Article

Attenuated sensitivity to neuroactive steroids in GABAA receptor δ-subunit knockout mice

University of Turku, Turku, Southwest Finland, Finland
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/1999; 96(22):12905-10. DOI: 10.1073/pnas.96.22.12905
Source: PubMed
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.

Full-text

Available from: Timothy M Delorey
Attenuated sensitivity to neuroactive steroids in
-aminobutyrate type A receptor delta subunit
knockout mice
Robert M. Mihalek
a
, Pradeep K. Banerjee
b
, Esa R. Korpi
c
, Joseph J. Quinlan
a
, Leonard L. Firestone
a
, Zhi-Ping Mi
d
, Carl Lagenaur
d
,
Verena Tretter
e
, Werner Sieghart
e
, Stephan G. Anagnostaras
f
, Jennifer R. Sage
f
, Michael S. Fanselow
f
, Alessandro Guidotti
g
,
Igor Spigelman
h
, Zhiwei Li
b,h
, Timothy M. DeLorey
j
, Richard W. Olsen
b,k
, and Gregg E. Homanics
a,i,l
Departments of
a
AnesthesiologyCritical Care Medicine,
i
Pharmacology, and
d
Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA
15261;
j
Molecular Research Institute, 2495 Old Middlefield Way, Mountain View, CA 94043;
c
Department of Pharmacology and Clinical Pharmacology,
University of Turku, FIN-20520 Turku, Finland;
e
University Clinic for Psychiatry, Section of Biochemical Psychiatry, Wa¨hringer Gu¨rtel 18–20, A-1090 Vienna,
Austria;
f
Department of Psychology, University of California, Los Angeles, CA 90095-1563;
g
Psychiatric Institute, Department of Psychiatry, University of
Illinois, Chicago, IL 60612;
h
Division of Oral Biology and Medicine, University of California School of Dentistry and Departments of
b
Molecular and Medical
Pharmacology and
k
Anesthesiology, 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 sedativehypnotic 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 sedativehypnotic responses and behavior and
provide insights into the physiology of neurosteroids.
I
nhibitory ion currents in the vertebrate central nervous system
primarily are carried by Cl
ions conducted via
-aminobutyric
acid type A receptors (GABA
A
-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).
GABA
A
-Rs are modulated by many drugs, including ethanol,
benzodiazepines, various anesthetics, and neuroactive steroids
(2–5). Specific roles for various GABA
A
-R subunits in mediating
anesthetic responses are beginning to be elucidated (6–9).
Additionally, GABA
A
-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
GABA
A
-Rs that exhibit a unique pharmacology. Such receptor
isoforms are benzodiazepine-insensitive (14), neuroactive ste-
roid-insensitive (15), and Zn
2
-sensitive (16). Nearly 30% of
cerebellar GABA
A
-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
GABA
A
-Rs (19, 20). Those synthesized in the brain, termed
neurosteroids, are believed to regulate anxiety, stress, and
neuronal excitability by modulating GABA
A
-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 GABA
A
-R
function and has been used clinically as an i.v. anesthetic. The
chemical analog ganaxalone (3
-hydroxy-3
-methyl-5
-
pregnan-20-one) was developed for improved bioavailability and
potential anxiolytic and anticonvulsant activity (22).
To investigate the contribution of
-containing GABA
A
-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 129SvJ 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. 1 A).
Three correctly targeted ES cell lines were microinjected into
C57BL6J blastocysts, two of which produced chimeric mice.
Highly chimeric males were mated to C57BL6J females (The
Jackson Laboratory). Agouti offspring that were heterozygous
for the targeted allele (
/
) were interbred to produce mice that
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 C57BL6J X strain 129SvSvJ, F
2
-F
5
generation.
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% formaldehyde1% agarose gel, blotted to Hybond-N
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: GABA,
-aminobutyric acid; GABA
A
-R, GABA type A receptor; mIPSC, min-
iature inhibitory postsynaptic current; PTZ,pentylenetetrazol; LORR, loss of righting reflex;
ES, embryonic stem.
l
To whom reprint requests should be addressed at: University of Pittsburgh School of
Medicine, Departments of AnesthesiologyCritical Care Medicine and Pharmacology,
W1356 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: homanics@smtp.anes.
upmc.edu.
