showing that mice lacking the &bgr;3 subunit33 or the &agr;1 subunit6 are less sensitive to the hypnotic effects of midazolam and etomidate, we found that &agr;3KO mice are likewise less sensitive to these agents, collectively indicating that the &agr;1, &agr;3, and &bgr;3 subunits are involved in mediating the hypnotic response to GABAergic anesthetics. In contrast, &agr;3KO mice showed an increase in the sensitivity to the hypnotic, immobilizing, and hypothermic effects of pentobarbital. This is astonishing because GABAAreceptors are a major target for pentobarbital.34 Consistent with this assumption &agr;1KO mice6 and &bgr;3(N265M) point-mutant mice32 are less sensitive to the hypnotic effects of pentobarbital; moreover, the immobilizing response to pentobarbital is completely absent in &bgr;3(N265M) point-mutant mice.32 However, the GABAAreceptor antagonist gabazine attenuates the hypnotic response to pentobarbital to a much lesser extent than the hypnotic response to muscimol and propofol, pointing to the potential relevance of non-GABAAreceptor targets for pentobarbital actions.35 Furthermore, in contrast to etomidate some effects of pentobarbital such as respiratory depression are similar in WT and &bgr;3(N265M) point-mutant mice.32 Together with the fact that pentobarbital additionally acts on other drug targets such as nicotinic acetylcholine receptors, &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, kainate receptors, and glycine receptors36 it seems likely that at least some of the anesthetic effects of pentobarbital are mediated through non-GABAergic mechanisms. It is therefore possible that the increased sensitivity to pentobarbital in &agr;3KO mice results from alterations in non-GABAergic pathways such as the cholinergic system where &agr;3 subunit expression has been described37 or potentially from up-regulation of non-GABAergic targets of pentobarbital or other compensatory mechanisms.
On the basis of the initial observation that ketamine/xylazine anesthesia is prolonged in &agr;3KO mice and actions of such compounds would normally be expected to be unaltered in a GABAAreceptor subunit KO mouse, investigating the contribution of N  -methyl-D-aspartate receptor antagonism and of &agr;2-adrenergic receptor agonism to this phenomenon was a major goal in this study. We therefore examined the action of ketamine and of the &agr;2-adrenergic agonist medetomidine. Medetomidine was chosen for two reasons: (1) Essential pharmacologically relevant data, for example, with respect to binding to &agr;2-adrenergic receptor subtypes were not available for xylazine, and (2) there is a significant body of experimental studies on the sedative, hypnotic, and hypothermic actions with medetomidine.38 Strikingly, our studies revealed a clear dissociation between the sedative, hypothermic, and hypnotic effects induced by medetomidine. Although the sedative and hypothermic effects were indistinguishable between WT and &agr;3KO mice, &agr;3KO mice were more sensitive to the hypnotic effects of medetomidine. However, it is interesting to note that the unspecific blockade of all GABAAreceptor subtypes by the antagonist gabazine attenuates the hypnotic effects of medetomidine.16 This indicates that the hypnotic response to medetomidine may be modulated in different directions by different GABAAreceptor subtypes. Interestingly, &agr;3KO mice did not differ from WT mice in their hypnotic response to ketamine. However, the decrease in body temperature was more pronounced. Even though ketamine does not typically exhibit synergistic effects in combination with other anesthetic agents,39 our findings suggest that the increased sensitivity to the hypnotic effects of &agr;2-adrenergic agents combined with the increased sensitivity to the hypothermic effects of ketamine might cause the significant phenotype observed in &agr;3KO mice in response to the drug combination ketamine/xylazine in a synergistic manner.
