Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice.
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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ABSTRACT: Rapid activation of postsynaptic GABAA receptors (GABAARs) is crucial in many neuronal functions, including the synchronization of neuronal ensembles and controlling the precise timing of action potentials. Although the γ2 subunit is believed to be essential for the postsynaptic clustering of GABAARs, synaptic currents have been detected in neurons obtained from γ2(-/-) mice. To determine the role of the γ2 subunit in synaptic GABAAR enrichment, we performed a spatially and temporally controlled γ2 subunit deletion by injecting Cre-expressing viral vectors into the neocortex of GABAARγ2(77I)lox mice. Whole-cell recordings revealed the presence of miniature IPSCs in Cre(+) layer 2/3 pyramidal cells (PCs) with unchanged amplitudes and rise times, but significantly prolonged decays. Such slowly decaying currents could be evoked in PCs by action potentials in presynaptic fast-spiking interneurons. Freeze-fracture replica immunogold labeling revealed the presence of the α1 and β3 subunits in perisomatic synapses of cells that lack the γ2 subunit. Miniature IPSCs in Cre(+) PCs were insensitive to low concentrations of flurazepam, providing a pharmacological confirmation of the lack of the γ2 subunit. Receptors assembled from only αβ subunits were unlikely because Zn(2+) did not block the synaptic currents. Pharmacological experiments indicated that the αβγ3 receptor, rather than the αβδ, αβε, or αβγ1 receptors, was responsible for the slowly decaying IPSCs. Our data demonstrate the presence of IPSCs and the synaptic enrichment of the α1 and β3 subunits and suggest that the γ3 subunit is the most likely candidate for clustering GABAARs at synapses in the absence of the γ2 subunit.The Journal of neuroscience : the official journal of the Society for Neuroscience. 07/2014; 34(31):10219-10233.
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ABSTRACT: Allopregnanolone and its equipotent stereoisomer, pregnanolone (together termed ALLO), are neuroactive steroids that positively and allosterically modulate the action of gamma-amino-butyric acid (GABA) at GABAA receptors. Levels of ALLO are reduced in the cerebrospinal fluid of female premenopausal patients with post-traumatic stress disorder (PTSD), a severe, neuropsychiatric condition that affects millions, yet is without a consistently effective therapy. This suggests that restoring downregulated brain ALLO levels in PTSD may be beneficial. ALLO biosynthesis is also decreased in association with the emergence of PTSD-like behaviors in socially isolated (SI) mice. Similar to PTSD patients, SI mice also exhibit changes in the frontocortical and hippocampal expression of GABAA receptor subunits, resulting in resistance to benzodiazepine-mediated sedation and anxiolysis. ALLO acts at a larger spectrum of GABAA receptor subunits than benzodiazepines, and increasing corticolimbic ALLO levels in SI mice by injecting ALLO or stimulating ALLO biosynthesis with a selective brain steroidogenic stimulant, such as S-norfluoxetine, at doses far below those that block serotonin reuptake, reduces PTSD-like behavior in these mice. This suggests that synthetic analogs of ALLO, such as ganaxolone, may also improve anxiety, aggression, and other PTSD-like behaviors in the SI mouse model. Consistent with this hypothesis, ganaxolone (3.75-30 mg/kg, s.c.) injected 60 min before testing of SI mice, induced a dose-dependent reduction in aggression toward a same-sex intruder and anxiety-like behavior in an elevated plus maze. The EC50 dose of ganaxolone used in these tests also normalized exaggerated contextual fear conditioning and, remarkably, enhanced fear extinction retention in SI mice. At these doses, ganaxolone failed to change locomotion in an open field test. Therefore, unlike benzodiazepines, ganaxolone at non-sedating concentrations appears to improve dysfunctional emotional behavior associated with deficits in ALLO in mice and may provide an alternative treatment for PTSD patients with deficits in the synthesis of ALLO. Selective serotonin reuptake inhibitors (SSRIs) are the only medications currently approved by the FDA for treatment of PTSD, although they are ineffective in a substantial proportion of PTSD patients. Hence, an ALLO analog such as ganaxolone may offer a therapeutic GABAergic alternative to SSRIs for the treatment of PTSD or other disorders in which ALLO biosynthesis may be impaired.Frontiers in Cellular Neuroscience 09/2014; 8:256. · 4.47 Impact Factor
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ABSTRACT: Objective Extrasynaptic γ‐aminobutyric acid type A receptors that contain the δ subunit (δGABAA receptors) are highly expressed in the dentate gyrus (DG) subfield of the hippocampus, where they generate a tonic conductance that regulates neuronal activity. GABAA receptor‐dependent signaling regulates memory and also facilitates postnatal neurogenesis in the adult DG; however, the role of the δGABAA receptors in these processes is unclear. Accordingly, we sought to determine whether δGABAA receptors regulate memory behaviors, as well as neurogenesis in the DG. Methods Memory and neurogenesis were studied in wild‐type (WT) mice and transgenic mice that lacked δGABAA receptors (Gabrd−/−). To pharmacologically increase δGABAA receptor activity, mice were treated with the δGABAA receptor‐preferring agonist 4,5,6,7‐tetrahydroisoxazolo(5,4‐c)pyridin‐3‐ol (THIP). Behavioral assays including recognition memory, contextual discrimination, and fear extinction were used. Neurogenesis was studied by measuring the proliferation, survival, migration, maturation, and dendritic complexity of adult‐born neurons in the DG. ResultsGabrd−/− mice exhibited impaired recognition memory and contextual discrimination relative to WT mice. Fear extinction was also impaired in Gabrd−/− mice, although the acquisition of fear memory was enhanced. Neurogenesis was disrupted in Gabrd−/− mice as the migration, maturation, and dendritic development of adult‐born neurons were impaired. Long‐term treatment with THIP facilitated learning and neurogenesis in WT but not Gabrd−/− mice. InterpretationδGABAA receptors promote the performance of certain DG‐dependent memory behaviors and facilitate neurogenesis. Furthermore, δGABAA receptors can be pharmacologically targeted to enhance these processes. Ann Neurol 2013;74:611–621Annals of Neurology 01/2013; 74(4). · 11.19 Impact Factor
Attenuated sensitivity to neuroactive steroids in
?-aminobutyrate type A receptor delta subunit
Robert M. Mihaleka, Pradeep K. Banerjeeb, Esa R. Korpic, Joseph J. Quinlana, Leonard L. Firestonea, Zhi-Ping Mid, Carl Lagenaurd,
Verena Trettere, Werner Siegharte, Stephan G. Anagnostarasf, Jennifer R. Sagef, Michael S. Fanselowf, Alessandro Guidottig,
Igor Spigelmanh, Zhiwei Lib,h, Timothy M. DeLoreyj, Richard W. Olsenb,k, and Gregg E. Homanicsa,i,l
Departments ofaAnesthesiology?Critical Care Medicine,iPharmacology, anddNeurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA
15261;jMolecular Research Institute, 2495 Old Middlefield Way, Mountain View, CA 94043;cDepartment of Pharmacology and Clinical Pharmacology,
University of Turku, FIN-20520 Turku, Finland;eUniversity Clinic for Psychiatry, Section of Biochemical Psychiatry, Wa ¨hringer Gu ¨rtel 18–20, A-1090 Vienna,
Austria;fDepartment of Psychology, University of California, Los Angeles, CA 90095-1563;gPsychiatric Institute, Department of Psychiatry, University of
Illinois, Chicago, IL 60612;hDivision of Oral Biology and Medicine, University of California School of Dentistry and Departments ofbMolecular and Medical
Pharmacology andkAnesthesiology, University of California School of Medicine, Los Angeles, CA 90095
Edited by Erminio Costa, University of Illinois, Chicago, IL, and approved September 8, 1999 (received for review July 21, 1999)
?-Aminobutyric acid (GABA) type A receptors mediate fast inhib-
itory synaptic transmission and have been implicated in responses
to sedative?hypnotic agents (including neuroactive steroids), anx-
iety, and learning and memory. Using gene targeting technology,
we generated a strain of mice deficient in the ? 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. Elec-
trophysiological recordings from hippocampal slices revealed a
significantly faster miniature inhibitory postsynaptic current decay
time in null mice, with no change in miniature inhibitory postsyn-
aptic current amplitude or frequency. Learning and memory as-
sessed with fear conditioning were normal. These results begin to
illuminate the novel contributions of the ? subunit to GABA
pharmacology and sedative?hypnotic responses and behavior and
provide insights into the physiology of neurosteroids.
acid type A receptors (GABAA-Rs). These ligand-gated recep-
tors are believed to be pentamers, with subunits selected from
(at least) 15 possible variants (?1–6, ?1–4, ?1–3, ?, and ?) (1).
GABAA-Rs are modulated by many drugs, including ethanol,
benzodiazepines, various anesthetics, and neuroactive steroids
(2–5). Specific roles for various GABAA-R subunits in mediating
anesthetic responses are beginning to be elucidated (6–9).
Additionally, GABAA-Rs have been shown to be involved in
epilepsy (4, 10, 11), various behavioral states such as depression
and anxiety (12), and learning and memory (11, 13).
Several studies have shown that the ? subunit participates in
GABAA-Rs that exhibit a unique pharmacology. Such receptor
isoforms are benzodiazepine-insensitive (14), neuroactive ste-
roid-insensitive (15), and Zn2?-sensitive (16). Nearly 30% of
cerebellar GABAA-Rs appear to contain the ? subunit; levels of
? mRNA are highest in the cerebellar granule cells, secondarily
in hippocampus and thalamus (17, 18).
Neuroactive steroids are naturally occurring metabolites of
endogenous steroid hormones, or synthetic analogs, which exert
very rapid, nongenomic effects on membrane-bound
GABAA-Rs (19, 20). Those synthesized in the brain, termed
neurosteroids, are believed to regulate anxiety, stress, and
neuronal excitability by modulating GABAA-R function in vivo
(5, 21). Examples include metabolites of progesterone and
corticosterone that enhance GABA inhibition and preg-
nenolone (3?-hydroxy-5?-pregnan-20-one) sulfate that inhibits
GABA action (13, 19–21). The synthetic analog alphaxalone
(3?-hydroxy-5?-pregnan-11,20-dione) enhances GABAA-R
function and has been used clinically as an i.v. anesthetic. The
chemical analog ganaxalone (3?-hydroxy-3?-methyl-5?-
nhibitory ion currents in the vertebrate central nervous system
pregnan-20-one) was developed for improved bioavailability and
potential anxiolytic and anticonvulsant activity (22).
To investigate the contribution of ?-containing GABAA-R
isoforms to behavior and various drug responses, we used gene
targeting in embryonic stem (ES) cells to create a strain of mice
lacking a functional ?-subunit gene. Here we report the char-
acterization of the ? knockout mice.
