Effect of GABA on GnRH Neurons Switches from

Depolarization to Hyperpolarization at Puberty in the

Female Mouse
SEONG-KYU HAN, ISTVAN M. ABRAHAM, AND ALLAN E. HERBISON
Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom
The amino acid -aminobutyric acid (GABA) plays an impor-
tant role in the regulation of the GnRHneurons. We examined
whether GABA depolarizes or hyperpolarizes GnRH neurons
over postnatal development using gramicidin, perforated-
patch electrophysiology combined with GnRH-LacZ trans-
genic mice in whom GnRH neurons can be made to fluoresce.
The basic membrane properties and GABA responsiveness of
GnRH neurons were not altered by transgene expression or
fluorescence. Ten of 12 immature GnRH neurons (1017 d)
were depolarized by GABA in a direct and dose-dependent
manner that was blocked by a GABA
A
receptor antagonist. In
peripubertal GnRH neurons (2530 d), GABA exerted depo-
larizing (4/11) as well as hyperpolarizing (5/11) effects on
GnRHneurons. Inadult female mice, GABAwas foundtoexert
exclusively hyperpolarizing actions on GnRH neurons (9/10)
that were direct and mediated by the GABA
A
receptor. GABA
switched from depolarizing to hyperpolarizing actions
around postnatal d 31, the time of vaginal opening. Uniden-
tified preoptic area neurons exhibited predominantly hyper-
polarizing responses to GABA at all three postnatal stages.
These findings demonstrate that GnRH neurons display an
unusually late postnatal switch in their response to GABA.
They also provide the first direct evidence that GABAinhibits
the electrical activity of postpubertal GnRH neurons. (Endo-
crinology 143: 14591466, 2002)
G
nRH NEURONS REPRESENT the final output neurons
of the neuronal network controlling fertility in all
mammals. As such, an understanding of the neural control
of the GnRH neurons is critical. The amino acid transmitter
-aminobutyric acid (GABA) is thought to be one of the most
important neural regulators of GnRH neurons and has been
proposed to play key roles in the developmental migration
(1), pubertal activation (2), pulsatility, and gonadal steroid
feedback regulation (3) of the GnRH neurons. During em-
bryogenesis, GABA depolarizes GnRH neurons through the
direct activation of GABA
A
receptors (4), and this is likely to
aid in their correct temporal and spatial pattern of migration
and biosynthetic activity (57). Following birth, GABA
A
re-
ceptor signaling changes in GnRH neurons during the late
postnatal period (8), and studies in the monkey indicate that
alterations in GABAergic input are involved in the pubertal
activation of the GnRH neurons (9, 10). In the adult, inves-
tigations in several species have suggested that GABA acts
through GABA
A
receptors located within the vicinity of the
GnRH perikarya to modulate their pulsatile activity (1113).
Furthermore, a fall in local GABA release is implicated in the
generation of the preovulatory GnRH/LH surge in the fe-
male (1418), and steroid-dependent changes in GABAergic
transmission are believed to help bring about negative feed-
back in both the male and female (1922).
Despite the wealth of data indicating a critical role for
GABAinthe neural control of the GnRHneurons, several key
questions remain to be answered. Arguably, the most im-
portant of these is that of determining whether GABAexcites
or inhibits the electrical activity of postnatal GnRH neurons.
Although recent studies in normal (10, 23) and transgenic
(24) mice have shown that all GnRHneurons express GABA
A
receptors, the experimental conditions used in these studies
did not allow the investigators to determine whether GABA
depolarized or hyperpolarized these cells. It is well estab-
lished that GABA acts through GABA
A
receptors to excite
embryonic neurons but, under normal circumstances, then
switches in the first to second postnatal weeks to exert its
inhibitory actions (25). Although this depolarization-to-
hyperpolarization pattern of GABAergic influence is be-
lieved to occur in the great majority of neurons within the
brain, some neuronal phenotypes continue to exhibit depo-
larizing responses to GABA in the adult brain (2628). In
relation to the GnRHneurons, the GT1 immortalized cell line
is depolarized by GABA
A
receptor activation (29, 30), but
whether this reflects the embryonic nature of the GT1 cells
or is, instead, a true reflection of adult GnRH neurons is
unknown. Furthermore, despite the great majority of in vivo
studies reportinginhibitoryactions of GABAonLHsecretion
in adult mammals (3), it is intriguing that the very early
investigations all showedstimulatory effects of GABAon LH
release (31, 32).