Another objective of this study was to evaluate whether &agr;3-containing GABAAreceptors on noradrenergic neurons constrain ketamine/xylazine action. For this purpose, we generated “rescue” mice expressing the &agr;3 subunit exclusively in noradrenergic neurons. In these mice, the &agr;3 subunit is expressed under the control of its endogenous promoter, thus limiting expression to cells in which expression occurs naturally. Compared with the WT allele the expression level of the rescue allele was approximately 70%. We assume that transcriptional processes may be partially disrupted due to the presence of foreign DNA (i.e.  , plasmid backbone of the targeting vector) in the allele. Presumably &agr;3 expression levels in noradrenergic neurons will not reach WT expression levels in the &agr;3-DBH-Rescue mice either, although this has not been formally examined. As a consequence a rescue of the &agr;3KO phenotype may not restore function to WT levels. Indeed, this is what we observed in this study: the presence of &agr;3-containing GABAAreceptors on noradrenergic neurons partially abolishes the increased hypnotic and immobilizing actions of ketamine/xylazine observed in the &agr;3KO mice. The increased hypothermic actions, however, were not affected. This indicates that although noradrenergic neurons are involved in modulating the hypnotic and immobilizing actions of ketamine/xylazine, they may not be involved in modulating their hypothermic action.
Our findings are consistent with a model in which the noradrenergic neurons naturally express both &agr;2-adrenergic receptors and &agr;3-containing GABAAreceptors.11 Activation of both receptors would be predicted to decrease the activity of the noradrenergic neurons. Similar to previous findings in the reticular nucleus of the thalamus,40 currently unexplained compensations to preserve inhibitory function may be present in &agr;3KO mice. At the same time, increased sensitivity to &agr;2-adrenergic agonists might occur in &agr;3KO mice to compensate for the lack of GABAergic inhibition and designed to keep the inhibitory modulation of these neurons constant. This would explain why the hypnotic and hypothermic actions of compounds such as midazolam and etomidate, which act mainly or exclusively via  GABAAreceptors without any effect on &agr;2-adrenergic receptors, are reduced in the &agr;3KO mice, whereas the hypnotic action of medetomidine, which has no effect on GABAAreceptors, is increased.
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Fig. 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t  tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t  test). (B  ) Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250 mg/kg ketamine, 75 mg/kg midazolam, and 30 mg/kg etomidate. t  Tests with Bonferroni correction revealed that the temperature decrease was more pronounced in &agr;3KO mice compared with WT mice 60 min and 120 min after administration of ketamine and pentobarbital, respectively, and that the temperature decrease was less pronounced in &agr;3KO mice compared with WT mice 90 min after administration of midazolam or etomidate. Data represent the mean ± SEM; *P  < 0.05, **P  < 0.01 and ***P  < 0.001, n = 9 WT and n = 7 &agr;3KO for ketamine; n = 9 for pentobarbital; n = 11 for etomidate and midazolam. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on (A 
	) duration of LORR and (B 
	) core body temperature. (A 
	) Duration of LORR was significantly increased in &agr;3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t 
	tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t 
	test). (B 
	) Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250 mg/kg ketamine, 75 mg/kg midazolam, and 30 mg/kg etomidate. t 
	Tests with Bonferroni correction revealed that the temperature decrease was more pronounced in &agr;3KO mice compared with WT mice 60 min and 120 min after administration of ketamine and pentobarbital, respectively, and that the temperature decrease was less pronounced in &agr;3KO mice compared with WT mice 90 min after administration of midazolam or etomidate. Data represent the mean ± SEM; *P 
	< 0.05, **P 
	< 0.01 and ***P 
	< 0.001, n = 9 WT and n = 7 &agr;3KO for ketamine; n = 9 for pentobarbital; n = 11 for etomidate and midazolam. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t  tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t  test). (B  ) Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250 mg/kg ketamine, 75 mg/kg midazolam, and 30 mg/kg etomidate. t  Tests with Bonferroni correction revealed that the temperature decrease was more pronounced in &agr;3KO mice compared with WT mice 60 min and 120 min after administration of ketamine and pentobarbital, respectively, and that the temperature decrease was less pronounced in &agr;3KO mice compared with WT mice 90 min after administration of midazolam or etomidate. Data represent the mean ± SEM; *P  < 0.05, **P  < 0.01 and ***P  < 0.001, n = 9 WT and n = 7 &agr;3KO for ketamine; n = 9 for pentobarbital; n = 11 for etomidate and midazolam. KO = knockout; LORR = loss of righting reflex; WT = wild type.