Materials and Methods
Generation of Mutant Mice. A 9.5-kb PstI restriction fragment was
isolated from a strain 129?SvJ P1 phage library (Genome
Systems, St. Louis) with ?-specific primers from exon 2 (5?-
CAGGGCAATGAATGACATTG-3?) and exon 3 (5?-
CAAGCGCCACATTCACAG-3?). A replacement-type DNA
targeting vector was constructed in which the MC1Neo gene
(Stratagene) was blunt-end ligated into a blunted HindIII site in
exon 4 (Fig. 1A). The targeting vector was linearized with ClaI
before electroporation into R1 ES cells (23). G418-resistant ES
cell clones were screened for targeting by Southern blot analysis
of BamHI-digested genomic DNA hybridized with a 3? probe
(probe D) (Fig. 1A).
Three correctly targeted ES cell lines were microinjected into
C57BL?6J blastocysts, two of which produced chimeric mice.
Highly chimeric males were mated to C57BL?6J females (The
Jackson Laboratory). Agouti offspring that were heterozygous
were wild type (??/?), ??/?, and homozygous null (??/?). The
mice used for the studies reported here were derived primarily
from the ?#767 ES cell line. A more limited analysis of the
?#773 ES cell line yielded similar results. The genetic back-
ground of all mice was C57BL?6J X strain 129Sv?SvJ, F2-F5
Northern Blot Analysis. Total RNA was isolated from adult mouse
brain by using Trizol reagent (Life Technologies, Grand Island,
NY). Approximately 10 ?g of total RNA was electrophoresed in
a 1.9% formaldehyde?1% agarose gel, blotted to Hybond-N
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: GABA, ?-aminobutyric acid; GABAA-R, GABA type A receptor; mIPSC, min-
ES, embryonic stem.
lTo whom reprint requests should be addressed at: University of Pittsburgh School of
Medicine, Departments of Anesthesiology?Critical Care Medicine and Pharmacology,
W1356 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: firstname.lastname@example.org.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
October 26, 1999 ?
vol. 96 ?
no. 22 ?
(Amersham Pharmacia) and hybridized as described (24). After
the membrane was hydridized with probe D, it was rehybridized
with a human ?-actin cDNA probe (CLONTECH) as a control
for RNA loading in each lane.
Western Blot Analysis. Antibody production, gel electrophoresis,
and blotting were performed as in ref. 25. Briefly, the ?(1–44)R5
polyclonal antibody (26) was prepared by immunizing rabbits
with a maltose binding protein-?(1–44)-7His fusion protein and
the ? subunit and does not precipitate ?1?3?2 receptors (26).
Equal amounts of cerebellar membrane proteins were subject to
SDS?PAGE and immunoblotted onto poly(vinylidene difluo-
ride) membranes. Membranes were incubated with digoxigenin-
labeled antibodies and treated with anti-digoxigenin-alkaline
phosphatase Fab fragments (Boehringer Mannheim). Proteins
Bedford, MA). Blots were exposed to Kodak X-Omat S film and
recorded with a DocuGel 2000i gel system using RFLPSCAN
software (MWG Biotec, Ebersberg, Germany).
GABAA-R Ligand Binding. [3H]Ro15–4513 and [3H]muscimol bind-
ing to whole brain homogenates was determined exactly as
described (7). To visualize brain regional distribution of GABA
and benzodiazepine binding sites, 14-?m horizontal sections
were cut from ??/?and ??/?brains by using a Microm cryostat,
thaw-mounted onto gelatin-coated object glasses, and used for
[3H]Ro 15–4513 (a benzodiazepine site ligand) and [3H]musci-
mol (a GABA site ligand) autoradiography as described in detail
by Ma ¨kela ¨ et al. (27).
Electrophysiology. Transverse slices of dorsal hippocampus were
obtained by using standard techniques (28). Pharmacologically
isolated miniature inhibitory postsynaptic currents (mIPSCs)
were recorded from cells located in the upper blade of the
dentate gyrus at 34.5°C during perfusion with artificial cerebro-
spinal fluid (ACSF) composed of 125 mM NaCl, 2.5 mM KCl,
2 mM CaCl2, 2 mM MgCl2, 26 mM NaHCO3, and 10 mM
dextrose. The ACSF was continuously bubbled with a 95%?5%
mix of O2?CO2to ensure adequate oxygenation of slices and a
pH of 7.4. Patch pipettes contained 135 mM Cs gluconate, 2 mM
and 0.2 mM Na2GTP, pH to 7.5 with CsOH. mIPSCs were
recorded in the presence of 0.5 mM tetrodotoxin, 40 mM
D(-)-2-amino-5-phosphonopentanoate, 10 mM 6-cyano-7-
nitroquinoxaline-2,3-dione, and 1 mM CGP 54,626. Signals were
recorded with an amplifier (Axoclamp 2A, Axon Instruments,
Foster City, CA). Data were acquired with PCLAMP 7 (Axon
Instruments) software, digitized at 20 kHz (Digidata 1200B,
Axon Instruments), and analyzed by using the Mini Analysis
Program (version 4.1.1, Jaejin Software, Leonia, NJ).
Behavioral Characterization. All animal experiments were ap-
proved by the Institutional Animal Care and Use Committee.
Mice were grouped together by age, weight, and generation such
that they were all between 8 and 16 weeks old and 15–35 g. Mice
of both sexes were used for all studies.