In the current series of studies, we have investigated the
effects of GABA
A
receptor activation upon the electrical ac-
tivity of GnRH neurons over the course of postnatal devel-
opment using gramicidin perforated-patch electrophysiol-
ogy in the female mouse. This method represents the only
patch-clamp technique through which the electrophysiolog-
ical responses of a neuron can be assessed without altering
Abbreviations: ACSF, Artificial cerebrospinal fluid; gal, -galacto-
sidase; CMFDG, 5-chloromethyl-fluorescein di--d-galactopyranosi-
dase; DIC, differential interference contrast; E
Cl
, chloride equilibrium
potential; GABA, -aminobutyric acid; GNLZ, GnRH-LacZ; MS, medial
septum; OVLT, organum vasculosum of the lamina terminalis; rACSF,
recording ACSF; rPOA, rostral preoptic area; TBS, Tris-buffered saline;
TTX, tetrodotoxin citrate.
0013-7227/02/$15.00/0 Endocrinology 143(4):14591466
Printed in U.S.A. Copyright 2002 by The Endocrine Society
1459
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the intracellular chloride environment; a critical point when
assessing GABA
A
receptor responses (3335). In the past, we
had undertaken electrophysiological investigations on
GnRH neurons in the nontransgenic mice through morpho-
logical identificationandpostrecording gene profiling (8, 23).
However, the gramicidin approach does not easily permit us
to extract the recorded cells cytoplasmic contents for post-
recording GnRH genotyping. In this study, we have there-
fore used GnRH-LacZ (GNLZ) transgenic mice (7), in asso-
ciation with fluorescein di--d-galactosidase compounds
(36), to preidentify GnRH neurons through fluorescence in
the acute brain slice procedure.
Materials and Methods
GNLZ transgenic mice
The production and analysis of the 5.5-GNLZ-3.5 lines of mice have
been reported in detail elsewhere (7). In brief, transgenic mice (C57/
Bl6J CBA/Ca) were produced by pronuclear injection of fertilized
mouse eggs with a construct comprising all the introns and exons of
GnRH-1, in addition to 5.5 kb of upstream 5 and 3.5 kb of downstream
3 sequence, into whicha nuclear-localizedLacZcassette was engineered
into exon 2 sequences. As assessed by the presence of -galactosidase
(gal) immunoreactivity, approximately 80% of postnatal GnRH neu-
rons in these mice express nuclear-located transgene (7). Homozygous
and heterozygous 5.5-GNLZ-3.5 mice, which appear normal in all re-
spects including their fertility, were bred and housed under conditions
of 12 h of light (lights on at 0700 h) with constant access to foodandwater
and treated in accordance with UK Home Office requirements under
projects 80/1475. The day of vaginal opening occurs between postnatal
d 28 and 31 in female mice under these housing conditions. Vaginal
smears were taken from adult female mice to determine the stage of the
estrous cycle.
Brain slice electrophysiology
Immature (postnatal d 1017), peripubertal (postnatal d 2530), and
adult (postnatal d 3655) female 5.5-GNLZ-3.5 and wild-type mice were
killed by cervical dislocation, decapitated between 1000 and 1200 h, their
brains rapidly removed, and placed in ice-cold, bicarbonate-buffered,
artificial cerebrospinal fluid (ACSF) of the following composition (in
mm): 118 NaCl, 3 KCl, 0.5 CaCl
2
, 6.0 MgCl
2
, 11 d-glucose, 10 HEPES, and
25 NaHCO
3
(pH 7.4 when bubbled with 95% O
2
and 5% CO
2
). Brains
were blocked and glued with cyanoacrylate to the chilled stage of a
vibratome and 150- to 200-m-thick coronal slices containing the medial
septum and preoptic area prepared. Slices from transgenic mice were
then incubated in ACSF containing 1040 m 5-chloromethyl-fluores-
cein di--d-galactopyranosidase (CMFDG, Molecular Probes, Inc., Eu-
gene, OR) at room temperature in the dark. The slices were then incu-
bated at 30 C for 30 min in oxygenated recording ACSF (rACSF)
consisting of (in mm): 118 NaCl, 3 KCl, 2.5 CaCl
2
, 1.2 MgCl
2
, 11 d-
glucose, 10 HEPES, and 25 NaHCO
3
, and thereafter kept at room tem-
perature in rACSF for at least 1 h before recording. Slices were trans-
ferred to the recording chamber, held submerged, and continuously
superfused with rACSF at a rate of 45 ml/min. The slices were viewed
with an upright fluorescence Axioskop FS microscope (Carl Zeiss, Jena,
Germany) with 10 or 40 immersion objectives (Achroplan 0.75W,
Ph2, Carl Zeiss) and either fluorescence illumination using the fluores-
cein isothiocyanate filter block 15 (H546) (Carl Zeiss) or Normaski dif-
ferential interference contrast (DIC) optics. Slices were initially exam-
ined under fluorescence with the low-power objective to determine the
distribution of fluorescent cells. A single GnRH neuron was then
brought into focus under the high-power objective using fluorescence
for 510 sec before switching to DIC optics. Following patching of the
cell under DIC optics, it was then briefly (5 sec) examined under fluo-
rescence again to confirm its fluorescent identity. All recordings were
made at room temperature (2023 C).