×
Fig. 2. Effects of medetomidine on locomotor activity (A  ), duration of LORR (B  ) and body temperature (C  ) in &agr;3KO and wt mice. (A  ) Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 min time period (insert) and looking at 5-min time bins. (B  ) Duration of LORR was significantly increased in &agr;3KO mice by 250 µg/kg medetomidine compared with WT mice (Sheffé post hoc  tests). (C  ) Body temperature was comparable between &agr;3KO mice and WT mice. Data represent the mean ± SEM; n = 9 per genotype. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 2. Effects of medetomidine on locomotor activity (A 
	), duration of LORR (B 
	) and body temperature (C 
	) in &agr;3KO and wt mice. (A 
	) Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 min time period (insert) and looking at 5-min time bins. (B 
	) Duration of LORR was significantly increased in &agr;3KO mice by 250 µg/kg medetomidine compared with WT mice (Sheffé post hoc 
	tests). (C 
	) Body temperature was comparable between &agr;3KO mice and WT mice. Data represent the mean ± SEM; n = 9 per genotype. KO = knockout; LORR = loss of righting reflex; WT = wild type.
Fig. 2. Effects of medetomidine on locomotor activity (A  ), duration of LORR (B  ) and body temperature (C  ) in &agr;3KO and wt mice. (A  ) Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 min time period (insert) and looking at 5-min time bins. (B  ) Duration of LORR was significantly increased in &agr;3KO mice by 250 µg/kg medetomidine compared with WT mice (Sheffé post hoc  tests). (C  ) Body temperature was comparable between &agr;3KO mice and WT mice. Data represent the mean ± SEM; n = 9 per genotype. KO = knockout; LORR = loss of righting reflex; WT = wild type.
×
Fig. 3. Schematic of genetic rescue of the &agr;3KO in noradrenergic neurons. (A  ) &agr;3KO and rescue alleles. The &agr;3KO allele (top  ) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety.18 It carries an artificial exon (light green  , 5*) flanked by lox  P sites (red  ), targeting vector-derived homologous genomic sequences (light brown  ), and the plasmid backbone of the targeting vector (blue  ). Genomic sequences that were not included in the targeting vector are shown as dark brown  . The most relevant feature is that the &agr;3KO allele contains the endogenous exon 5 (dark green  , 5) and an artificial exon 5 (light green  , 5*), which results in mRNA degradation, which is the likely mechanism of the &agr;3KO.18 In the rescue allele, elimination of the artificial exon by Cre-lox  P-mediated recombination results in &agr;3 mRNA (orange  ) being made and thus the expression of the &agr;3 subunit being restored. (B  ) Exonic structure of predicted gene products from WT, &agr;3KO, and rescue allele. Presence of the artificial exon 5, 5*, in the &agr;3KO results in mRNA breakdown and thus a functional KO. The exonic structure of WT and rescue alleles is identical. KO = knockout; mRNA = messenger RNA; WT = wild type.
Fig. 3. Schematic of genetic rescue of the &agr;3KO in noradrenergic neurons. (A 
	) &agr;3KO and rescue alleles. The &agr;3KO allele (top 
	) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety.18It carries an artificial exon (light green 
	, 5*) flanked by lox 
	P sites (red 
	), targeting vector-derived homologous genomic sequences (light brown 
	), and the plasmid backbone of the targeting vector (blue 
	). Genomic sequences that were not included in the targeting vector are shown as dark brown 
	. The most relevant feature is that the &agr;3KO allele contains the endogenous exon 5 (dark green 
	, 5) and an artificial exon 5 (light green 
	, 5*), which results in mRNA degradation, which is the likely mechanism of the &agr;3KO.18In the rescue allele, elimination of the artificial exon by Cre-lox 
	P-mediated recombination results in &agr;3 mRNA (orange 
	) being made and thus the expression of the &agr;3 subunit being restored. (B 
	) Exonic structure of predicted gene products from WT, &agr;3KO, and rescue allele. Presence of the artificial exon 5, 5*, in the &agr;3KO results in mRNA breakdown and thus a functional KO. The exonic structure of WT and rescue alleles is identical. KO = knockout; mRNA = messenger RNA; WT = wild type.