Sleep Time Assay. Alphaxalone (8 mg?kg and 16 mg?kg), preg-
nanolone (16 mg?kg), midazolam (25 mg?kg), and propofol (50
mg?kg) were administered i.v. Etomidate (20 mg?kg), pento-
barbital (45 mg?kg), and ketamine (150 mg?kg) were given by
i.p. injection. Injection volumes were 5 ?l?g body weight for i.v.
and 20 ?l?g for i.p. Neuroactive steroids [alphaxalone (Research
Biochemicals, Natick, MA) and pregnanolone (Sigma)] were
dissolved in a 22.5% (wt?vol) solution of 2-hydroxypropyl-?-
cyclodextrin (Research Biochemicals). Pentobarbital (Abbott)
and ketamine (Sigma) were dissolved in sterile saline. Midazo-
lam HCl (Roche Clinical Laboratories, Burlington, NC) was
dissolved in 0.8% NaCl, 0.01% EDTA, and 1% benzyl alcohol.
Sleep times were determined as follows: after drug injection
and loss of the righting reflex, mice were placed on their backs
in a V-shaped trough and a timer was started. The sleep time
period ended when the animal was able to flip over six times in
45 sec (neuroactive steroids) or three times in 30 sec (all other
drugs). Normothermia was maintained with the aid of a heat
lamp. All assays were performed by an investigator who was
unaware of the genotypes of the individual mice being tested.
Effect of genotype was compared by Student’s t test.
Loss of Righting Reflex (LORR) and Tail-Clamp?Withdrawal Assay.
LORR and tail-clamp?withdrawal using volatile anesthetics
were performed as described (7). Briefly, mice were placed in
small, cylindrical wire-mesh cages attached to a carousel housed
in a sealed Plexiglas box. Anesthetics diluted with oxygen were
delivered from an anesthetic-specific vaporizer. After 15-min
equilibration at each concentration, the carousel was rotated five
times while the mice were observed. Scoring was quantal; mice
that passively rolled over twice were scored as positive for
LORR. For tail-clamp, the carousel was removed and a 10-
compartment divider was installed. After a 15-min exposure to
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
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 ?-actin probe for loading control. (D)
Western blot analysis of total cerebellar protein from ??/?and ??/?with
?(1–44) polyclonal antibody. The 54-kDa ? protein present in wild-type lanes
is completely absent from ??/?lanes. Higher molecular mass bands of non-
specific binding show equal loading of samples in all lanes.
Gene targeting and molecular characterization. (A) Structure of ?
www.pnas.orgMihalek et al.
If any motor activity occurred as a result of the stimulus, the
concentration was scored as one that permitted a positive
Elevated Plus-Maze Test. The anxiolytic effect of the synthetic
neuroactive steroid ganaxolone (CoCensys, Irvine, CA) was
tested by using the elevated plus-maze. The plus-maze was
constructed as described (29). A stock solution of ganaxolone
was made in DMSO and diluted with saline (pH 7.1) before
systemic administration. The final concentration of DMSO was
0.1%, a concentration known not to interfere with GABAA-R
Ganaxolone (10 mg?kg, i.p.) or an equal volume of saline was
injected 10 min before testing (n ? 9?group). To start the 5-min
test session, a mouse was placed on the central platform of the
maze, facing an open arm. Open-arm and closed-arm entries and
the cumulative time spent on the open and closed arms was
recorded. A mouse was considered to be on the central platform
when all four paws were within its perimeter. The ratio of
open?total arm entries (% open-arm entries) was calculated and
expressed as mean % open-arm entries ? SEM. Data were
analyzed by using one-way ANOVA, and ganaxolone-treated
group means were compared with their respective saline-treated
controls by using Bonferroni’s post-hoc test.
Low-Dose Pentylenetetrazol (PTZ) Seizures. Pretreatment of ??/?
and ??/?mice with ganaxolone (10 mg?kg, i.p.) or saline
occurred 10 min before PTZ (Sigma) (20 mg?kg, i.p.) injection.
The behavior induced by saline?PTZ versus ganaxolone?PTZ
was observed and recorded for 2–3 h post-PTZ treatment. Data
are expressed as mean duration of hypoactivity ? SEM (n ?
9?group). One-way ANOVA followed by Bonferroni’s post-hoc
test was used to compare multiple group means.
Pavlovian Fear Conditioning. Associative memory was examined
with context and tone-dependent fear conditioning (31, 32).
Briefly, a tone conditional stimulus was paired with an aversive
unconditional foot shock stimulus in a novel context. Mice
trained in this manner develop a fear of both tone and context,
which was measured as freezing, an adaptive defense reaction.
One day after conditioning, mice were returned to the training
context for an 8-min context test. One day after the context test,
mice were placed in a novel context for an 8-min tone fear test.
After a 2-min baseline period, the training tone was played for
both tests. To assess exploratory activity, crossovers were scored
during the 3-min period before tone-shock pairing on the
conditioning day. To assess pain sensitivity, velocity was com-
puter-tracked for 2 sec immediately before and for 2 sec during
the first foot shock (33). Consolidation and retention of contex-
tual fear was examined by repeating the context test 50 days after
Production of Mice. Of 307 ES cell clones analyzed for gene
targeting, three displayed the predicted BamHI restriction frag-
ment length polymorphism indicative of correct gene targeting
at the ? locus. Two cell lines (?#767 and ?#773) yielded
germ-line competent chimeric males. As indicated in Fig. 1B,
probe D hybridized to a 6.6-kb BamHI fragment from the
allele. The ? locus was examined with 12 restriction enzymes,
three unique genomic probes, and a neo probe. All results (data
not shown) were consistent with the targeting event depicted in
Northern and Western blots were used to examine ? gene
expression. Hybridization of adult cerebellar total RNA with
probe D showed an abundant 1.9-kb band in the ??/?mice. In
contrast, samples from ??/?mice revealed the absence of the
wild-type 1.9-kb mRNA (Fig. 1C). In addition, in mice bearing
a targeted allele, a novel 2.3-kb band was observed. Rehybrid-
ization of the same blot with a neo probe revealed that only the
2.3-kb message from ??/?and ??/?mice contained neo-
homologous sequences (data not shown). These results suggest
that the targeting event resulted in a ? allele that was transcribed
as a chimeric ??neo mRNA.