Patch pipettes were pulled from thin-wall borosilicate glass capillary
tubing (GC150TF, 1.5 mm outer diameter, Harvard Apparatus Ltd.,
Edenbridge, UK) on a Flaming/Brown puller (P-97; Sutter Instruments
Co., Novato, CA). The patch pipette solution was passed through a
disposable 0.22-m filter before use and contained (in mm): 130 KCl, 5
NaCl, 0.4 CaCl
2
, 1 MgCl
2
, 10 HEPES, 1.1 EGTA, with pH adjusted to 7.3
with KOH. Gramicidin (Sigma, St. Louis, MO) was first dissolved in
dimethylsulfoxide (Sigma) to a concentration of 2.55 mg/ml and then
diluted in the pipette solution just before use to a final concentration of
2.55 g/ml and sonicated for 10 min. Before backfilling the electrode
with the gramicidin-containing solution, the tip of the electrode was
always loaded with a small volume of gramicidin-free pipette solution.
The gramicidin-perforated patch clamp recordings were performed us-
ing an Axoclamp-2B amplifier (probe gain, 0.1 MU with HS-2 probe;
Axon Instruments, Foster City, CA) operating in bridge mode. The tip
resistance of the electrodes was 68 M. The junction potential between
the patch pipettes and bath solution was nulled before giga-seal for-
mation. Experiments began 1520 min after giga-seal formation when
the resting membrane potential of the cell reached a stable level below
45 mV (33). Spontaneous activities were sampled online using a
Digidata 1200 interface (Axon Instruments) connected to an IBM
PC/AT clone (Evesham, Cambridge, UK). Signals were filtered (3
kHz, Bessel filter of Axoclamp-2B) before digitizing at a rate of 2 kHz.
Acquisition and subsequent analysis of the acquired data were per-
formed using the Clampex7 suite of software (Axon Instruments).
Traces were plotted using Origin 5 computer software (MicroCal
Software, Northampton, MA).
Test compounds were dissolved in the rACSF solution and tested by
adding to the perfusing rACSF at known concentrations. Previous stud-
ies in the mouse have shown that GABAresponses are mediated entirely
by the GABA
A
receptor (8, 2224). Accordingly, we used GABA (10, 30,
100 m) to test GnRH neuron responses and bicuculline methiodide
(2050 m) to confirm the GABA
A
receptor dependency. Tetrodotoxin
citrate (TTX, 0.5 m) was used to demonstrate direct effects of GABAon
recorded cells. All compounds were purchased from Tocris Cookson
(Bristol, UK).
Immunocytochemistry
Slices treated with CMFDG as above were placed in 4% paraformal-
dehyde in phosphate-buffered solution (pH 7.6) for 20 h at 4 C. Fol-
lowing fixation, slices were rinsed several times in Tris-buffered saline
(TBS) at room temperature and then incubated with a polyclonal rabbit
antibody specific for GnRH (LR1, 1:5000; gift of Dr. R. Benoit, Montreal,
Canada) for 48 h at 4 C. After washing in TBS, slices were incubated in
Texas Red-labeled, antirabbit immunoglobulins (1:200, Vector Labora-
tories, Inc., Peterborough, UK) for 2 h at room temperature, washed,
mounted onto slides, and coverslipped with Vectashield (Vector Lab-
oratories, Inc.). All antibodies were dissolved in TBS containing 0.3%
Triton X-100 and 0.3% BSA. Slices were viewed using a DMRB fluores-
cence microscope (Leica Corp., Nussloch, Germany) with Texas and I3
filter blocks (Leica Corp.) at 40 or 100 objective magnification to
determine whether the cells were double labeled.
Statistical analysis
Experimental data were expressed as mean sem and the number
of neurones tested and analyzed was presented by n. The relationship
between postnatal days and membrane potential changes was deter-
mined by nonlinear regression analysis using Genstat 3.2 statistical
software (Lawes Agricultural Trust, Rothamsted, UK). One-way
ANOVA was performed to test the spontaneous firing frequency and
resting membrane potential differences among the postnatal age groups.
One population t test was used to assess significant changes in GABA-
induced membrane potential in immature, peripubertal, and adult
groups. A level of P less than 0.05 was considered to be significant.
