Full text of DOI:10.1007/BF03343970

J. Endocrinol. Invest. 25: 96-100, 2002
Interactions between GABAergic and aminoacidergic
pathways in the control of gonadotropin and GH
secretion in pre-pubertal female rats
L. Pinilla, L.C. González, M. Tena-Sempere, and E. Aguilar
Department of Physiology, Faculty of Medicine, Córdoba University, Córdoba, Spain
ABSTRACT. Present experiments were carried out
in 23-day-old female rats to analyze the interac-
tion between excitatory amino acids (EAAs) and
gamma-aminobutyric acid (GABA) in the control
of gonadotropin and GH secretion. For this pur-
pose, serum concentrations of LH, FSH and GH
were measured after injection of different ago-
nists of EAA receptor subtypes [N-methyl-D-as-
partate (NMDA); kainic acid (KA), -ꢀ-amino-3-hy-
droxy-5-methylisoxazole-4-propionic acid (AMPA)],
antagonists of GABA receptors (bicuculline, pha-
clofen) or the combined administration of both
types of drugs. The results obtained indicated
that: 1) GABA has a minor physiological role in the
control of LH and GH secretion, since neither LH
nor GH serum concentrations changed after ad-
ministration of bicuculline (antagonist of GABAA
receptors) or phaclofen (antagonist of GABAB re-
ceptors); 2) GABA has a sex-specific physiological
role in the control of FSH secretion in female rats,
in which FSH secretion increases after phaclofen
administration; 3) GH secretion was enhanced af-
ter administration of NMDA, KA and AMPA, while
LH increased only after activation of NMDA re-
ceptors; 4) the stimulatory effect of NMDA on LH
secretion was counteracted by administration of
phaclofen; and 5) bicuculline and phaclofen re-
duced the ability of NMDA and AMPA to stimu-
late GH secretion. In conclusion, present experi-
ments evidenced a physiological role of GABA,
mediated by GABAB receptors, in the control of
FSH secretion and a cross-talk between excitato-
ry and inhibitory amino acids in the control of an-
terior pituitary secretion.
(J. Endocrinol. Invest. 25: 96-100, 2002)
©2002, Editrice Kurtis
INTRODUCTION
mental model used (8-12), while activation of AMPA
receptors inhibited PRL secretion (6, 7). Gamma ami-
nobutyric acid (GABA) is also involved in the control of
anterior pituitary hormone secretion, and stimulatory
or inhibitory actions of GABA on GH, gonadotropins
and PRL secretion have been described (13).
The hypothalamic control of anterior pituitary hor-
mone secretion includes complex networks in which
many neurotransmitters are involved (1). Excitatory
amino acids (EAAs) play a crucial role in the control of
hypothalamic-pituitary function acting through their
interaction with different receptor subtypes such as
N-methyl-D-aspartate (NMDA); kainic acid (KA), -ꢀ-
amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA), and metabotropic receptors (2). Activation
of AMPA, NMDA and KA receptors stimulates GH se-
cretion (3, 4). NMDA stimulated LH secretion in male
and female rats, whereas KA was only stimulatory in
males (5), and AMPA was ineffective in both sexes (6-
7). Activation of NMDA and KA receptors stimulated
or inhibited PRL secretion depending on the experi-
Physiological interactions between different neuro-
transmitters involved in the control of hypothalamic
or pituitary hormone secretion have been described.
For example, bicuculline (antagonist of GABAA re-
ceptors) and phaclofen (antagonist of GABAB re-
ceptors) blocked glutamic acid-evoked LHRH secre-
tion by arcuate nucleus-median eminence (AC-ME)
preparations (14). Similarly, the stimulatory effect of
muscimol (agonist of GABAA receptors) on LH se-
cretion was blocked by MK-801 (antagonist of NM-
DA receptors) (15). In this sense, we recently des-
cribed the cross-talk between excitatory and in-
hibitory amino acids in the regulation of GH secretion
in neonatal rats, since the stimulatory effect of GABA
on GH secretion was abolished by pretreatment with
MK-801 (an antagonist of NMDA receptors) (16).
Present experiments were carried out to analyze the
Key-words: GABA, NMDA, KA, AMPA, gonadotropins, GH.
Correspondence: Prof. Enrique Aguilar, Department of Cellular Biology, Phy-
siology and Immunology, Ayda Menendez, Pidal s/n 14004, Cordoba, Spain.
E-mail: fi1agbee@uco.es
Accepted June 6, 2001.