Fig. 3. Schematic of genetic rescue of the &agr;3KO in noradrenergic neurons. (A  ) &agr;3KO and rescue alleles. The &agr;3KO allele (top  ) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety.18 It carries an artificial exon (light green  , 5*) flanked by lox  P sites (red  ), targeting vector-derived homologous genomic sequences (light brown  ), and the plasmid backbone of the targeting vector (blue  ). Genomic sequences that were not included in the targeting vector are shown as dark brown  . The most relevant feature is that the &agr;3KO allele contains the endogenous exon 5 (dark green  , 5) and an artificial exon 5 (light green  , 5*), which results in mRNA degradation, which is the likely mechanism of the &agr;3KO.18 In the rescue allele, elimination of the artificial exon by Cre-lox  P-mediated recombination results in &agr;3 mRNA (orange  ) being made and thus the expression of the &agr;3 subunit being restored. (B  ) Exonic structure of predicted gene products from WT, &agr;3KO, and rescue allele. Presence of the artificial exon 5, 5*, in the &agr;3KO results in mRNA breakdown and thus a functional KO. The exonic structure of WT and rescue alleles is identical. KO = knockout; mRNA = messenger RNA; WT = wild type.
×
Fig. 4. Localization and quantification of the &agr;3KO in global rescue mice. (A  ) Generation of global rescue mice. An &agr;3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse.19 Some of the offspring will carry both the &agr;3KO allele and the EIIa-Cre transgene. This mouse is bred with a WT mouse to breed out the EIIa-Cre transgene (depicted by 2 sequential horizontal arrows  ) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the &agr;3 subunit is expected to be expressed in all cells in which it is naturally expressed. (B  ) Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: &agr;3 subunit distribution pattern is equivalent in WT and global rescue mice. Representative sections from two WT and two global rescue mice are shown. (C  ) Left panel: Representative Western Blot: &agr;3 subunit expression level is decreased in global rescue mice compared with WT mice. Right panel: Quantification of Western blot signal: signals were normalized to the &agr;3 subunit signal at 10 &mgr;g protein in WT mice (100%). &agr;3 subunit expression level in global rescue mice is 70 ± 7% of WT expression. Data represent the mean ± SEM of five experiments. KO = knockout; WT = wild type.
Fig. 4. Localization and quantification of the &agr;3KO in global rescue mice. (A 
	) Generation of global rescue mice. An &agr;3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse.19Some of the offspring will carry both the &agr;3KO allele and the EIIa-Cre transgene. This mouse is bred with a WT mouse to breed out the EIIa-Cre transgene (depicted by 2 sequential horizontal arrows 
	) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the &agr;3 subunit is expected to be expressed in all cells in which it is naturally expressed. (B 
	) Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: &agr;3 subunit distribution pattern is equivalent in WT and global rescue mice. Representative sections from two WT and two global rescue mice are shown. (C 
	) Left panel: Representative Western Blot: &agr;3 subunit expression level is decreased in global rescue mice compared with WT mice. Right panel: Quantification of Western blot signal: signals were normalized to the &agr;3 subunit signal at 10 &mgr;g protein in WT mice (100%). &agr;3 subunit expression level in global rescue mice is 70 ± 7% of WT expression. Data represent the mean ± SEM of five experiments. KO = knockout; WT = wild type.
Fig. 4. Localization and quantification of the &agr;3KO in global rescue mice. (A  ) Generation of global rescue mice. An &agr;3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse.19 Some of the offspring will carry both the &agr;3KO allele and the EIIa-Cre transgene. This mouse is bred with a WT mouse to breed out the EIIa-Cre transgene (depicted by 2 sequential horizontal arrows  ) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the &agr;3 subunit is expected to be expressed in all cells in which it is naturally expressed. (B  ) Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: &agr;3 subunit distribution pattern is equivalent in WT and global rescue mice. Representative sections from two WT and two global rescue mice are shown. (C  ) Left panel: Representative Western Blot: &agr;3 subunit expression level is decreased in global rescue mice compared with WT mice. Right panel: Quantification of Western blot signal: signals were normalized to the &agr;3 subunit signal at 10 &mgr;g protein in WT mice (100%). &agr;3 subunit expression level in global rescue mice is 70 ± 7% of WT expression. Data represent the mean ± SEM of five experiments. KO = knockout; WT = wild type.
