The Journal of Neuroscience, September 1, 2000, 20(17):6431–6441 ␣ Phenotypic Characterization of an 4 Neuronal Nicotinic Acetylcholine Receptor Subunit Knock-Out Mouse Shelley A. Ross,1 John Y. F. Wong,1 Jeremiah J. Clifford,3 Anthony Kinsella,4 Jim S. Massalas,1 Malcolm K. Horne,1 Ingrid E. Scheffer,1,5 Ismail Kola,2 John L. Waddington,3 Samuel F. Berkovic,5 and John Drago1 1Neurosciences Group, Monash University Department of Medicine and 2Institute of Reproduction and Development, Monash Medical Centre, Clayton, Victoria, 3168, Australia, 3Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2, Ireland, 4Department of Mathematics, Dublin Institute of Technology, Dublin 8, Ireland, and 5Department of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, Victoria, 3084, Australia Neuronal nicotinic acetylcholine receptors (nAChR) are present in tions; conversely, heightened levels of behavioral topographies in high abundance in the nervous system (Decker et al., 1995). Mt were reduced by nicotine in the late phase of the unhabitu- There are a large number of subunits expressed in the brain that ated condition. Ligand autoradiography confirmed the lack of combine to form multimeric functional receptors. We have gen- high-affinity binding to radiolabeled nicotine, cytisine, and epiba- ␣ erated an 4 nAChR subunit knock-out line and focus on defining tidine in the thalamus, cortex, and caudate putamen, although the behavioral role of this receptor subunit. Homozygous mutant binding to a number of discrete nuclei remained. The study ␣ mice (Mt) are normal in size, fertility, and home-cage behavior. confirms the pivotal role played by the 4 nAChR subunit in the Spontaneous unconditioned motor behavior revealed an etho- modulation of a number of constituents of the normal mouse gram characterized by significant increases in several topogra- ethogram and in anxiety as assessed using the plus-maze. Fur- phies of exploratory behavior in Mt relative to wild-type mice (Wt) thermore, the response of Mt to nicotine administration suggests over the course of habituation to a novel environment. Further- that persistent nicotine binding sites in the habenulo- more, the behavior of Mt in the elevated plus-maze assay was interpeduncular system are sufficient to modulate motor activity consistent with increased basal levels of anxiety. In response to in actively exploring mice. ␣ nicotine, Wt exhibited early reductions in a number of behavioral Key words: 4 ; nicotinic receptor; homologous recombination; topographies, under both unhabituated and habituated condi- anxiety; knock-out; behavioral topography Nicotine is one of the most widely consumed psychoactive drugs Despite detailed characterization of nAChR subunits at the and exerts a number of pharmacological actions in the CNS and molecular level, not much is known about the in vivo functional PNS (Decker et al., 1995). Neuronal nicotinic acetylcholine recep- role of individual subunits. Most nAChR ligands show similar tors (nAChR) constitute a heterogenous family of pentameric patterns of high-affinity labeling that resembles the distribution of oligomers with contributions from 11 subunits (Le Novere and ␣ /␤ subunits. A number of nAChR agonists that bind to the ␣ /␤ ␣ ␣ ␣ 4 2 4 2 Changeux, 1995). Five types of subunits ( 2– 6) and three types receptor configuration in vitro are known to have an effect on ␤ ␤ ␤ ␣ ␤ of subunits ( 2– 4) permit combinations of and type subunits anxiety (Pomerleau, 1986; Gilbert et al., 1989; Brioni et al., 1993), to form a number of functional receptors with subunits of two or attention (Brioni et al., 1997), and antinociception (Tripathi et al., more different types (Changeux et al., 1998), whereas subunits 1982; Damaj et al., 1998), implicating the ␣ /␤ receptor in the ␣ ␣ ␣ 4 2 7– 9 can form -bungarotoxin-sensitive homopentameric recep- mediation of a number of physiological processes. Analysis of an tors (Corringer et al., 1995). The topography of nAChR subunits ␣ independently generated line of 4 nAChR subunit knock-out mice varies (Deneris et al., 1989; Duvoisin et al., 1989; Wada et al., 1989, (Marubio et al., 1999) validated the significant role played by this 1990; Hill et al., 1993; Elgoyhen et al., 1994; Court and Perry, 1995; receptor subunit in nociception. Loss of nicotinic binding sites and ␣ Le Novere et al., 1996; Brioni et al., 1997; Zoli et al., 1998), with 4 a decrease of nAChR protein expression has been shown in pa- ␤ and 2 transcripts being found in a large number of CNS nuclei, tients with Alzheimer’s disease and patients with Parkinson’s dis- ␣ ␣ ␣ ␣ ␤ ␤ whereas 2, 3, 5, 6, 3, and 4 mRNAs are restricted to a few ␣ ␤ ease with cognitive dysfunction (Giacobini, 1991; Brioni et al., cholinergic pathways, which also express 4 and 2. ␣ 1997). Furthermore, several mutations in the 4 nAChR subunit have been identified in autosomal dominant nocturnal frontal lobe Received Jan. 10, 2000; revised June 9, 2000; accepted June 14, 