Growth is seasonally regulated in goldﬁsh, and the highest
growth rates are found in the early summer after the spring
breeding period (Marchant and Peter, 1986). Growth hormone
secretion from the goldﬁsh anterior pituitary also varies
seasonally (Marchant and Peter, 1986; Trudeau et al., 1992).
Serum growth hormone levels increase during gonadal
development in late autumn and winter, a time when somatic
growth is lowest (Marchant and Peter, 1986; Trudeau et al.,
1992). This increased release of growth hormone probably acts
in concert with gonadotropin-II (GTH-II; the luteinizing-
hormone-like molecule in ﬁsh) to stimulate steroidogenesis
(Van Der Kraak et al., 1990; Le Gac et al., 1993) during
seasonal redevelopment of the gonad.
The control of growth hormone release involves both
stimulatory and inhibitory mechanisms. Growth hormone
release in the goldﬁsh is stimulated by bombesin (Himick and
Peter, 1994), dopamine (Chang et al., 1985, 1990; Wong et al.,
1993), thyrotropin-releasing hormone (TRH; Trudeau et al.,
1992), neuropeptide Y (NPY; Peng et al., 1993), growth
hormone-releasing hormone (GHRH; Vaughan et al., 1992),
gonadotropin-releasing hormone (GnRH; Marchant et al.,
1989a), cholecystokinin (Himick et al., 1993) and pituitary
adenylate cyclase-activating polypeptide (PACAP; Wong et
al., 1998). In contrast, growth hormone release is inhibited by
insulin-like growth factor-1 (IGF-1; Weil et al., 1999),
norepinephrine (Chang et al., 1985), serotonin (Somoza and
Peter, 1991) and somatostatin (SRIF; Marchant et al., 1987).
Interactions among these stimulatory and inhibitory
neuroendocrine systems drive seasonal cyclicity in serum
growth hormone levels (for reviews, see Peter and Marchant,
1995; Trudeau, 1997).
In contrast to the peptidergic and aminergic regulation of
growth hormone release in ﬁsh, little information is available
concerning the involvement of amino acid neurotransmitters.
Glutamate, which is converted to γ-aminobutyric acid (GABA)
by two molecular forms of glutamic acid decarboxylase in the
The Journal of Experimental Biology 203, 1477–1485 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
Double-labelling studies at the electron microscopic
level demonstrated that
-aminobutyric acid (GABA)-
immunoreactive nerve endings are associated with growth-
hormone-secreting cells in the proximal pars distalis of the
goldﬁsh pituitary gland, suggesting that GABA may be
important for the control of growth hormone release in this
species. An in vitro assay for GABA-transaminase activity
demonstrated that the pituitary is a site for the metabolism
of GABA to succinic acid. In vitro, GABA or the GABA
antagonists bicuculline and saclofen did not affect the rate
of growth hormone release from dispersed pituitary cells
in static incubation. In contrast, intracerebroventricular
injection of GABA reduced serum growth hormone levels
within 30min. During the seasonal gonadal cycle,
intraperitoneal injection of GABA was without effect in
sexually regressed goldﬁsh, but caused a signiﬁcant
decrease in serum growth hormone levels in sexually
recrudescent animals. Intraperitoneal implantation of solid
silastic pellets containing oestradiol increased serum GH
levels ﬁvefold in sexually regressed and recrudescent
goldﬁsh; in both groups, GABA suppressed the oestradiol-
stimulated increase in circulating growth hormone levels.
The effect of oestradiol on basal serum growth hormone
levels was speciﬁc since progesterone and testosterone
were without effect. However, in recrudescent animals
treated with progesterone and testosterone, the inhibitory
effects of GABA on serum growth hormone levels were
absent, indicating a differential role for these steroids in
growth hormone release. Taken together, these results
demonstrate that GABA has an inhibitory effect on growth
hormone release in goldﬁsh.
Key words: GABA, γ-aminobutyric acid, immunocytochemistry,
growth hormone, oestradiol, goldﬁsh, Carassius auratus.