Results
CMFDG fluorescence in GNLZ mice
The identity of fluorescent cells located within the medial
septum(MS) and rostral preoptic area (rPOA) was examined
by fixing CMFDG-treated brain slices and processing them
for GnRHimmunofluorescence (n 5). The pattern of GnRH
immunofluorescence was identical with that reported pre-
1460 Endocrinology, April 2002, 143(4):14591466 Han et al. Direct Effects of GABA on GnRH Neurons
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viously in the adult mouse brain (37). The CMFDGtreatment
resulted in the fluorescence of two populations of neurons:
one that overlapped that of the GnRHneurons in the MS and
rPOAand another in the lateral septum. Approximately 30%
of all GnRH-immunofluorescent neurons exhibited CMFDG
fluorescence regardless of their location within the MS and
rPOA (Fig. 1). The CMFDG fluorescent cells detected in the
lateral septum lacked GnRH immunofluorescence. The dis-
tribution of these lateral septal cells was the same as that
reported for gal expression in 5.5-GNLZ-3.5 mice (7) and all
other GnRH-LacZ transgenic mice (38) and represent cells
that continue to express gal despite the cessation of GnRH
biosynthesis in the adult (37). In relation to the CMFDG
fluorescence, all cells located within the midline MS, and
more than 90% of those around the organum vasculosum of
the lamina terminalis (OVLT) in the rPOA, were also found
to be immunofluorescent for GnRH (Fig. 1). Within the re-
gion of the OVLT, approximately one CMFDG-fluorescent
cell that did not exhibit detectable GnRH immunofluores-
cence was identified in each brain slice. It is unclear whether
these cells represent GnRHneurons with lowlevels of GnRH
peptide or are cells expressing the transgene in an ectopic
manner. The CMFDG fluorescence was equivalent in het-
erozygous and homozygous mice, but brain slices from non-
transgenic mice did not exhibit any CMFDG fluorescence.
Electrophysiological recordings from GnRH neurons
To evaluate whether the CMFDG treatment and/or brief
fluorescence exposure had any effects on GnRHneurons, we
made recordings from four immature CMFDG-fluorescent
GnRH neurons under whole-cell, patch-clamp conditions
identical with those used previously to investigate nontrans-
genic immature GnRH neurons (8). Equivalent values for
nontransgenic immature GnRH neurons (n 1021; 8) are
given in parentheses. All cells were spontaneously active
with mean spontaneous firing rates of 0.340.86 Hz (0.021.0
Hz) and a mean resting membrane potential of 63 1.2 mV
(64 2 mV). To assess GABA
A
receptor responses, we
examined the effect of 30 m GABA and found it to evoke a
mean membrane depolarization of 37 5 mV (27 6 mV).
These observations suggest that there are no major delete-
rious effects of the fluorescence procedure and that GABA
responsivity is maintained.
Using the gramicidin-perforated patch technique, record-
ings were made from 12 immature, 11 peripubertal, and 10
adult GnRH neurons located within the rPOA. In nearly all
cases, only a single recorded cell was obtained from each
mouse and animal n numbers were 11, 10, and 9, respec-
tively. The majority of recorded cells were adjacent to the
OVLT, and the rest were located dorsally in the midline
extending up into the MS. Under perforated patch, the mean
resting membrane potential was 51.8 2.2, 49.9 1.4,
and 49.0 1.7 mV; action potential amplitude 14.7 1.9,
13.4 3.0, and 13.2 1.3 mV; and mean firing frequency
3.9 0.5, 3.1 0.7, and 3.6 0.7 Hz, respectively. No
significant differences (ANOVA) were detected in any of
these parameters across the postnatal age groups examined.
In immature mice (postnatal d 1017), a 1-min exposure to
10, 30, and/or 100 m GABA was found to evoke 4.818.1
mV membrane depolarizations in 10 of 12 GnRH neurons
that were dose dependent, blocked completely by bicucul-
line, and repeatable (Fig. 2). In all cases, cell firing was re-
duced following the membrane depolarization although a
short-lived, transient increase in firing was usually observed
immediately following the onset of the depolarization (Fig.
2). The depolarizing effect of GABA on immature GnRH
neurons was maintained in the presence of TTX (n 2). In
two cells, GABA was not found to exert any effect on mem-
brane potential but reduced the action potential firing of one
cell.