96

GABA and EAAs interactions
interactions between GABA and EAAs pathways in
the control of gonadotropin and GH secretion in pre-
pubertal female rats. Specifically, we tried to analyze
whether the stimulatory effect of EAAs receptor ago-
nists on GH and LH secretion was modified by pre-
treatment with antagonists of GABA receptors.
phaclofen+AMPA. Animals were killed by decapita-
tion and trunk blood was collected. Doses and timing
for drug administration were selected on the basis of
previous studies (5-13). Since we observed an unex-
pected increase in FSH levels after phaclofen admin-
istration, the effects of bicuculline and phaclofen were
also tested in 23-day-old male rats.
The experiment design was approved by the Cór-
doba University Ethics Committee for animal expe-
rimentation and was conducted in accordance with
the European Union normative for care and use of
experimental animals. At the doses used no be-
havioral effects were observed.
MATERIAL AND METHODS
Animals
Female Wistar rats born in our laboratory were kept
under controlled conditions of light (12 h light/12 h
darkness, lights on at 07:00 h) and temperature (22
C), with free access to pelleted food (Pacsa Sanders,
Seville, Spain) and tap water. On day 1 of life, each
dam was left with eight pups. The pups were sepa-
rated from their mothers immediately before start-
ing treatments and they were kept warm by a heat-
ing source next to their cage.
Hormonal determinations
After centrifugation (1600 g at 4 C for 20 min),
serum was collected, frozen and stored at -20 C un-
til use. The concentrations of LH, FSH and GH were
measured in 10-25 μl by means of a double anti-
body RIA method using RIA kits supplied by the
National Institutes of Health (NIH, Bethesda, MD,
U.S.A.). Rat-LH-I-9, Rat-FSH-I-8 and Rat-GH-I-6 were
labeled with 125I by the chloramine T method (17)
and hormone concentrations were expressed using
reference preparation (RP) LH-RP-3, FSH-RP-2 and
GH-RP-2 as standards. Intra-assay coefficients of
variation were below 8%. The sensitivity of the as-
says were 3.75, 50 and 20 pg/tube for LH, FSH and
GH, respectively. For each hormone, all samples
were measured in the same assay.
Drugs
The following drugs were used:
EAA receptors: agonist of NMDA receptors, of KA
receptors, of AMPA receptors (Research Biochemical
Inc., RBI, M.A., U.S.A.). The drugs were dissolved in
saline (NMDA and KA) or dimethylsulfoxide (DMSO)-
saline (AMPA) and injected intraperitoneally (ip) at
a dose of 15 mg/kg (NMDA) or 2.5 mg/kg (KA and
AMPA) 15 min prior to sacrifice.
Antagonists of GABA receptors: bicuculline (an-
tagonist of GABAA receptors, RBI); phaclofen (an-
tagonist of GABAB receptors, RBI). The drugs were
dissolved in saline and injected ip at a dose of 5
mg/kg (bicuculline) and 1 mg/kg (phaclofen), 30
min prior to sacrifice.
Statistics
Values are expressed as means SE. Differences be-
tween groups were analyzed using one-or two-way
ANOVA followed by Tukey’s test.
Experimental design
RESULTS
On day 23 of age, female rats were randomly divid-
ed in groups of 8-10 animals and injected (30 and 15
min prior to sacrifice) with 1) vehicle+vehicle; 2) ve-
hicle+NMDA; 3) vehicle+KA; 4) vehicle+ AMPA; 5)
bicuculline+vehicle; 6), phaclofen+vehicle; 7) bicu-
culline+NMDA; 8) bicuculline+KA; 9) bicuculline+AM-
PA; 10) phaclofen+NMDA; 11) phaclofen+KA; 12)
Blockade of GABAA receptors with bicuculline did
not affect serum concentrations of LH, FSH and GH.
Administration of phaclofen (an antagonist of GABAB
receptors) selectively increased FSH secretion in fe-
males. This effect was sex-dependent and was not
observed in male rats (Table 1).
Table 1 - Effects of bicuculline and phaclofen on LH, FSH, and GH secretion (ng/ml) in male and female rats.
Treatment
Females
FSH
Males
FSH
LH
GH
LH
GH
Vehicle (10)
0.50 0.09
0.17 0.05
0.45 0.16
8.43 1.43
8.06 1.26
1.59 0.38
0.50 0.05
2.81 0.43
0.14 0.02
0.16 0.02
0.14 0.03
5.63 0.82
3.76 0.56
6.74 1.26
3.21 0.42
3.03 0.36
2.73 0.34
Bicuculline (10)
Phaclofen (10)
15.14 1.30*
Values are expressed as mean SE, number of animals in brackets. *pꢁ0.01 vs vehicle-injected group (ANOVA followed by Tukey’s test).