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.
PNAS
October 26, 1999
vol. 96
no. 22
12905–12910
NEUROBIOLOGY
Page 1
(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
purifying by affinity chromatography. The antibody is specific for
the
subunit and does not precipitate
1
3
2 receptors (26).
Equal amounts of cerebellar membrane proteins were subject to
SDSPAGE 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
were detected by fluorescence using the CSPD substrate (Tropix,
Bedford, MA). Blots were exposed to Kodak X-Omat S film and
recorded with a DocuGel 2000i gel system using
RF LPSCAN
software (MWG Biotec, Ebersberg, Germany).
GABA
A
-R Ligand Binding. [
3
H]Ro15–4513 and [
3
H]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
[
3
H]Ro 15–4513 (a benzodiazepine site ligand) and [
3
H]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 CaCl
2
, 2 mM MgCl
2
, 26 mM NaHCO
3
, and 10 mM
dextrose. The ACSF was continuously bubbled with a 95%5%
mix of O
2
CO
2
to ensure adequate oxygenation of slices and a
pH of 7.4. Patch pipettes contained 135 mM Cs gluconate, 2 mM
MgCl
2
, 1 mM CaCl
2
, 11 mM EGTA, 10 mM Pipes, 2 mM K
2
ATP,
and 0.2 mM Na
2
GTP, 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 mgkg and 16 mgkg), preg-
nanolone (16 mgkg), midazolam (25 mgkg), and propofol (50
mgkg) were administered i.v. Etomidate (20 mgkg), pento-
barbital (45 mgkg), and ketamine (150 mgkg) were given by
i.p. injection. Injection volumes were 5
lg body weight for i.v.
and 20
lg for i.p. Neuroactive steroids [alphaxalone (Research
Biochemicals, Natick, MA) and pregnanolone (Sigma)] were
dissolved in a 22.5% (wtvol) 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-ClampWithdrawal Assay.
LORR and tail-clampwithdrawal 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
Fig. 1. Gene targeting and molecular characterization. (A) Structure of
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
-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.
12906
www.pnas.org Mihalek et al.
Page 2
each concentration of anesthetic, a tail-clamp stimulus was given.
If any motor activity occurred as a result of the stimulus, the
concentration was scored as one that permitted a positive
response.
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 GABA
A
-R
function (30).
Ganaxolone (10 mgkg, i.p.) or an equal volume of saline was
injected 10 min before testing (n 9group). 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
opentotal 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 mgkg, i.p.) or saline
occurred 10 min before PTZ (Sigma) (20 mgkg, i.p.) injection.
The behavior induced by salinePTZ versus ganaxolonePTZ
was observed and recorded for 2–3 h post-PTZ treatment. Data
are expressed as mean duration of hypoactivity SEM (n
9group). 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
6 min. Freezing (% time SEM) was scored continuously during
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
training.
Results
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
wild-type
gene and a 7.7-kb BamHI fragment from the targeted
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
Fig. 1 A.
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.
Western blots from cerebellar membranes (the neuronal tissue
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
allele.
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 F
2
and F
5
pups 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
5% of
/
pups die before weaning. To determine whether the
knockout had any effect on fecundity, true breeding F
3
lines
were established. These breeding experiments revealed that
/
breeding pairs produced statistically fewer pups per litter than
the
/
breeding pairs [
/
matings: 7.6 0.4 pupslitter (n
47 litters) vs.
/
matings: 6.3 0.3 pupslitter (n 50 litters)]
(P 0.01).
Pharmacological Characterization. Ligand binding in whole brain
homogenates from
/
and
/
mice was studied with
[
3
H]Ro15–4513 and [
3
H]muscimol. The slopes of the binding
isotherms were comparable (Table 1), allowing valid comparison
of the K
d
values. Maximal receptor number and binding affinity
for [
3
H]muscimol in homogenates from
/
brains were similar
to previous reports (7, 34). However, the maximal binding for
muscimol was markedly decreased in
/
homogenates (Table
1). The binding affinity for [
3
H]Ro15–4513 in homogenates from
/
was similar to previous reports (7). The maximal receptor
number and affinity constants for [
3
H]Ro15–4513 were not
different between
/
and
/
brain homogenates (Table 1).