In peripubertal female mice (postnatal d 2530), a range of
different effects of 10, 30, and 100 m GABA were observed
in GnRH neurons. Four of the 11 cells responded to GABA
with membrane depolarization, five exhibited membrane
hyperpolarizations, and two displayed no change in mem-
brane potential. One of the depolarized GnRH neurons in-
FIG. 1. Low- and high-power photomicro-
graphs of fluorescent GnRH neurons in
acute brain slices of the preoptic area pre-
pared from GnRH-LacZ mice. The CMFDG
fluorescence is green and the GnRH immu-
nofluorescence is red. Note in A and B that
the five fluorescent cells (arrowheads) lying
at different focal planes in the slice are all
immunofluorescent for GnRH, although one
GnRH neuron (arrow) is clearly not. Scale
bars represent 10 m.
Han et al. Direct Effects of GABA on GnRH Neurons Endocrinology, April 2002, 143(4):14591466 1461
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creased its firing rate, but the other depolarized cell and all
hyperpolarized cells reduced their overall firing frequency
during the GABA application.
Inadult female mice (diestrus or estrus, postnatal d3655),
testing with 10, 30, and/or 100 m GABA resulted in 2.87.0
mV hyperpolarizations of the membrane potential (Fig. 3) in
9 of 10 cells with the remaining cell showing no response. The
membrane hyperpolarization was always associated with a
decrease in action potential firing and was dose dependent
(Fig. 4A), blocked completely by bicuculline, and repeatable
(Fig. 3). As for the membrane depolarizations in younger
mice, the GABA-evoked hyperpolarizations persisted in the
presence of TTX (n 2). The stage of the estrous cycle did
not effect the hyperpolarizing response to GABA.
The plotting of mean data from the different age groups
(Fig. 4A) revealed that GABAevoked a clear dose-dependent
depolarization of membrane potential in immature GnRH
neurons in parallel to the hyperpolarization of adult cells, but
no overall directional effect of GABAon membrane potential
was recorded from peripubertal GnRH neurons. Nonlinear
regression analysis of the membrane response of each indi-
vidual GnRH neuron indicated that the time point at which
GABA responses were switching from depolarization to hy-
perpolarization within the recorded population of GnRH
neurons was around postnatal d 3031 (Fig. 4B).
Electrophysiological recordings from non-GnRH neurons
To assess the specificity of the late postnatal switch in
GABA response displayed by GnRH neurons, we also made
gramicidin perforated-patch recordings from unidentified
non-GnRH neurons located within the preoptic area of both
CMFDG-treated transgenic slices and wild-type mice at the
same postnatal time points. Under perforated patch, the
mean resting membrane potential of these cells was 49.2
2.7, 49.4 1.3, and 48.8 2.1 mV, respectively. In im-
mature mice, four of nine (44%) preoptic neurons were hy-
perpolarized by GABA (Fig. 5A), one was depolarized, and
the remaining four neurons were unresponsive. No signifi-
cant effect of GABA on membrane potential was detected
when the results were averaged (Fig. 6). All hyperpolarized
neurons responded with a decrease in firing (Fig. 5A). In
peripubertal animals, 8 of 13 (62%) preoptic neurons were
hyperpolarized by GABA, two were depolarized, and three
were unaffected. As a group, this resulted in a significant
(P 0.05) hyperpolarizing response to GABA (Fig. 6). In
adult animals, four of five (80%) preoptic neurons were hy-
perpolarized by GABA (Fig. 5B), and the remaining cell did
not respond. As a group, a significant (P 0.01) hyperpo-
larizing response was observed. Thus, in this heterogeneous
group of preoptic neurons, both the percentage of neurons
hyperpolarized by GABA(4480%) and the amplitude of the
hyperpolarizing effects of GABA (Fig. 6) were found to in-
crease with age. As with the responses in GnRHneurons, the
GABA-evoked hyperpolarizations were blocked completely
by bicuculline and persisted in TTX (n 3).
The differences in the membrane responses of GnRH and
FIG. 2. GABA depolarizes immature GnRHneurons. Gramicidin, per-
forated-patch recording of an immature GnRH neuron showing a
depolarizing response to 1-min exposure (black bar) to 30 M GABA
that is blocked completely by bicuculline (Bic) and repeatable after
washout. Resting membrane potential 53 mV.
FIG. 3. GABA hyperpolarizes adult GnRH neurons. Gramicidin, per-
forated-patch recording from an adult GnRH neuron showing a hy-
perpolarizing response to 1-min exposure (black bar) to 100 MGABA
that is blocked completely by bicuculline (Bic) and repeatable after
washout. Resting membrane potential 46 mV.
1462 Endocrinology, April 2002, 143(4):14591466 Han et al. Direct Effects of GABA on GnRH Neurons
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unidentified preoptic neurons to GABA are clearly evident
in the graph depicting mean membrane changes to 100 m
GABA throughout postnatal development (Fig. 6). Whereas
GABA significantly (P 0.01) depolarized immature GnRH
neurons, it had no significant effect on immature preoptic
cells. In contrast, in peripubertal animals, GABA had no
significant effects on GnRHneurons but hyperpolarized pre-
optic cells (P 0.05). In adults, GABA hyperpolarized (P
0.01) both GnRH and preoptic cells (Fig. 6).
