The Journal of Neuroscience, May 14, 2008 • 28(20):5383–5393 • 5383 Cellular/Molecular Ketamine, But Not Phencyclidine, Selectively Modulates ␣ ␦ Cerebellar GABAA Receptors Containing 6 and Subunits Wulf Hevers,1,3 Stephen H. Hadley,2 Hartmut Lu¨ddens,1 and Jahanshah Amin2 1Carl-Ludwig Department of Physiology, University of Leipzig, D-04103 Leipzig, Germany, 2Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida 33612, and 3Laboratory of Molecular Biology at the Department of Psychiatry, University of Mainz, D-55131 Mainz, Germany Phencyclidine (PCP) and ketamine are dissociative anesthetics capable of inducing analgesia, psychomimetic behavior, and a catatonic state of unconsciousness. Despite broad similarities, there are notable differences between the clinical actions of ketamine and PCP. Ketamine has a lower incidence of adverse effects and generally produces greater CNS depression than PCP. Both noncompetitively inhibit NMDA receptors, yet there is little evidence that these drugs affect GABAA receptors, the primary target of most anesthetics. ␣ ␤ ␦ 6 2/3 receptors are subtypes of the GABAA receptor family and are abundantly expressed in granular neurons within the adult cerebellum. Here, using an oocyte expression system, we show that at anesthetically relevant concentrations, ketamine, but not PCP, ␣ ␤ ␦ ␣ ␤ ␦ modulates 6 2 and 6 3 receptors. Additionally, at higher concentrations, ketamine directly activates these GABAA receptors. Comparatively, dizocilpine (MK-801 [(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]), a potent non- competitive antagonist of NMDA receptors that is structurally unrelated to PCP, did not produce any effect on ␣6␤2␦ receptors. Of the ␣ ␤ ␣ ␤ ␥ ␣ ␤ ␦ ␣ ␤ ␥ ␣ ␤ ␦ ␣ ␤ ␥ ␣ ␤ ␦ ␣ ␤ ␦ recombinant GABAA receptor subtypes examined ( 1 2, 1 2 2, 1 2 , 4 2 2, 4 2 , 6 2 2, 6 2 , and 6 3 ), the actions of ketamine were unique to ␣6␤2␦ and ␣6␤3␦ receptors. In dissociated granule neurons and cerebellar slice recordings, ketamine poten- ␣ tiated the GABAergic conductance arising from 6-containing GABAA receptors, whereas PCP showed no effect. Furthermore, ketamine potentiation was absent in cerebellar granule neurons from transgenic functionally null ␣6 ؊/؊ and ␦؊/؊ mice. These findings suggest that the higher CNS depressant level achieved by ketamine may be the result of its selective actions on ␣6␤2/3␦ receptors. ␣ ␤ ␦ Key words: ketamine; 6 2/3 GABAA receptors; granule neurons; slice recording; cerebellum; transgenic mice; tonic conductance Introduction It has been established that dissociative anesthetics are Ketamine and phencyclidine (PCP) are chemical congeners be- noncompetitive inhibitors of NMDA excitatory ligand-gated longing to a class of pharmacological agents known as dissocia- ion channels (Lodge and Anis, 1982; Anis et al., 1983; Mac- tive anesthetics. These drugs produce similar clinical actions, al- Donald et al., 1987, 1991; ffrench-Mullen and Rogawski, 1989; though the impact of ketamine on CNS functioning differs from Rogawski and Wenk, 2003). These drugs also modulate the that of PCP and its analogues (Greifenstein et al., 1958; Johnstone activity of nicotinic acetylcholine, muscarinic, and opioid re- et al., 1959; Collins et al., 1960; Chen, 1965; Domino et al., 1965; ceptors, and voltage-gated ion channels, but at significantly Corssen and Domino, 1966). Ketamine has a lower potency, a higher concentrations than needed to block NMDA receptors shorter duration of action, a faster rate of induction, results in a (Finck and Ngai, 1982; Ramoa et al., 1990; Hustveit et al., lower incidence of adverse emergence reactions, and is an effec- 1995; Hirota and Lambert, 1996; Scheller et al., 1996; Brau et tive anesthetic across different animal species (PCP shows species al., 1997; Furuya et al., 1999; Flood and Krasowski, 2000; Ya- selectivity). Furthermore, ketamine lacks the convulsive side ef- makura et al., 2000; Schnoebel et al., 2005). Studies conducted fects that PCP produces at higher doses. Collectively, these stud- at low doses of ketamine suggest that the inhibition of NMDA ies suggest that ketamine possesses a higher CNS depressant ac- receptors results in its psychomimetic and analgesic proper- tivity and produces a better quality of anesthesia than PCP ties (Tricklebank et al., 1987; Klepstad et al., 1990; Oye et al., (McCarthy et al., 1965). 