with the highest level of ? expression) of three different ??/?and
three different ??/?mice were probed with the rabbit polyclonal
antibody ?(1–44) (26). The ? subunit exhibits an apparent
molecular mass of 54 kDa (Fig. 1D) and is readily apparent in the
??/?lanes. In contrast, samples from the ??/?mouse cerebel-
lums were completely devoid of the ? protein. Together, these
results demonstrated that the gene targeting event disrupted the
? locus and prevented production of ? protein, i.e., a true null
The size, histological appearance, and folding of the folia in
the cerebellum all appeared normal and indistinguishable be-
tween ??/?and ??/?mice, as did the staining patterns of
toluidine blue, RT97, and antisynaptophysin (data not shown).
Of 1,030 F2and F5pups genotyped at weaning from hetero-
zygote mating pairs, only 211 (20.5%) were null. This percent of
null pups is significantly below the expected Mendelian fre-
quency distribution of 25% (P ? 0.005). Thus it appears that
? knockout had any effect on fecundity, true breeding F3lines
were established. These breeding experiments revealed that ??/?
breeding pairs produced statistically fewer pups per litter than
the ??/?breeding pairs [??/?matings: 7.6 ? 0.4 pups?litter (n ?
47 litters) vs. ??/?matings: 6.3 ? 0.3 pups?litter (n ? 50 litters)]
(P ? 0.01).
Pharmacological Characterization. Ligand binding in whole brain
homogenates from ??/?and ??/?mice was studied with
[3H]Ro15–4513 and [3H]muscimol. The slopes of the binding
of the Kdvalues. Maximal receptor number and binding affinity
for [3H]muscimol in homogenates from ??/?brains were similar
to previous reports (7, 34). However, the maximal binding for
muscimol was markedly decreased in ??/?homogenates (Table
??/?was similar to previous reports (7). The maximal receptor
number and affinity constants for [3H]Ro15–4513 were not
different between ??/?and ??/?brain homogenates (Table 1).
Table 1. Binding of [3H]Ro15-4513 and [3H]muscimol to whole
Wild type Null
Maximal binding (pmol?mg)
Maximal binding (pmol?mg)
8.9 ? 1.2
1.4 ? 0.1
1.2 ? 0.2
9.1 ? 1.0
1.2 ? 0.1
1.3 ? 0.3
37.0 ? 9.9
4.5 ? 0.4
1.1 ? 0.2
21.0 ? 10.9
2.4 ? 0.4‡
1.1 ? 0.4
*These data were pooled from four separate binding experiments for each
for each [3H]Ro15-4513 concentration tested.
†These data were pooled from three separate binding experiments for each
genotype. Each experiment consisted of five specific binding determinations
at each [3H]muscimol concentration tested.
‡P ? 0.02.
Mihalek et al.PNAS ?
October 26, 1999 ?
vol. 96 ?
no. 22 ?
Using in situ autoradiography to examine the abundance and
distribution of GABA and benzodiazepine sites, it was observed
that [3H]muscimol binding was drastically reduced in most
regions of ??/?brains (Fig. 2). In contrast, the [3H]Ro 15–4513
binding was slightly elevated in several regions of the ??/?brain,
including the thalamus, striatum, and the cerebellar granule cell
Delta Knockout Mice Exhibit Altered Synaptic GABAA Currents.
mIPSCs were recorded in hippocampal slices from adult ??/?
and ??/?mice to directly investigate the possibility of altered
GABAA-R current kinetics in mutant mice. Table 2 illustrates
that the average amplitude, rise time, and frequency of mIPSCs
recorded at 0 mV did not differ between genotypes. However,
the decay time (?) of mIPSCs from ??/?mice was significantly
faster than that of ??/?controls.
Delta Knockout Mice Are Resistant to Neuroactive Steroids: Reduced
Sleep Time. Using a sleep time assay, significant differences were
found in response to i.v. injections of alphaxalone and preg-
in sleep time duration compared with ??/?at the two concen-
trations of alphaxalone used (8 mg?kg and 16 mg?kg), respec-
tively. Comparison of ??/?and ??/?after an 8 mg?kg injection
of pregnanolone revealed a 42% reduction in sleep time in ??/?
mice. Intraperitoneal injection of alphaxalone (75 mg?kg) also
revealed a similar difference (data not shown). Similar effects
were observed in ??/?mice derived from the ?#773 cell line
(data not shown). Wild-type and ??/?mice responded similarly
to etomidate, pentobarbital, midazolam, propofol, and ketamine
(an NMDA receptor antagonist) (Table 3).