Discussion
We report here the use of LacZ-based fluorescence to pre-
identify GnRH neurons in the acute brain slice procedure.
This method (36), which has been used previously in neu-
ronal preparations (39), provides an alternative to the use of
green fluorescent protein (24, 40) to identify GnRH neurons
in situ. A disadvantage of this method is that it permits us to
visualize only approximately 30% of the GnRH neuronal
population, compared with 6590% when using green flu-
orescent protein tagging (24, 40). Because more than 80% of
the GnRH neurons in GNLZ mice express immunoreactive
FIG. 4. Effect of GABA on GnRH neurons switches over postnatal
development. A, Plots showing the mean membrane potential re-
sponses of immature (n 12), peripubertal (n 11), and adult (n
10) GnRH neurons to 10, 30, and 100 M GABA exposure. Whereas
immature GnRH neurons display a dose-dependent membrane de-
polarization (open circles), peripubertal GnRHneurons showno over-
all response (open triangles) and adult GnRHneurons (filled squares)
exhibit a dose-dependent hyperpolarizing action of GABA. B, Scatter
plot showing the individual membrane potential changes recorded for
all GnRH neurons in response to 100 M GABA. The curve (y 50
64/[1 0.0089 * x]) was derived froma hyperbolic regression model
generated by the Genstat 3.2 statistical software package.
FIG. 5. Unidentified preoptic area neurons are hyperpolarized by
GABA in immature and adult mice. A, Gramicidin, perforated-patch
recording from a preoptic area neuron in a immature female mouse
showing a hyperpolarizing response to 100 M GABA (bar). Resting
membrane potential 47 mV. B, Gramicidin, perforated-patch re-
cording from a preoptic area neuron in an adult female mouse dis-
playing a hyperpolarizing response to 100 M GABA (bar). Resting
membrane potential 54 mV.
FIG. 6. Comparison of GABA responses between GnRH and uniden-
tified preoptic area neurons in the female mouse. Histograms show
mean ( SEM) membrane potential responses to 100 M GABA by
GnRH (open bars) and preoptic area (shaded) neurons recorded from
brain slices prepared from immature, peripubertal, and adult female
mice. *, P 0.05; **, P 0.01.
Han et al. Direct Effects of GABA on GnRH Neurons Endocrinology, April 2002, 143(4):14591466 1463
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gal, it seems most likely that the nonfluorescent GnRH
neurons have insufficient enzymatically active gal to cat-
alyze the CMFDG into its fluorescent derivatives. It is im-
portant to be aware that some cells targeted by GnRH pro-
moter transgenics do not represent authentic GnRHneurons
in the adult mouse brain (37, 38). However, these cells are
easily distinguishable from GnRH neurons through their
distinctive location in the lateral septum.
Using a protocol that involves minimal fluorescence illu-
mination, we show here that the membrane potential and
firing rate of fluorescent GnRH neurons, as well as the re-
sponsiveness of their GABA
A
receptors, are not altered when
compared with unmodified GnRH neurons (8). This is im-
portant evidence indicating that the transgene and/or
CMFDG fluorescence have no major deleterious effects on
the cell. When comparing fluorescent GnRH neurons re-
corded under different patch-clamp conditions, we did, un-
expectedly, find that their firing rate was substantially faster
in perforated patch mode, compared with whole-cell record-
ings. This suggests that intracellular constituents, which are
quickly replaced by the electrode solution in whole-cell
patch-clamp but not in perforated patch recordings, are
likely to be critical for GnRH neurons to fire at their normal
frequency. The less negative resting membrane potentials
found under perforated patch conditions is typical of this
mode and likely to be caused by the inevitable higher series
resistance encountered, compared with the whole-cell ap-
proach (33, 34).
We have found that GABA depolarizes GnRH neurons in
immature female mice but then switches about the time of
puberty to a hyperpolarizing action in the adult. Although
this pattern holds true for the great majority of GnRH neu-
rons we recorded, some cells were found that were unre-
sponsive to GABA at all three developmental stages. We
have encountered variability in GABA responses with im-
mature GnRH neurons in earlier recordings (8) and this may
reflect true heterogeneity within the GnRH population. Al-
ternatively, the heterogeneity may result from experimental
variability. For example, we have been unable to confirm
GnRH immunoreactivity in approximately 10% of fluores-
cent cells located in the OVLT region in our transgenic mice,
and it is possible that one or two of the 33 cells we recorded
were not GnRH neurons.