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Fig. 5. Recue of &agr;3 expression in defined neuronal cell types. (A  ) An &agr;3KO mouse is crossed with a DBH-iCre mouse to obtain DBH-iCre Rescue mice (in our studies hemizygous males) containing both the &agr;3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the &agr;3 KO allele functions as a “STOP” signal, resulting in messenger RNA degradation.18 In noradrenergic neurons of the Rescue mouse, this “STOP” signal is removed by cre-lox  P-mediated recombination. Thus, in the Rescue mice, the &agr;3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the DAT-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. (B  ) Neuron-specific rescue of &agr;3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that the &agr;3 subunit is highly expressed in the LC in WT mice. In &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) &agr;3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of &agr;3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. (C  ) Neuron-specific rescue of &agr;3 subunit expression in the SNpc: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that &agr;3 subunit expression is found in dopaminergic neurons of the SNpc of WT mice and in &agr;3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the SNpc of &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. DBH-iCre = dopamine &bgr; hydroxylase impoved Cre; DAT = dopamine transporter; WT = wild type; LC, locus coeruleus; SNpc = substantia nigra pars compacta.
Fig. 5. Recue of &agr;3 expression in defined neuronal cell types. (A 
	) An &agr;3KO mouse is crossed with a DBH-iCre mouse to obtain DBH-iCre Rescue mice (in our studies hemizygous males) containing both the &agr;3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the &agr;3 KO allele functions as a “STOP” signal, resulting in messenger RNA degradation.18In noradrenergic neurons of the Rescue mouse, this “STOP” signal is removed by cre-lox 
	P-mediated recombination. Thus, in the Rescue mice, the &agr;3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the DAT-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. (B 
	) Neuron-specific rescue of &agr;3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the &agr;3 subunit (red 
	) and tyrosine hydroxylase (green 
	) shows that the &agr;3 subunit is highly expressed in the LC in WT mice. In &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) &agr;3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of &agr;3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. (C 
	) Neuron-specific rescue of &agr;3 subunit expression in the SNpc: Immunofluorescence double-labeling of the &agr;3 subunit (red 
	) and tyrosine hydroxylase (green 
	) shows that &agr;3 subunit expression is found in dopaminergic neurons of the SNpc of WT mice and in &agr;3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the SNpc of &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. DBH-iCre = dopamine &bgr; hydroxylase impoved Cre; DAT = dopamine transporter; WT = wild type; LC, locus coeruleus; SNpc = substantia nigra pars compacta.
Fig. 5. Recue of &agr;3 expression in defined neuronal cell types. (A  ) An &agr;3KO mouse is crossed with a DBH-iCre mouse to obtain DBH-iCre Rescue mice (in our studies hemizygous males) containing both the &agr;3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the &agr;3 KO allele functions as a “STOP” signal, resulting in messenger RNA degradation.18 In noradrenergic neurons of the Rescue mouse, this “STOP” signal is removed by cre-lox  P-mediated recombination. Thus, in the Rescue mice, the &agr;3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the DAT-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. (B  ) Neuron-specific rescue of &agr;3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that the &agr;3 subunit is highly expressed in the LC in WT mice. In &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) &agr;3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of &agr;3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. (C  ) Neuron-specific rescue of &agr;3 subunit expression in the SNpc: Immunofluorescence double-labeling of the &agr;3 subunit (red  ) and tyrosine hydroxylase (green  ) shows that &agr;3 subunit expression is found in dopaminergic neurons of the SNpc of WT mice and in &agr;3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the SNpc of &agr;3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in &agr;3KO mice. Scale bar: 50 µm. DBH-iCre = dopamine &bgr; hydroxylase impoved Cre; DAT = dopamine transporter; WT = wild type; LC, locus coeruleus; SNpc = substantia nigra pars compacta.