A trivial explanation for the change in neuroactive steroid
response could be an unexpected pharmacokinetic effect. To
investigate this possibility, pregnanolone concentration in brain
tissue was determined (35) in a limited number of mice at waking
from a 16 mg?kg i.v. injection. After an average sleep time of
45.3 ? 7.4 min, ??/?brains had 5.5 ? 0.8 (n ? 2) ?g?g
pregnanolone, and after an average sleep time of 33.2 ? 4.9 min,
??/?brains had 7.3 ? 1.2 (n ? 3) ?g?g pregnanolone. The level
of endogenous allopregnanolone also was measured in the same
mice: ??/?, 514 ? 48 pg?g (n ? 2) and ??/?, 394 ? 118 pg?g (n ?
3). These preliminary results indicate no gross differences
between genotypes in neuroactive steroid pharmacokinetics or
A second explanation for the difference in sleep times is that
they result from the influence of genetic background. Therefore,
the two strains used to make the ??/?mice, C57BL?6J and strain
129?SvJ mice, were tested with alphaxalone (16 mg?kg) and
pregnanolone (16 mg?kg). Sleep times were not statistically
different between strains for either compound: alphaxalone,
C57BL?6J (8.1 ? 1.3 min., n ? 10) and strain 129?SvJ (9.1 ? 2.7
min., n ? 10) (P ? 0.34) and pregnanolone, C57BL?6J (38.4 ?
binding sites in horizontal brain sections of GABAA-R ??/?and ??/?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.
Autoradiographic distribution of [3H]muscimol and [3H]Ro 15–4513
Table 2. Hippocampal electrophysiology
Rise time (msec)
Decay, ? (msec)
1.53 ? 0.11
11.30 ? 1.05
3.04 ? 0.42
7.05 ? 0.39
1.49 ? 0.10
9.92 ? 0.65
2.85 ? 0.26
5.74 ? 0.18*
? is the time from 90% to 37% of decay phase of mIPSC.
*P ? 0.05.
Table 3. Sleep time assays
Sleep time, min
(mean ? SEM)
Alphaxalone (8 mg?kg) 18
3.5 ? 0.5
1.6 ? 0.4*
8.6 ? 0.8
5.3 ? 0.7†
24.1 ? 1.8
14.0 ? 2.2‡
56.4 ? 3.2
52.2 ? 3.6
14.1 ? 1.8
13.7 ? 2.5
14.4 ? 4.0
13.2 ? 4.8
55.3 ? 5.0
42.0 ? 4.5
40.1 ? 3.8
37.5 ? 2.6
Alphaxalone (16 mg?kg)
Pregnanolone (8 mg?kg)
Pentobarbital (45 mg?kg)
Propofol (50 mg?kg)
Midazolam (25 mg?kg)
Etomidate (20 mg?kg)
Ketamine (150 mg?kg)
*, P ? 0.02; †, P ? 0.01; ‡, P ? 0.002.
www.pnas.orgMihalek et al.
3.5 min., n ? 9) and strain 129?SvJ (43.8 ? 1.6 min., n ? 10) (P ?
0.16). Thus, the phenotype observed in the ??/?mice is not likely
caused by genetic background effects from the parental strains.
Reduced Anxiolysis. Using an elevated plus-maze assay, the ten-
dency of mice to enter or remain on the open arm of the maze
is increased by anxiolytic drugs (36). Basal levels of anxiety (i.e.,
anxiety levels in drug-naive mice) were not statistically different
between genotypes. With percent open-arm entries as a measure
of anxiolysis, a 10 mg?kg i.p. dose of ganaxolone in ??/?mice
0.01). No significant increase in open-arm entries occurred in
??/?mice. Thus, the well-characterized anxiolytic effect of
neuroactive steroids was absent in ??/?mice.
Reduced Pro-Absence Seizure Effect. Absence seizures are exacer-
bated by GABAA-R agonists, including neuroactive steroids
(37). Using a low-dose PTZ absence seizure model, ganaxolone
(10 mg?kg, i.p.) failed to prolong PTZ-induced absence-like
freezing in ??/?mice, whereas it increased absence-like freezing
74% in ??/?mice (Fig. 3B;**, P ? 0.001). PTZ-induced
hypoactivity was not statistically different between genotypes.
Normal Behavioral Responses to Volatile Anesthetics. Wild-type and
??/?mice were compared for their sensitivity toward the ob-
tunding (LORR) and pain suppression (tail-clamp?withdrawal)
effects of two halogenated volatile anesthetics. There was no
difference statistically in the tail-clamp response between ??/?
and ??/?mice in the EC50 value for either anesthetic or for
LORR with halothane (Table 4). Similar results were obtained
in our analyses of the ?#773 mice (data not shown).
Normal Fear Conditioning, Exploratory Activity, and Pain Sensitivity.
Fig. 4A depicts freezing during the 8-min context test. There
were no significant differences in context conditioning. Fig. 4B
depicts cued conditioning, during the 2-min baseline period
before and during the 6-min tone: there were no significant
differences. Mutant mice showed comparable levels of activity to
??/?during the baseline period on the conditioning day and
showed robust foot shock reactivity equivalent to controls (data
not shown). Finally, ??/?mice showed robust context freezing
during the 50-day posttraining memory test, not significantly
different than ??/?(data not shown).