In good agreement with previous results (8, 23, 24), the
effects of GABA on mouse GnRH neurons were blocked
completely by bicuculline. This indicates that the GABA
A
receptor mediates all fast GABA actions at the level of the
GnRH perikarya in this species. This appears to be different
in the guinea pig in whom mediobasal hypothalamic GnRH
neurons express functional GABA
B
receptors (41). Although
our results show that the GABA
A
receptor-mediated depo-
larizing-to-hyperpolarizing switch exists for GnRH neurons
in much the same way that it does for most other neurons
(25), a major difference is that it occurs relatively late in
neuronal development. In regions such as the cortex and
hippocampus, GABA becomes hyperpolarizing at different
times in specific neuronal phenotypes but is predominantly
inhibitory by the second postnatal week (25, 4244). Within
the hypothalamus, GABA loses its excitatory actions around
postnatal d 10 in arcuate nucleus neurons (45), and oxytocin
neurons switch from GABA-mediated excitation to inhibi-
tion in the second postnatal week (46). Indeed, we showhere
that unidentified preoptic neurons, residing in the same gen-
eral region as the GnRHneurons, are mostly hyperpolarized
by GABA even within the second postnatal week. Thus, the
depolarization-hyperpolarization switch observed here in
the fifth postnatal week with GnRHneurons is very unusual.
Chloride and bicarbonate are the principal permeant ions
of the GABA
A
receptor. Whereas bicarbonate has been im-
plicated in mediating depolarizing effects of the GABA
A
receptor under specific circumstances within the adult brain
(26), it is chloride that is considered the principal ionophore
responsible for the developmental shift fromdepolarizing to
hyperpolarizing actions of the receptor (25, 44). When intra-
cellular chloride ion concentrations within a cell result in a
chloride equilibrium potential (E
Cl
) above the resting mem-
brane potential of that cell, opening of the GABA
A
receptor
will result in an efflux of chloride ions and, consequently,
membrane depolarization. In contrast, when chloride ion
concentrations are relatively low, setting the E
Cl
below the
resting membrane potential of the cell, opening of the
GABA
A
receptor will result in an inward flux of chloride and
hyperpolarization of the cell. As such, the principal deter-
minant of whether GABA depolarizes or hyperpolarizes a
neuron is the relationship between its intracellular chloride
ion concentration and membrane potential. Accordingly, the
slow fall in intracellular chloride levels within neurons dur-
ing early postnatal life is thought to underlie the develop-
mental switch in GABA actions (25, 44). Because the resting
membrane potential of GnRHneurons (65 to 70 mV) does
not change across postnatal development (23), our present
data imply that E
Cl
approaches the resting membrane po-
tential of the majority of GnRH neurons around postnatal d
31 and thereafter falls below this level to result in GABA
A
receptor hyperpolarization in adulthood.
Of the molecules now established to play a role in deter-
mining chloride ion homeostasis in neurons, the chloride ion
extruder KCC2 and importer NKCC1, alongside the voltage-
dependent chloride ion extruder ClC2, are all thought to be
developmentally regulated (28, 47, 48). However, it seems
most likely that the induction of KCC2 expression during the
first postnatal week is the principal determinant of the slow
fall in intracellular chloride concentrations within neurons
and the eventual hyperpolarizing effects of GABA (28). Im-
portantly, Ganguly et al. (49) have recently demonstratedthat
it is the increasing GABA-mediated depolarization occurring
with development that initiates KCC2 expression within a
cell and the consequent switch to GABA hyperpolarization.
The identification and expression profiling of transporter
molecules determining chloride ion homeostasis in GnRH
neurons has yet to be established. However, it is intriguing
to speculate that the very delayed GABA depolarization-
hyperpolarization switch observed here in GnRH neurons
may result simply from a relatively delayed onset of exci-
tatory drive to the GnRH neurons and resultant absence of
KCC2 expression until puberty.
Froma physiological perspective, our present findings are
of importance to the understanding of the process of puberty
and the neural control of GnRHsecretion in the adult female.
Although GABA-evoked depolarization was observed to ul-
1464 Endocrinology, April 2002, 143(4):14591466 Han et al. Direct Effects of GABA on GnRH Neurons
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timately suppress GnRH neuron firing in prepubertal mice,
this inhibition is very likely to be an artifact and result from
the sustained concentrations of exogenous GABA elevating
the membrane potential into a range that inactivates the Na

channel and also allows shunting inhibition (50, 51). Many

immature GnRH neurons displayed an initial excitation to
GABA before the spike inhibition, and this excitation is very
likely to be the physiological effect of GABA in these cells.
