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. 1999 Oct 26;96(22):12905-10.
doi: 10.1073/pnas.96.22.12905.

Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice

R M Mihalek  1 , P K Banerjee, E R Korpi, J J Quinlan, L L Firestone, Z P Mi, C Lagenaur, V Tretter, W Sieghart, S G Anagnostaras, J R Sage, M S Fanselow, A Guidotti, I Spigelman, Z Li, T M DeLorey, R W Olsen, G E Homanics
Affiliations

Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice

R M Mihalek et al. Proc Natl Acad Sci U S A. .

Abstract

gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

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Figures

Figure 1
Figure 1
Gene targeting and molecular characterization. (A) Structure of δ locus. Numbered black boxes represent exons in genomic DNA. Probe D is 830-bp PCR product used for genotyping. The neo gene was flanked on the 5′ side by 8.3 kb of genomic DNA and 1.2 kb of 3′ genomic DNA. Probe D hybridizes to an ≈7.7-kb BamHI restriction fragment at a correctly targeted locus compared with a 6.6-kb BamHI fragment from the endogenous locus. (B) Southern blot analysis of BamHI digested DNA illustrating mice of all three genotypes. (C) Northern blot analysis using adult whole brain total RNA hybridized with probe D or human β-actin probe for loading control. (D) Western blot analysis of total cerebellar protein from δ+/+ and δ−/− with δ(1–44) polyclonal antibody. The 54-kDa δ protein present in wild-type lanes is completely absent from δ−/− lanes. Higher molecular mass bands of nonspecific binding show equal loading of samples in all lanes.
Figure 2
Figure 2
Autoradiographic distribution of [3H]muscimol and [3H]Ro 15–4513 binding sites in horizontal brain sections of GABAA-R δ+/+ and δ−/− mice. Representative images illustrate the reduction of [3H]muscimol binding and region-specific elevation of [3H]Ro 15–4513 binding. Gr, cerebellar granule cell layer; Th, thalamus; Str, striatum.
Figure 3
Figure 3
Anxiolytic (A) and pro-absence (B) effects of ganaxolone in δ+/+ and δ−/− mice. (A) Mice were injected with ganaxolone (10 mg/kg, i.p.) 10 min before testing on the elevated plus-maze assay (n = 9/group). Ganaxolone produced an ≈2-fold increase in the number of open-arm entries in δ+/+ (*, P < 0.01, Bonferroni’s post-hoc test), whereas no significant change was observed in δ−/−. (B) Mice were treated with ganaxolone (10 mg/kg, i.p.) or saline (n = 9/group) 10 min before an absence seizure-producing dose of PTZ (20 mg/kg). Behavior was observed for 2–3 h posttreatment. Ganaxolone significantly prolonged absence-like behavior in δ+/+ (**, P < 0.001, Bonferroni’s post-hoc test), but not in δ−/− mice (mean ± SEM).
Figure 4
Figure 4
Pavlovian fear conditioning. Mice were given three tone-shock pairings in a distinctive context. (A) Context fear. One day after training, mice were returned to conditioning chambers and contextual freezing (% time, mean ± SEM) was assessed for 8 min. Mutant mice exhibited normal levels of contextual fear. (B) Tone fear. One day after the context fear test, the mice were brought to a novel context, and after a 2-min baseline (BL) period, the conditioning tone was played for 6 min. Freezing (%time, mean ± SEM) was assessed for both periods.
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References

    1. Barnard E A, Skolnick P, Olsen R W, Mohler H, Sieghart W, Biggio G, Braestrup C, Bateson A N, Langer S Z. Pharmacol Rev. 1998;50:291–313. - PubMed
    1. Homanics G E, Quinlan J J, Mihalek R, Firestone L L. Toxicol Lett. 1998;100–101:301–307. - PubMed
    1. Lüddens H, Korpi E R. Neuroscientist. 1996;2:15–23.
    1. Olsen R W, DeLorey T M, Gordey M, Kang M-H. In: Jasper’s Basic Mechanisms of the Epilepsies: Advances in Neurology. 3rd Ed. Delgado-Escueta A V, Wilson W A, Olsen R W, Porter R J, editors. Vol. 79. Philadelphia: Lippincott; 1999. pp. 499–510.
    1. Olsen R W, Sapp D W. Adv Biochem Psychopharmacol. 1995;48:57–74. - PubMed

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