The absence of the ? subunit of the GABAA-R resulted in a
duration of alphaxalone?pregnanolone anesthesia and the anxi-
olytic effect and pro-absence seizure effect of ganaxolone all
were reduced?ablated. The reduced response of ??/?mice to
neuroactive steroids reveals a potential role of ?-containing
GABAA-Rs in modulating behavioral responses to endogenous
neuroactive steroids. This dramatic reduction in whole animal
neuroactive steroid sensitivity was specific, as deletion of ? had
no effect on response to several other sedative?hypnotic agents
in the sleep time assay or on the LORR or tail clamp?withdrawal
response after exposure to volatile anesthetics. The observed
difference does not appear to be confounded by pharmacoki-
netic or genetic background influences. These results show an
animal model that demonstrates a selective alteration in behav-
ioral responses to neuroactive steroids. If responses to endoge-
nous neuroactive steroids are similarly attenuated, these mice
??/?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
in ??/?. (B) Mice were treated with ganaxolone (10 mg?kg, i.p.) or saline (n ?
Behavior was observed for 2–3 h posttreatment. Ganaxolone significantly
prolonged absence-like behavior in ??/?(**, P ? 0.001, Bonferroni’s post-hoc
test), but not in ??/?mice (mean ? SEM).
Anxiolytic (A) and pro-absence (B) effects of ganaxolone in ??/?and
ings 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.
Pavlovian fear conditioning. Mice were given three tone-shock pair-
Table 4. Behavioral responses to volatile anesthetics
(atm % ? SEM)
(mean ? SEM)
1.55 ? 0.03
1.50 ? 0.04
2.43 ? 0.05
2.32 ? 0.05
10.71 ? 1.66
11.11 ? 1.72
11.11 ? 2.03
11.86 ? 2.09
0.72 ? 0.04
0.71 ? 0.04
8.83 ? 2.74
10.65 ? 1.47
Mihalek et al.PNAS ?
October 26, 1999 ?
vol. 96 ?
no. 22 ?
will be invaluable for elucidating the physiological mechanisms
of these enigmatic compounds.
These results support the findings that neuroactive steroids
have unique, noninteracting binding requirements on
GABAA-Rs distinct from those of benzodiazepines and barbi-
turates (19, 21). Additionally, the differential behavioral re-
sponse to sedative?hypnotic agents indicates that different drugs
have different molecular targets as suggested by Eger et al. (38)
and illustrated by the GABAA-R ?3 knockout (9). It remains to
be determined whether ? subunit-containing GABAA-Rs rep-
resent actual targets of steroid action or whether the behavioral
sensitivity is indirectly altered in the ??/?mice.
Although these results (i.e., reduced behavioral response to
neuroactive steroids in ??/?mice) may appear at odds to those
with Zhu et al. (15) (i.e., enhanced sensitivity to neuroactive
steroids by GABAA-Rs lacking ?), one must keep in mind that
in vivo behavioral changes may not necessarily be directly related
to in vitro findings using recombinant receptors, because of the
complexity of the nervous system, the circuits involved, pleio-
tropic changes in a chronic model (lifetime null mutation), etc.
Detailed in vitro studies will be needed to analyze the possible
mechanism(s) responsible for the altered neuroactive steroid
sensitivity. One possibility would be that the GABAA-Rs re-
maining in ??/?mice have reduced sensitivity to neuroactive
The dentate gyrus granule cell electrophysiology (chosen
because it is one of three major cell types that express ?) suggests
that there is no drastic reduction in synaptic inhibition in these
cells because of the lack of decrease in mIPSC amplitude and
frequency. Rather, there may be a change in the molecular
composition of the GABAA-Rs involved. The observed faster
decay of mIPSCs is consistent with such a possibility. The
electrophysiological data also suggest ?-subunit involvement in
normal synaptic transmission in the dentate gyrus.
Analysis of the expected Mendelian ratio of pups and the
fecundity of wild-type versus null breeding pairs indicated that
deletion of the ? subunit impacted reproduction: litters from
??/?parents had fewer than the expected number of null pups
and ??/?parents had fewer pups per litter than ??/?parents. The
reduced sensitivity to neuroactive steroids may affect multiple
aspects of reproduction and development, an intriguing possi-
bility that warrants further investigation.
In conclusion, we have shown that a global deletion of the ?
subunit of the GABAA-R resulted in a decrease in the sensitivity
of mice to the sedative?hypnotic, anxiolytic, and pro-absence
effects of neuroactive steroids, and this change was remarkably
specific. Together, these results reveal a central involvement of
?-containing GABAA-Rs in neuroactive steroid action in mul-
tiple behavioral modalities.
We thank Carolyn Ferguson, JoAnn Steinmiller, Frank Kist, Janey
Whalen, and Jodi Daggett for expert technical assistance. This work was
supported by the University Anesthesiology and Critical Care Medicine
Foundation, the National Institutes of Health (Grants AA10422 to
G.E.H., GM52035 to L.L.F., and NS28772 to R.W.O.), and the National
Science Foundation (Grant IBN 9723295 to M.S.F.).
1. Barnard, E. A., Skolnick, P., Olsen, R. W., Mohler, H., Sieghart, W., Biggio,
G., Braestrup, C., Bateson, A. N. & Langer, S. Z. (1998) Pharmacol. Rev. 50,
2. Homanics, G. E., Quinlan, J. J., Mihalek, R. & Firestone, L. L. (1998) Toxicol.
Lett. 100–101, 301–307.
3. Lu ¨ddens, H. & Korpi, E. R. (1996) Neuroscientist 2, 15–23.
4. Olsen, R. W., DeLorey, T. M., Gordey, M. & Kang, M.-H. (1999) in Jasper’s
Basic Mechanisms of the Epilepsies: Advances in Neurology, eds. Delgado-
Escueta, A. V., Wilson, W. A., Olsen, R. W. & Porter, R. J. (Lippincott,
Philadelphia), 3rd Ed., Vol. 79, pp. 499–510.