1992). However, the mechanism by which ketamine produces a higher CNS depression than PCP at high doses has remained an enigma (Kress, 1997). Received Dec. 10, 2007; revised April 10, 2008; accepted April 10, 2008. GABA receptors are the prime target for the hypnotic class of This work was supported by grants from the German Science Foundation (Deutsche Forschungsgemeinschaft Lu A 319/2–1) (H.L.), the MAIFOR program of the University of Mainz (W.H.), and American Heart Established Investiga- anesthetic compounds (Franks and Lieb, 1994; Macdonald and tor Awards (J.A.). We thank Blake Singletary for help with editing, Stacey Kolar for help with Figures, and Ingo Olsen, 1994). A number of studies also implicated GABAA recep- Bohme for genotyping the mouse lines. tors in ketamine anesthesia (Scholfield, 1980; Gage and Robert- Correspondence should be addressed to Jahanshah Amin, Department of Molecular Pharmacology and Physiol- son, 1985; Bennett et al., 1988; Irifune et al., 1992, 2000; Lin et al., ogy, University of South Florida, Tampa, FL 33612. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.5443-07.2008 1992; Wakasugi et al., 1999), although these provided either in- Copyright © 2008 Society for Neuroscience 0270-6474/08/285383-11$15.00/0 direct evidence for the action of ketamine on GABAA receptors, • ␣ ␦ 5384 • J. Neurosci., May 14, 2008 • 28(20):5383–5393 Hevers et al. Ketamine Acts on GABAA Receptors Containing 6 and Subunits or the concentration of ketamine used was significantly higher than the anesthetically relevant concentration. ␣ ␤ ␦ The GABAA receptor subtypes 6 2/3 are expressed at high levels exclusively within mature cerebellar granule neurons (Laurie et al., 1992; Wisden et al., 1996) and show high sensitivity to GABA (Saxena and Macdonald, 1996; Storustovu and Ebert, 2006; Hadley and Amin, 2007). We investigated the effects of ketamine, PCP, and MK-801 [(ϩ)-5-methyl-10,11- dihydro-5H-dibenzo [a,d] cyclohepten- 5,10-imine maleate] (a potent noncompet- itive inhibitor of NMDA receptor) on ␣6- ␦ and -containing GABAA receptors. We show that ketamine, but not PCP or MK- 801, within an anesthetically relevant con- centration range, potentiated the GABA current arising from ␣6- and ␦-containing GABAA receptors in oocytes, dissociated granule neurons, and cerebellar slices iso- lated from rodents. The potential role of ␣ ␤ ␦ GABAA 6 2/3 receptors in ketamine an- esthesia is discussed. Figure 1. Ketamine potentiation of GABA-induced currents in ␣6␤2␦ receptors. A, B, GABA concentration–response rela- Materials and Methods tionship of ␣6␤2␦ receptors. A, GABA-induced current traces for ␣6␤2␦ receptors. B, Data points for GABA concentration– cRNA quantitation and oocyte injection. Xenopus response relationship. The ␣6␤2␦ receptors show two distinct and separable states of agonist affinity, one exhibiting ␮M and laevis frogs were anesthetized by bathing in a the other nM affinities for GABA. The high-affinity state is associated with a significant level of constitutive channel activity. C, solution containing 0.1% MS-222 (tricaine ␣ ␤ ␦ Representative current traces for ketamine-dependent potentiation of EC1,GABA,EC3,GABA,EC8,GABA, and EC16,GABA for 6 2 methane sulfonate; Sigma-Aldrich). Before ␮ receptors.D,Bargraphdepictingtheaverageof10,20,50,and100 M ketaminepotentiationatEC1,GABA,EC3,GABA,EC8,GABA,and ovariectomy, the toe of the frog was pinched to EC16,GABA. Error bars indicate SEM. assess the state of anesthesia. After surgery, the frog was killed by decapitation according to the The cerebellar vermis was trimmed and then glued to the stage of an protocol approved by the Institutional Animal Care and Use Committee. IntegraSlice 7550MM (cyanoacrylate glue; Campden Instruments). Sag- Oocyte preparation, in vitro transcription of complementary rat RNA ittal slices of ϳ200 or ϳ500 ␮m thickness were prepared and maintained (cRNA), and the drug perfusion system have been described previously in a continuously carboxygenated perfusion chamber at room tempera- (Walters et al., 2000). The quality of cRNA was determined by electro- ture (21°C) containing artificial CSF (ACSF) (see below, Solutions and phoresis of the cRNA on a 1% formaldehyde-containing agarose gel and drugs) for 1 h. Single slices (ϳ200 ␮m) were transferred to a recording then quantified spectrophotometrically. Two to four preparations of chamber (RC-26GLP; Warner Instruments), placed under an upright cRNAs were tested for each subunit. The cRNA mixture (in ϳ diethylpyrocarbonate-treated water) was injected into Xenopus laevis oo- fixed stage microscope (Axioskop FS; Zeiss), and perfused at 4 ml/min cytes using a Picospritzer II (General Valve Corporation). The ratio of the with carboxygenated ACSF. Using Nomarski optics and an enhanced coinjected subunits ␣:␤:␥/␦ was1:1:1.8 except for expression of ␣6␤2␦ infrared video system (Luigs and Neumann), whole-cell patch-clamp receptors in the low-affinity state where the amount of ␤2 was relatively recordings of individual cells within the second or third layer of the inner decreased to one-tenth (1:0.1:1.8) (Hadley and Amin, 2007). Five to