2000. epilepsy (Steinlein et al., 1995, 1997). The large number of subunits This work is supported in part by the National Health and Medical Research suggests a potential for considerable diversity in nAChR func- Council of Australia (NHMRC), the Australian Commonwealth Department of Vet- tion(s). Defining the specific role played by individual subunits in eran’s Affairs, and an unrestricted educational grant from Parke-Davis (Australia). S.R. was supported by a Peter Bladin Scholarship from the Epilepsy Society of determining spontaneous motor behavior and responses to drug Australia. J.D. is a Monash University Logan Research Fellow. J.J.C. was supported challenge will be aided by ongoing analysis of nAChR subunit gene by the NHMRC Brain Network into Mental Health Diseases. J.L.W. and J.J.C. are knock-out mice (Picciotto et al., 1995; Orr-Urtreger et al., 1997; supported by a Galen Fellowship from the Irish Brain Research Foundation, the Marubio et al., 1999; Xu et al., 1999). Higher Education Authority of Ireland, and the Royal College of Surgeons in Ireland. We thank Prof. Bevyn Jarrott (Department of Pharmacology, Monash University) for Nicotine is known to reduce anxiety in both chronic smokers the gift of iodinated ␣-bungarotoxin. We also thank Dr. Ian Simpson for his help with (Gilbert, 1979; Pomerleau, 1986; Gilbert et al., 1989) and nonsmok- organ histology. ers (Hutchinson and Emley, 1973). Anxiolytic-like effects of nico- J.Y.F.W. and J.J.C. contributed equally to this work. tine and a select number of neuronal nicotinic receptor agonists Correspondence should be addressed to Dr. J. Drago, Department of Medicine, 5th Floor E Block, Monash Medical Centre, Clayton Road, Clayton, Victoria, 3168, have also been documented in experimental animals (Costall et al., Australia. E-mail: [email protected]. 1989; Brioni et al., 1993; Cao et al., 1993). The differential behav- Copyright © 2000 Society for Neuroscience 0270-6474/00/206431-11$15.00/0 ioral profile of neuronal nicotinic agonists implies that the anxio- • ␣ 6432 J. Neurosci., September 1, 2000, 20(17):6431–6441 Ross et al. 4 Neuronal Nicotinic Receptor Knock-Out Mice Figure 1. Construction of targeting vector and Southern blot analysis of ES clones. Representation of the genomic map of ␣ the 4 nAChR gene (A), the targeting vector (B), and the expected allelic disruption after homologous recombination (C). The restriction sites shown are abbreviated as follows: N, NotI; H, HindIII; Bg, BglII; Sc, ScaI; X, XhoI; E, EcoRI; B, BamHI. NEO represents the neomycin phosphotransferase resistance gene, and TK represents the thymidine kinase gene. The origin of the probes used for homologous recom- bination screening are also shown. Probe pE1 is a HindIII- BglII fragment used to confirm 5Ј recombination, and probe pE2 is a HindIII-BamHI fragment used for confirmation of 3Ј recombination. D shows the Southern blot of HindIII- digested ES cell genomic DNA probed with pE1 (Wt allele is 6 kb, and the recombinant allele is 4.2 kb), showing correctly targeted clones (C3, C44, C55, and C59); C67 is a randomly selected nontargeted clone. E shows the Southern blot of BamHI-digested ES cell genomic DNA probed with pE2; the Wt allele is 12 kb, and the recombinant allele is 2.5 kb (clones C3, C44, C55, and C59 are correctly targeted). F shows a Southern blot of a BamHI-digested probed with a NEO- specific probe. Clones C3, C44, C55, and C59 have a single band at the expected size of 8 kb, confirming a single inser- tion event in all the clones examined. lytic actions of nicotine may be mediated by a specific subunit by probing a BamHI Southern blot with a neomycin phosphotransferase ␣ (NEO) gene cDNA probe (Fig. 1). Four recombinants were identified by configuration of the nAChR. We generated an 4 nAChR knock- ␣ Southern blotting (Fig. 1), one of which (C3) was injected into BALB/C out line to test the hypothesis that mice lacking 4 nAChR subunits blastocysts, and chimeras were generated. Chimeras were mated with CF1 would show behavioral features consistent with heightened basal mice, and a single heterozygous mouse (Hz) was obtained. The Hz founder levels of anxiety. was used to generate the entire colony by subsequent crossing with C57/ BL6 mice. Hz progeny, obtained after two backcrosses with C57/BL6 mice, MATERIALS AND METHODS were mated to establish a number of mutant mice (Mt) mating pairs and wild-type mice (Wt) mating pairs. All Mt mice used in this study were Animals. All procedures involving the use of live animals conformed to the therefore derived from Mt intermatings, and all Wt mice were derived National Health and Medical Research Council (NHMRC) code of from Wt intermatings. Maximal diversity in the genetic background was practice. maintained by randomly interchanging breeders within a given genotype. Cloning. A 1.0 kb cDNA probe encoding the putative second transmem- ϭ ϭ ϭ brane domain of the ␣ nAChR receptor subunit was cloned by PCR Tissue preparation.