THE INHIBITORY EFFECTS OF
-AMINOBUTYRIC ACID (GABA) ON GROWTH
HORMONE SECRETION IN THE GOLDFISH ARE MODULATED BY SEX STEROIDS
V. L. TRUDEAU
*, O. KAH
, J. P. CHANG
, B. D. SLOLEY
, P. DUBOURG
, E. J. FRASER
AND R. E. PETER
Department of Biology, University of Ottawa, PO Box 450, Station A, Ottawa, Ontario, Canada K1N 6N5,
Endocrinologie Moléculaire de la Reproduction, UPRES-A, CNRS-6026, Campus de Beaulieu, Rennes Cedex
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9,
C. V. Technologies Inc., Edmonton, Alberta, Canada T6G 2C2 and
Neurocytochimie Fonctionelle, URA-CNRS,
339 Avenue des Facultés, Talence Cedex 33405, France
Accepted 17 February; published on WWW 6 April 2000
brain (Bosma et al., 1999; Pinal and Tobin, 1998), is localized
in nerve terminals innervating the goldﬁsh anterior pituitary.
Activation of the N-methyl-D-glutamate (NMDA)-type
glutamate receptor rapidly inhibits growth hormone release in
this species (Trudeau et al., 1996). The effects of GABA on
growth hormone release have not been studied in any species
of ﬁsh; however, cell bodies containing GABA are located in
hypophysiotrophic areas of the goldﬁsh brain known to be
involved in the control of growth hormone release (Martinoli
et al., 1990). Moreover, GABA is an important neuroendocrine
regulator of the release of other pituitary hormones in ﬁsh:
GABA has prominent stimulatory effects on GTH-II release
(Kah et al., 1992; Sloley et al., 1991; Trudeau et al., 1993a,b;
Mañanos et al., 1999) and inhibitory effects on prolactin
release (Prunet et al., 1993). In mammals and birds, GABA has
been reported to have both stimulatory and inhibitory effects
on growth hormone release. In the rat, for example, early
reports indicated that central injection of GABA can either
stimulate (McCann and Rettori, 1988) or inhibit (Fiók et al.,
1984) growth hormone release in adults, depending on the site
of application. In addition, GABA has a direct stimulatory
effect on growth hormone release in vitro from pituitaries of
young rats, whereas GABA has no effect or only slightly
stimulates growth hormone release in adult rats (Fiók et al.,
1984; Ács et al., 1990). The direct stimulatory effect of GABA
in neonatal rats is independent of adrenal, gonadal and thyroid
hormones (Ács et al., 1993). In the chicken, in vitro tissue
culture experiments show that GABA inhibits growth hormone
release in the presence of hypothalamic tissue but not directly
at the pituitary (Hall et al., 1984). These interesting and
conﬂicting reports led us to test whether GABA has any role
in regulating growth hormone release in goldﬁsh.
Materials and methods
Common goldﬁsh (Carassius auratus) weighing 15–40g
were purchased throughout the year from commercial
suppliers. Fish were acclimated to 17°C, and fed and
maintained on a simulated natural photoperiod as reported
previously (Trudeau et al., 1991). Fish were anaesthetized by
immersion in 0.05% tricaine methane sulphonate (TMS) prior
to any handling for drug injection, steroid pellet implantation
and blood sampling. Blood samples were taken by puncture of
the caudal vasculature using a 25 gauge needle attached to a
1ml syringe. Blood was allowed to clot for 16–24h, and serum
was collected by centrifugation.
Localization of GABA in the pituitary
Animals were anaesthetized with TMS and decapitated. The
pituitaries were rapidly dissected, ﬁxed with 6.5%
glutaraldehyde in phosphate buffer and cut at a thickness of
40µm with a vibratome. The sections were processed for
GABA pre-embedding immunohistochemistry as described
previously (Kah et al., 1987a, 1992). For electron microscopy,
the sections were ﬂat-embedded, and fragments of interest
were cut to obtain ultrathin sections. These sections were
exposed to salmon growth hormone antibodies and, after
rinsing, to a secondary antibody coupled to 20nm gold
particles (Kah et al., 1987a, 1992).