An increase in GnRH neuron firing is observed when all-
pregnanolone, an allosteric enhancer of GABA
A
receptor
activity (22), is applied to gramicidin-patched immature
GnRHneurons (Han, S.-K., and A. E. Herbison, unpublished
data). Thus, GABA
A
receptor activation will excite immature
GnRHneurons, whereas postpubertal GnRHneurons will be
uniformly inhibitedby GABA. As a consequence, it wouldbe
predicted that the direct activation of GABA
A
receptors on
GnRH perikarya would stimulate LH release in prepubertal
mice but inhibit LHin adults. Interestingly, a series of in vivo
studies by Moguilevsky et al. (52) and Szwarcfarb et al. (53)
have demonstrated precisely this phenomenon in the female
rat; an elevation in whole-brain GABA levels increases LH
release in immature rats but decreases LHsecretion in adults.
The transition phase for that effect was around postnatal d
25, the time from which there is likely to be a progressive
increase in the frequency of GnRH pulses occurring within
the median eminence (54).
Precisely how GABA may participate in the activation of
the GnRH neurons at puberty in the mouse remains unclear.
If the final postnatal maturation of neural inputs to GnRH
neurons follows a similar course to that of other neuronal
phenotypes (55), albeit at a much later postnatal time point,
then GABA may have an important role. In that scenario,
GABA would provide the principal stimulatory input to the
prepubertal GnRHneurons right up into the fourth postnatal
week and then switch to an inhibitory role as glutamate
becomes the dominant excitatory transmitter. It is known
that N-methyl-d-aspartate receptor expression in GnRHneu-
rons is increased greatly between postnatal d 5 and 15 in the
mouse (56). If correct, this would mean that the process of
GnRH secretory activation in the mouse at puberty is fun-
damentally different from that occurring in the primate in
which a release from an inhibitory GABA restraint is clearly
involved in the female monkey (2, 9, 10).
In terms of understanding the role of GABA in regulating
the electrical activity of mature GnRH neurons, this study
provides evidence that GABA
A
receptor activation is indeed
inhibitory. The suppressive influence of GABA on LH se-
cretion reported in the past (920) is therefore likely to have
resulted from direct activation of GABA
A
receptors located
on GnRH cell bodies. In contrast, the stimulatory effects of
GABA reported for GT1 cells (29, 30) probably reflect their
embryonic nature. Proof of the inhibitory effect of GABA on
adult female GnRH neurons is critical for the current hy-
pothesis that gonadal steroid negative feedback involves an
increased GABAergic influence on GnRH cell bodies (3, 19
22) as well as the idea that GABAergic disinhibition of GnRH
neurons is important for the generation of the GnRH/LH
surge (1418). The physiological significance and role of
GABA acting directly or indirectly through GABA
A
and/or
GABA
B
receptors at the level of the GnRH terminal in the
regulation LH secretion remains unclear (57, 58).
In summary, using gramicidin, perforated-patch electro-
physiology, we show here that GnRH neurons in the female
mouse are depolarized in a direct manner by GABA
A
re-
ceptor activation up until the time of puberty whereupon
GABA switches to exerting a hyperpolarizing action. The
time of onset of this depolarization-hyperpolarization switch
in the fifth postnatal week is very delayed, compared with
other developing neurons, but in keeping with the other late
changes in GABA
A
receptor signaling that occur in mouse
GnRH neurons (8). We show that unidentified preoptic area
neurons, residing in the same region as the GnRH neurons,
are predominantly hyperpolarized by GABA from the sec-
ond postnatal week onward. The late onset of the GABA
A
receptor-mediated depolarization-hyperpolarization is most
likely because of delayed maturation of chloride ion ho-
meostasis within the developing GnRH neuron. Although
the contribution of these changes to the onset of puberty in
the mouse may only be speculated on at present, the dem-
onstration here that GABA
A
receptor activation does indeed
inhibit the firing of adult female GnRHneurons is critical for
our understanding of the neural regulation of these impor-
tant neurons.
Acknowledgments
Drs. John Bicknell and Martin Todman are thanked for critical ap-
praisal of the manuscript.
Received October 11, 2001. Accepted December 3, 2001.
Address all correspondence and requests for reprints to: Dr. Allan E.
Herbison, Laboratory of Neuroendocrinology, The Babraham Institute,
Cambridge CB2 4AT, United Kingdom. E-mail: allan.herbison@
bbsrc.ac.uk.
This work was supported by the Biotechnology and Biological Sci-
ences Research Council (UK) and a Marie Curie Fellowship (IMA) of the
European Community Human Potential Programme under contract
number HPMF-CT-2000-00512.
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