97

L. Pinilla, L.C. González, M. Tena-Sempere, et al.
a
LH secretion was increased by NMDA but not by
KA or AMPA (Fig. 1). Serum LH concentrations
were similar in animals treated with any of the ag-
onists of EAA receptors alone or combined with
bicuculline. In contrast, the stimulatory effect of
NMDA on LH secretion was partially blocked by
pretreatment with phaclofen, which also induced
a reduction on LH secretion in female rats treated
with KA (Fig. 1).
FSH secretion remained unchanged after activa-
tion of AMPA, NMDA and KA receptors (Fig. 2).
In bicuculline-pre-treated rats KA stimulated
significantly (p<0.01) FSH secretion. Co-adminis-
tration of NMDA+phaclofen or KA+bicuculline in-
creased FSH secretion when compared with con-
trol animals (Fig. 2).
24
16
8
a
**
GH secretion was stimulated by NMDA, KA and
AMPA. The stimulatory effect of AMPA was signif-
icantly reduced by co-administration of phaclofen
and bicuculline. The reduction of NMDA-stimulat-
ed GH secretion by bicuculline or phaclofen was in
the limits of significance. In contrast, phaclofen and
bicuculline potentiated the releasing effect of KA
on GH secretion (Fig. 3).
0
Vehicle
Vehicle
Bicuculline
NMDA
PHA
KA
AMPA
Fig. 2 - Serum concentrations of FSH in pre-pubertal female rats
injected with N-methyl-D-aspartate (NMDA), kainic acid (KA),
and -ꢀ-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA) alone or in combination with bicuculline or phaclofen.
**pꢁ0.01 respectively vs vehicle-injected group. a=pꢁ0.05
(ANOVA followed by Tukey’s test).
a
6
b
**
DISCUSSION
Present results indicate that in pre-pubertal female
rats: 1) GABA has a sex-specific physiological role in
the control of FSH secretion which increases after
phaclofen administration; 2) GH secretion is activated
by all subtypes of receptors for EAAs; 3) some inter-
actions between GABA and EAA pathways in the
control of gonadotropin secretion occur, since pha-
clofen reduced the stimulatory effect of NMDA on
LH release and coadministration of NMDA and pha-
clofen or KA and bicuculline stimulated FSH release;
4) the stimulatory effect of AMPA on GH secretion is
counteracted by bicuculline and phaclofen which po-
tentiated the KA-stimulated GH release.
**
4
*
2
The role of GABA in the control of gonadotropin se-
cretion is complex and depends on sex and age stud-
ied. In gonadectomized adult female rats, adminis-
tration of bicuculline and phaclofen (antagonists of
GABAA and GABAB receptors) increases LH secretion,
suggesting a GABAergic inhibition of the release of
LHRH (18), GABAB receptor antagonists being more
effective than the GABAA receptor antagonists (19).
Baclofen (an agonist of GABAB receptors) inhibited in
vitro the effects of LHRH on LH release (20). In 16-day-
0
Vehicle
Vehicle
Bicuculline
NMDA
PHA
KA
AMPA
Fig. 1 - Serum concentrations of LH in pre-pubertal female rats in-
jected with N-methyl-D-aspartate (NMDA), kainic acid (KA) and
ꢀ-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
alone or in combination with bicuculline or phaclofen. *pꢁ0.05
and **pꢁ0.01, respectively, vs corresponding vehicle-injected
group. a=pꢁ0.01; b=pꢁ0.05 (ANOVA followed by Tukey’s test).
-
98

GABA and EAAs interactions
b
ies have repeatedly proposed the existence of a se-
lective FSH-releasing factor (24, 25), which could me-
diate the effects of phaclofen on FSH. It should also be
considered that the selective release of both gonado-
tropins is related to the frequency and amplitude of
LHRH pulses. Pituitary stimulation with a high frequen-
cy of LHRH pulses increases LH secretion, while a stim-
ulation with low frequency of LHRH pulses increases
selectively FSH secretion (26, 27), it being possible
that phaclofen stimulated selectively FSH secretion
through a change in the frequency of LHRH pulses.