Metabolism of GABA in the pituitary
An in vitro enzyme assay was used to determine whether the
pituitary was a site for GABA metabolism. Extraction
procedures and determination of GABA-transaminase activity
were performed as described by Sloley and McKenna (1993)
except that a pooled homogenate of 10 pituitaries was
incubated for 1h before separation of
using a tri-octylamine solution. Radioactivity was measured by
pipetting 35µl of supernatant into 4ml of scintillation ﬂuid
(Packard Pico-Fluor 40) and counting in a Packard Tri-Carb
liquid scintillation analyser. The speciﬁcity of GABA-
transaminase activity was determined (in triplicate) by
incubating pituitary extracts in the presence of increasing
) of the GABA-transaminase
inhibitor γ-vinyl-GABA (GVG; a gift from Hoechst Marion
Roussel). GVG was added to pituitary extracts (on ice),
incubated for 10min at 37°C and then for 5min on ice before
addition of [
H]GABA (Amersham). The effectiveness of
GVG in inhibiting GABA-transaminase activity was
determined by calculating the dose giving 50% inhibition
) using the Prism 2 program (GraphPad Software, Inc.).
The effects of GABA on growth hormone release from
dispersed pituitary cells in vitro
To test whether the effects of GABA in inhibiting growth
hormone release were direct at the level of the pituitary, static
incubation of pituitary cells was carried out as described by
Chang et al. (1990). For a given experiment, 40 pituitaries were
collected from mixed populations of male and female goldﬁsh,
and their cells were dispersed by controlled trypsinization.
Dispersed cells were resuspended in culture medium (Medium
199 with Earle’s salts, Gibco, and containing 2.2gl
Hepes and 1% horse serum, pH7.2) and
) overnight in 24-well culture
plates, with or without 100000unitsl
streptomycin. Cells were incubated at 28°C, under
and at saturated humidity. On the following day, the
culture medium was replaced with testing medium (Medium
199 with Hank’s salts, Gibco, and containing 2.2gl
Hepes and 0.1% bovine serum albumin,
pH7.2), with or without 100000unitsl
streptomycin. A 0.1moll
GABA stock solution
was made up in distilled deionized water and diluted in testing
medium to give ﬁnal concentrations of 0.001–100µmoll
immediately prior to use. In another experiment, cells were
incubated with 10µmoll
GABA, in the presence or absence
of the GABA
antagonist bicuculline or
of the GABA
antagonist saclofen. GABAergic
drugs for cell culture and in vivo experiments were purchased
from Research Biochemicals International (RBI). Following an
additional 2h of incubation under the conditions described
V. L. TRUDEAU AND OTHERS
1479GABA inhibits growth hormone secretion
above, 750–800µl of medium was carefully removed from
each well and stored at −20°C until the growth hormone
contents were measured by radioimmunoassay. Treatments
were usually tested in triplicate. Growth hormone levels from
replicate experiments were normalized by expressing the data
as a percentage of basal growth hormone release. The viability
and responsiveness of cells were conﬁrmed in parallel
experiments with the same cell preparations using dopamine,
the protein kinase C activator 4-β-tetradecanoyl phorbol
acetate and cGnRH-II, agents known to stimulate growth
hormone release (Chang et al., 1994).