Table 1. Binding of [
3
H]Ro15-4513 and [
3
H]muscimol to whole
brain homogenates
Wild type Null
[
3
H]Ro15-4513 binding*
Apparent K
d
(nM) 8.9 1.2 9.1 1.0
Maximal binding (pmolmg) 1.4 0.1 1.2 0.1
Slope 1.2 0.2 1.3 0.3
[
3
H]Muscimol binding
Apparent K
d
(nM) 37.0 9.9 21.0 10.9
Maximal binding (pmolmg) 4.5 0.4 2.4 0.4
Slope 1.1 0.2 1.1 0.4
*These data were pooled from four separate binding experiments for each
genotype. Each experiment consisted of five specific binding determinations
for each [
3
H]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 [
3
H]muscimol concentration tested.
P 0.02.
Mihalek et al. PNAS
October 26, 1999
vol. 96
no. 22
12907
NEUROBIOLOGY
Page 3
Using in situ autoradiography to examine the abundance and
distribution of GABA and benzodiazepine sites, it was observed
that [
3
H]muscimol binding was drastically reduced in most
regions of
/
brains (Fig. 2). In contrast, the [
3
H]Ro 15–4513
binding was slightly elevated in several regions of the
/
brain,
including the thalamus, striatum, and the cerebellar granule cell
layer.
Delta Knockout Mice Exhibit Altered Synaptic GABA
A
Currents.
mIPSCs were recorded in hippocampal slices from adult
/
and
/
mice to directly investigate the possibility of altered
GABA
A
-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-
nanolone (Table 3). The
/
mice had a 54% and 38% reduction
in sleep time duration compared with
/
at the two concen-
trations of alphaxalone used (8 mgkg and 16 mgkg), respec-
tively. Comparison of
/
and
/
after an 8 mgkg injection
of pregnanolone revealed a 42% reduction in sleep time in
/
mice. Intraperitoneal injection of alphaxalone (75 mgkg) 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
froma16mgkg i.v. injection. After an average sleep time of
45.3 7.4 min,
/
brains had 5.5 0.8 (n 2)
gg
pregnanolone, and after an average sleep time of 33.2 4.9 min,
/
brains had 7.3 1.2 (n 3)
gg pregnanolone. The level
of endogenous allopregnanolone also was measured in the same
mice:
/
, 514 48 pgg(n 2) and
/
, 394 118 pgg(n
3). These preliminary results indicate no gross differences
between genotypes in neuroactive steroid pharmacokinetics or
endogenous levels.
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, C57BL6J and strain
129SvJ mice, were tested with alphaxalone (16 mgkg) and
pregnanolone (16 mgkg). Sleep times were not statistically
different between strains for either compound: alphaxalone,
C57BL6J (8.1 1.3 min., n 10) and strain 129SvJ (9.1 2.7
min., n 10) (P 0.34) and pregnanolone, C57BL6J (38.4
Fig. 2. Autoradiographic distribution of [
3
H]muscimol and [
3
H]Ro 15–4513
binding sites in horizontal brain sections of GABA
A
-R
/
and
/
mice.
Representative images illustrate the reduction of [
3
H]muscimol binding and
region-specific elevation of [
3
H]Ro 15–4513 binding. Gr, cerebellar granule
cell layer; Th, thalamus; Str, striatum.
Table 2. Hippocampal electrophysiology
Genotype Wild type Null
Rise time (msec) 1.53 0.11 1.49 0.10
Amplitude (mV) 11.30 1.05 9.92 0.65
Frequency (Hz) 3.04 0.42 2.85 0.26
Decay,
(msec) 7.05 0.39 5.74 0.18*
# Cellsmice 1614 126
is the time from 90% to 37% of decay phase of mIPSC.
*P 0.05.