Present experiments confirmed and extended previ-
ous results from our laboratory indicating that GH se-
cretion is activated by NMDA, KA and AMPA recep-
tors, whereas LH increased only after administration
of NMDA and FSH was unaffected (3, 5). These find-
ings evidenced a distinct role of EAA receptor sub-
types in the control of different pituitary hormones.
Interactions between excitatory and inhibitory amino
acids in the control of gonadotropin secretion are ev-
ident from present experiments. The reduction of
NMDA-stimulated LH secretion after phaclofen treat-
ment indicates that GABAB receptors play a permis-
sive role in the effects of NMDA on LH secretion. It
has previously been described that NMDA stimulates
LH via LHRH (28) and phaclofen decreased the stim-
ulatory effect of NMDA on LHRH release by median
eminence-arcuate nucleus (14). The experiments re-
ported herein indicated that the interactions between
NMDA and GABAB receptors in the control of LHRH
observed in vitro (14) are also operative in vivo.
In recent experiments (16), we demonstrated that the
stimulatory effect of GABA on GH secretion was coun-
teracted by MK-801 (an antagonist of NMDA recep-
tors). Present results indicated that, inversely, NMDA
and AMPA effects were partially counteracted by pha-
clofen (an antagonist of GABAB receptors), whereas
KA effect was potentiated by bicuculline. As a whole,
these interactions suggest that GABA receptors dif-
ferently modulate the functional role of different sub-
types of EAA receptors in the control of GH secretion.
In conclusion, our results suggest that GABAB recep-
tors play a selective physiological role in the control of
FSH secretion in pre-pubertal female rats and that a
cross-talk between excitatory and inhibitory amino
acids operates in the control of gonadotropin and GH
secretion in pre-pubertal female rats. In contrast with
recent results demonstrating that the stimulatory effect
of GABA on GH secretion required the activation of
NMDA receptors (16), present experiments suggest
that the full stimulatory effect of AMPA receptors on
GH secretion requires the activation of GABAA and
GABAB receptors. As a whole, the results obtained
suggest that the stimulatory effect of EAAs in the con-
trol of GH are modulated by GABA receptors.
b
b
200
150
100
50
b
*
*
*
*
*
*
*
*
*
0
Vehicle
Vehicle
Bicuculline
NMDA
PHA
KA
AMPA
Fig. 3 - Serum concentrations of GH in pre-pubertal female rats
injected with N-methyl-D-aspartate (NMDA), kainic acid (KA),
and -ꢀ-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA) alone or in combination with bicuculline or phaclofen.
*pꢁ0.01 vs corresponding vehicle-injected group. b=pꢁ0.05
(ANOVA followed by Tukey’s test).
old female rats, LH secretion is stimulated by musci-
mol (a GABAA agonist) and inhibited by baclofen (a
GABAB agonist) (15). The role of GABAA receptors in
the control of LH changes during pubertal develop-
ment, and GABAA receptor agonists stimulated LH
secretion in pre-pubertal rats (16-day-old) and inhibi-
ted it in peripubertal ones (30-day-old) (21). Our re-
sults indicate that LH secretion in 23-day-old females
is independent of activation of GABAA or GABAB re-
ceptors, since neither bicuculline nor phaclofen chan-
ged serum LH concentrations. The selective increase
in FSH secretion in female rats after phaclofen ad-
ministration was unexpected. However, our results re-
semble those obtained after administration of 5-hy-
droxytryptophan (5-HTP: precursor of serotonin syn-
thesis) in pre-pubertal rats. Administration of 5-HTP
increases FSH secretion in male but not in female rats
(22) and the increase in FSH levels was not accompa-
nied by concomitant increases in LH secretion (23).
The possibility that phaclofen increased LHRH secre-
tion is difficult to reconcile with the absence of
changes in LH secretion. Alternatively, different stud-
99

L. Pinilla, L.C. González, M. Tena-Sempere, et al.
ACKNOWLEDGEMENTS
tion of luteinizing hormone-releasing hormone secretion.
Endocrinology 1992, 131: 1559-1561.
The authors are very grateful to Mr. Rocio Campon and Ms. Inma-
culada Aguilar for their technical assistance. This work was suppor-
ted by grants PM98-0163 (CICYT, Spain) and 1FD97-0696-02 (FED-
ER). The NIH supplied the materials for hormone determinations.
15. Scacchi P., Carbone S., Szwarcfarb B., et al. Interactions be-
tween GABAergic and serotoninergic systems with excita-
tory amino acid neurotransmission in the hypothalamic con-
trol of gonadotropin secretion in prepubertal female rats.
Brain Res. Dev. 1998, 105: 51-58.
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