5. Olsen, R. W. & Sapp, D. W. (1995) Adv. Biochem. Psychopharmacol. 48, 57–74.
6. Gu ¨nther, U., Benson, J., Benke, D., Fritschy, J., Reyes, G., Knoflach, F.,
Crestani, F., Aguzzi, A., Arigoni, M., Lang, Y., et al. (1995) Proc. Natl. Acad.
Sci. USA 92, 7749–7753.
C., Mi, Z. P., Wang, X. H., Grayson, D. R. & Firestone, L. L. (1997) Mol.
Pharmacol. 51, 588–596.
8. Mihic, S., Ye, Q., Wick, M., Koltchine, V., Finn, S., Krasowski, M., Hanson, K.,
Mascia, M., Valenzuela, C., Greenblatt, E., et al. (1997) Nature (London) 389,
9. Quinlan, J. J., Homanics, G. E. & Firestone, L. L. (1998) Anesthesiology 88,
10. Huntsman, M. M., Porcello, D. M., Homanics, G. E., DeLorey, T. M. &
Huguenard, J. R. (1999) Science 283, 541–543.
11. DeLorey, T. M., Handforth, A., Anagnostaras, S. G., Homanics, G. E.,
Minassian, B. A., Asatourian, A., Fanselow, M. S., Delgado-Escueta, A.,
Ellison, G. D. & Olsen, R. W. (1998) J. Neurosci. 18, 8505–8514.
C., Fritschy, J.-M., Luscher, B. & Mohler, H. (1999) Nat. Neurosci. 2, 833–839.
13. Flood, J. F., Morley, J. E. & Roberts, E. (1992) Proc. Natl. Acad. Sci. USA 89,
14. Shivers, B. D., Killisch, I., Sprengel, R., Sontheimer, H., Kohler, M., Schofield,
P. R. & Seeburg, P. H. (1989) Neuron 3, 327–337.
15. Zhu, W. J., Wang, J. F., Krueger, K. E. & Vicini, S. (1996) J. Neurosci. 16,
16. Saxena, N. & Macdonald, R. (1996) Mol. Pharmacol. 49, 567–579.
17. Laurie, D. J., Seeburg, P. H. & Wisden, W. (1992) J. Neurosci. 12, 1063–1076.
18. Laurie, D. J., Wisden, W. & Seeburg, P. H. (1992) J. Neurosci. 12, 4151–4172.
Cell. Mol. Neurobiol. 16, 155–174.
20. Harrison, N. L. & Simmonds, M. A. (1984) Brain Res. 323, 287–292.
21. Morrow, A. L., Pace, J. R., Purdy, R. H. & Paul, S. M. (1990) Mol. Pharmacol.
22. Gasior, M., Carter, R. B., Goldberg, S. R. & Witkin, J. M. (1997) J. Pharmacol.
Exp. Ther. 282, 543–553.
23. Nagy, A., Cocza, E., Merenties Diaz, E., Prideaux, V. R., Ivanyi, E., Markkula,
M. & Rossant, J. (1990) Development (Cambridge, U.K.) 110, 815–821.
24. Homanics, G. E. (1991) Dev. Genet. 12, 371–379.
25. Jones, A., Korpi, E. R., McKernan, R. M., Pelz, R., Nusser, Z., Makela, R.,
Mellor, J. R., Pollard, S., Bahn, S., Stephenson, F. A., et al. (1997) J. Neurosci.
26. Jechlinger, M., Pelz, R., Tretter, V., Klausberger, T. & Sieghart, W. (1998)
J. Neurosci. 18, 2449–2457.
27. Ma ¨kela ¨, R., Uusi-oukari, M., Homanics, G. E., Quinlan, J. J., Firestone, L. L.,
Wisden, W. & Korpi, E. R. (1997) Mol. Pharmacol. 52, 380–388.
28. Spigelman, I., Zhang, L. & Carlen, P. L. (1992) J. Neurophysiol. 68, 55–69.
29. Pellow, S., Chopin, P., File, S. E. & Briley, M. (1985) J. Neurosci. Methods 14,
30. Nakahiro, M., Arakawa, O., Narahashi, T., Ukai, S., Kato, Y., Nishinuma, K.
& Nishimura, T. (1992) Neurosci. Lett. 138, 5–8.
31. Anagnostaras, S. G., Maren, S. & Fanselow, M. S. (1999) J. Neurosci. 19,
(1994) Cell 79, 59–68.
33. DeLorey, T. M., Handforth, A., Homanics, G. E., Minassian, B. A., Anag-
nostaras, S. G., Asatourian, A., Ellison, G., Fenslow, M. S., Delgado- Escueta,
A. V. & Olsen, R. W. (1998) J. Neurosci. 18, 8505–8514.
34. Wang, Y., Salvaterra, P. & Roberts, E. (1979) Biochem. Pharmacol. 28,
35. Matsumoto, K., Uzunova, V., Pinna, G., Taki, K., Uzunov, D., Mienville, J.,
Guidotti, A. & Costa, E. (1999) J. Neuropharmacol. 38, 955–963.
36. Lister, R. G. (1987) Psychopharmacology 92, 180–185.
37. Snead, O. C. III (1998) Ann. Neurol. 44, 688–691.
38. Eger, E. I., Koblin, D. D., Harris, R. A., Kendig, J. J., Pohorille, A., Halsey,
M. J. & Trudell, J. R. (1997) Anesth. Analg. 84, 915–918.
www.pnas.orgMihalek et al.