Table 3. Sleep time assays
Drug (dose) n Genotype
Sleep time, min
(mean SEM)
Alphaxalone (8 mgkg) 18 Wild type 3.5 0.5
11 Null 1.6 0.4*
Alphaxalone (16 mgkg) 17 Wild type 8.6 0.8
13 Null 5.3 0.7
Pregnanolone (8 mgkg) 18 Wild type 24.1 1.8
12 Null 14.0 2.2
Pentobarbital (45 mgkg) 25 Wild type 56.4 3.2
21 Null 52.2 3.6
Propofol (50 mgkg) 16 Wild type 14.1 1.8
11 Null 13.7 2.5
Midazolam (25 mgkg) 22 Wild type 14.4 4.0
16 Null 13.2 4.8
Etomidate (20 mgkg) 19 Wild type 55.3 5.0
20 Null 42.0 4.5
Ketamine (150 mgkg) 6 Wild type 40.1 3.8
6 Null 37.5 2.6
*
, P 0.02; †, P 0.01; ‡, P 0.002.
12908
www.pnas.org Mihalek et al.
Page 4
3.5 min., n 9) and strain 129SvJ (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 mgkg i.p. dose of ganaxolone in
/
mice
caused open-arm entries to increase nearly 2-fold (Fig. 3A;
*
, P
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 GABA
A
-R agonists, including neuroactive steroids
(37). Using a low-dose PTZ absence seizure model, ganaxolone
(10 mgkg, 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-clampwithdrawal)
effects of two halogenated volatile anesthetics. There was no
difference statistically in the tail-clamp response between
/
and
/
mice in the EC
50
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).
Discussion
The absence of the
subunit of the GABA
A
-R resulted in a
significant decrease in the sensitivity to neuroactive steroids. The
duration of alphaxalonepregnanolone anesthesia and the anxi-
olytic effect and pro-absence seizure effect of ganaxolone all
were reducedablated. The reduced response of
/
mice to
neuroactive steroids reveals a potential role of
-containing
GABA
A
-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 sedativehypnotic agents
in the sleep time assay or on the LORR or tail clampwithdrawal
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
Fig. 3. Anxiolytic (A) and pro-absence (B) effects of ganaxolone in
/
and
/
mice. (A) Mice were injected with ganaxolone (10 mgkg, i.p.) 10 min
before testing on the elevated plus-maze assay (n 9group). Ganaxolone
produced an 2-fold increase in the number of open-arm entries in
/
(
*
, P
0.01, Bonferroni’s post-hoc test), whereas no significant change was observed
in
/
.(B) Mice were treated with ganaxolone (10 mgkg, i.p.) or saline (n
9group) 10 min before an absence seizure-producing dose of PTZ (20 mgkg).
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).
Fig. 4. Pavlovian fear conditioning. Mice were given three tone-shock pair-
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.
Table 4. Behavioral responses to volatile anesthetics
Genotype n Anesthetic
EC
50
(atm % SEM)
Slope
(mean SEM)
Tail clamp
Wild type 30 Halothane 1.55 0.03 10.71 1.66
Null 29 Halothane 1.50 0.04 11.11 1.72
Wild type 20 Enflurane 2.43 0.05 11.11 2.03
Null 20 Enflurane 2.32 0.05 11.86 2.09
LORR assay
Wild type 19 Halothane 0.72 0.04 8.83 2.74
Null 15 Halothane 0.71 0.04 10.65 1.47
Mihalek et al. PNAS
October 26, 1999
vol. 96
no. 22
12909
NEUROBIOLOGY
Page 5
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
GABA
A
-Rs distinct from those of benzodiazepines and barbi-
turates (19, 21). Additionally, the differential behavioral re-
sponse to sedativehypnotic agents indicates that different drugs
have different molecular targets as suggested by Eger et al. (38)
and illustrated by the GABA
A
-R
3 knockout (9). It remains to
be determined whether
subunit-containing GABA
A
-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 GABA
A
-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 GABA
A
-Rs re-
maining in
/
mice have reduced sensitivity to neuroactive
steroids.
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 GABA
A
-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 GABA
A
-R resulted in a decrease in the sensitivity
of mice to the sedativehypnotic, anxiolytic, and pro-absence
effects of neuroactive steroids, and this change was remarkably
specific. Together, these results reveal a central involvement of
-containing GABA
A
-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,
291–313.
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.
7. Homanics, G. E., Ferguson, C., Quinlan, J. J., Daggett, J., Snyder, K., Lagenaur,
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,
385–389.