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Fig. 6. Effect of ketamine (139 mg/kg)/xylazine (21 mg/kg) on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice compared with WT mice, whereas mice expressing the &agr;3 subunit exclusively in noradrenergic neurons (DBH-Rescue) and mice carrying the iCre transgene (DBH-iCre) did not differ from WT mice (one-way ANOVA followed by post hoc  Dunnett t  tests). (B  ) Body temperature was significantly lower in &agr;3KO mice compared with WT mice 150 min and 180 min after injection. Body temperature in DBH-Rescue mice was significantly lower 180 min after injection compared with WT mice (two-way repeated measures ANOVA followed by post hoc t  tests with Bonferroni correction). Data represent the mean ± SEM; ***P  < 0.001 (&agr;3KO compared with WT), *P  < 0.05 (&agr;3KO compared with WT), #P  < 0.05 (DBH-Rescue compared with WT); n = 15 WT and DBH-iCre, n = 14 &agr;3KO and DBH-Rescue. LORR = loss of righting reflex; WT = wild type; DBH = dopamine &bgr; hydroxylase; iCre = improved cre.
Fig. 6. Effect of ketamine (139 mg/kg)/xylazine (21 mg/kg) on (A 
	) duration of LORR and (B 
	) core body temperature. (A 
	) Duration of LORR was significantly increased in &agr;3KO mice compared with WT mice, whereas mice expressing the &agr;3 subunit exclusively in noradrenergic neurons (DBH-Rescue) and mice carrying the iCre transgene (DBH-iCre) did not differ from WT mice (one-way ANOVA followed by post hoc 
	Dunnett t 
	tests). (B 
	) Body temperature was significantly lower in &agr;3KO mice compared with WT mice 150 min and 180 min after injection. Body temperature in DBH-Rescue mice was significantly lower 180 min after injection compared with WT mice (two-way repeated measures ANOVA followed by post hoc t 
	tests with Bonferroni correction). Data represent the mean ± SEM; ***P 
	< 0.001 (&agr;3KO compared with WT), *P 
	< 0.05 (&agr;3KO compared with WT), #P 
	< 0.05 (DBH-Rescue compared with WT); n = 15 WT and DBH-iCre, n = 14 &agr;3KO and DBH-Rescue. LORR = loss of righting reflex; WT = wild type; DBH = dopamine &bgr; hydroxylase; iCre = improved cre.
Fig. 6. Effect of ketamine (139 mg/kg)/xylazine (21 mg/kg) on (A  ) duration of LORR and (B  ) core body temperature. (A  ) Duration of LORR was significantly increased in &agr;3KO mice compared with WT mice, whereas mice expressing the &agr;3 subunit exclusively in noradrenergic neurons (DBH-Rescue) and mice carrying the iCre transgene (DBH-iCre) did not differ from WT mice (one-way ANOVA followed by post hoc  Dunnett t  tests). (B  ) Body temperature was significantly lower in &agr;3KO mice compared with WT mice 150 min and 180 min after injection. Body temperature in DBH-Rescue mice was significantly lower 180 min after injection compared with WT mice (two-way repeated measures ANOVA followed by post hoc t  tests with Bonferroni correction). Data represent the mean ± SEM; ***P  < 0.001 (&agr;3KO compared with WT), *P  < 0.05 (&agr;3KO compared with WT), #P  < 0.05 (DBH-Rescue compared with WT); n = 15 WT and DBH-iCre, n = 14 &agr;3KO and DBH-Rescue. LORR = loss of righting reflex; WT = wild type; DBH = dopamine &bgr; hydroxylase; iCre = improved cre.
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Table 1. Anesthetic Properties (Ketamine, Pentobarbital, Midazolam, Etomidate)
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Table 1. Anesthetic Properties (Ketamine, Pentobarbital, Midazolam, Etomidate)
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Table 2. Anesthetic Endpoints with Ketamine (139 mg/kg)/ Xylazine (21 mg/kg)
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Table 2. Anesthetic Endpoints with Ketamine (139 mg/kg)/ Xylazine (21 mg/kg)
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