9. Quinlan, J. J., Homanics, G. E. & Firestone, L. L. (1998) Anesthesiology 88,
775–780.
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.
12. Crestani, F., Lorez, M., Baer, K., Essrich, C., Benke, D., Laurent, J. P., Belzung,
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,
1567–1571.
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,
66486656.
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.
19. Lambert, J. J., Belelli, D., Hill-Venning, C., Callachan, H. & Peters, J. A. (1996)
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.
37, 263–270.
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.
17, 1350–1362.
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,
149–167.
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,
1106–1114.
32. Bourtchuladze, R., Frenguelli, B., Blendy, J., Cioffi, D., Schutz, G. & Silva, A. J.
(1994) Cell 79, 5968.
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,
1123–1128.
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, 688691.
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.
12910
www.pnas.org Mihalek et al.
Page 6
  • Source
    • "subunit knockout mice (Mihalek et al., 1999). These results thus suggest that ganaxolone may be useful in clinical practice for a subpopulation of patients in whom anxiety or PTSD symptoms are related to deficient ALLO biosynthesis. "
    [Show abstract] [Hide abstract] 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 posttraumatic 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 minutes before testing of SI mice, induced a dose-dependent reduction in aggression toward a same-sex intrude 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
    Full-text · Article · Sep 2014 · Frontiers in Cellular Neuroscience
  • Source
    • "the d subunit, are insensitive to a variety of benzodiazepines, but are sensitive to allopregnanolone [113,118]. Accordingly, d subunit GABA A receptors knockout mice show reduced sensitivity to allopregnanolone [118,119] . In addition to the differential sensitivity linked to sub-unit composition, there are differences linked to the regional brain localisation. "
    [Show abstract] [Hide abstract] ABSTRACT: Progesterone is a well-known steroid hormone, synthesized by ovaries and placenta in females, and by adrenal glands in both males and females. Several tissues are targets of progesterone and the nervous system is a major one. Progesterone is also locally synthesized by the nervous system and qualifies, therefore, as a neurosteroid. In addition, the nervous system has the capacity to bio-convert progesterone into its active metabolite allopregnanolone. The enzymes required for progesterone and allopregnanolone synthesis are widely distributed in brain and spinal cord. Increased local biosynthesis of pregnenolone, progesterone and 5α-dihydroprogesterone may be a part of an endogenous neuroprotective mechanism in response to nervous system injuries. Progesterone and allopregnanolone neuroprotective effects have been widely recognized. Multiple receptors or associated proteins may contribute to the progesterone effects: classical nuclear receptors (PR), membrane progesterone receptor component 1 (PGRMC1), membrane progesterone receptors (mPR), and γ-aminobutyric acid type A (GABAA) receptors after conversion to allopregnanolone. In this review, we will succinctly describe progesterone and allopregnanolone biosynthetic pathways and enzyme distribution in brain and spinal cord. Then, we will summarize our work on progesterone receptor distribution and cellular expression in brain and spinal cord; neurosteroid stimulation after nervous system injuries (spinal cord injury, traumatic brain injury, and stroke); and on progesterone and allopregnanolone neuroprotective effects in different experimental models including stroke and spinal cord injury. We will discuss in detail the neuroprotective effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABAA receptors.
    Full-text · Article · Sep 2014 · The Journal of Steroid Biochemistry and Molecular Biology
  • Source
    • "Given that direct intra-hippocampal ALLO produced a similar degree of object memory impairment as observed after systemic administration , the results are consistent with the view that the deleterious effects of ALLO on memory are a consequence of an inhibition of dorsal hippocampal neurons during critical periods of information processing. The δ subunit of the GABA-A receptor is richly expressed throughout the hippocampus and the dentate gyrus, and this subunit can influence neurosteroid sensitivity at GABA-A receptors (Belelli et al., 2002; Mihalek et al., 1999; Spigelman et al., 2003). Neurosteroids, such as ALLO, likely modulate neuronal signaling and synaptic plasticity by augmenting fast GABAergic neurotransmission (for a review see Belelli and Lambert, 2005 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.
    Full-text · Article · May 2014 · Hormones and Behavior
Show more