The effects of injection of GABA into the third brain ventricle
on growth hormone release in vivo
For injections into the third brain ventricle, 50µg of GABA
was delivered in 2µl of saline to sexually regressed female
goldﬁsh. These doses of GABA were chosen because we have
shown that they stimulate GTH-II release in vivo in goldﬁsh
(Trudeau et al., 1993b). To control for possible non-speciﬁc
effects, the amino acid taurine (50µg per 2µl of saline) was
also injected intracerebroventricularly. Taurine is abundant in
the brain and can stimulate GTH-II release in goldﬁsh (Sloley
et al., 1991); it shares some of the characteristic inhibitory
effects of GABA on neurotransmission (Huxtable, 1989). In
addition, the relative molecular masses of taurine and GABA
are similar and are, respectively, 125 and 103. Animals
(25–35g body mass) were anaesthetized with TMS and placed
in a goldﬁsh stereotaxic apparatus (Peter and Gill, 1975); the
skull was then opened using a circular dental saw. The
injection syringe was gently lowered into the third brain
ventricle, and 2µl of injection ﬂuid was expelled by light
pressure. The syringe was removed, and the ﬁsh was returned
to the experimental tank for recovery (<5min) from the
anaesthetic. Blood samples were drawn 30min after
The effects of GABA and sex steroids on growth hormone
Preliminary studies indicated that the inhibitory effects of
GABA on growth hormone were dependent on gonadal
status, implicating sex steroids in the control of growth
hormone release. Gonad-intact, sexually regressed or sexually
recrudescent goldﬁsh were implanted for 5 or 10 days with
control or silastic pellets containing progesterone, testosterone
or oestradiol (100µgg
bodymass), as reported by Trudeau et
al. (1991). On the day of experimentation, GABA dissolved in
bodymass; purchased from RBI) or saline
bodymass) was injected intraperitoneally, and
blood samples were taken 30min later. This dose of GABA
and sampling times were based on a preliminary study (not
shown) and also on our previous work in goldﬁsh in which
similar doses of GABA stimulated GTH-II release within
30min (Trudeau et al., 1993b).
Serum or culture medium growth hormone levels were
measured using a double-antibody radioimmunoassay (RIA)
(Murthy et al., 1993) with common carp growth hormone as
standard. All samples were assayed in duplicate.
Growth hormone concentrations in serum or in vitro
incubation medium were analysed by one-way analysis of
variance or Student’s t-test; treatment group means were
considered statistically different at P<0.05.
Immunohistochemical localization of GABA in the pituitary
The anterior pituitary of teleosts has the unique property
of receiving direct innervation, which is the functional
equivalent of the median eminence of tetrapods (Peter et al.,
1990). Furthermore, endocrine cells of the same type are
more or less grouped in speciﬁc portions of the anterior
pituitary, allowing the nature of the innervation of a given
cell type to be easily determined. At the light microscope
level (not shown), GABA-immunoreactive ﬁbres were
observed entering the pituitary stalk and digitating in all
lobes of the pituitary, notably the proximal pars distalis,
which contains mainly gonadotrophs and somatotrophs,
conﬁrming our previous studies (Kah et al., 1987a, 1992). At
the electron microscope level (Fig. 1), these ﬁbres were
detected in the neurohypophyseal digitations, but also in
direct contact with the secretory cells. Double-staining
studies at the electron microscope level demonstrated that
GABA-immunoreactive nerve proﬁles were frequently
associated with growth-hormone-positive cells. These nerve
proﬁles contained positive neurosecretory granules 60–80 nm
in diameter and, although no clear synaptic differentiation
could be detected apposed to the growth hormone cells,
membrane thickenings facing secretory cells and synaptic-
like vesicles were occasionally detected in the positive nerve
Metabolism of GABA in the pituitary
The presence of GABAergic nerve terminals in the pars
distalis suggested that GABA may be released and metabolized
in the pituitary. A sensitive in vitro assay demonstrated
GABA-transaminase activity in the goldﬁsh pituitary. γ-Vinyl-
GABA (GVG), previously characterised as a GABA
metabolism inhibitor in vivo in the goldﬁsh (Sloley et al.,
1991), effectively inhibited speciﬁc GABA-transaminase
activity in vitro (Fig. 2). The IC
for GVG inhibition of
goldﬁsh pituitary GABA-transaminase was 1.4µmoll
100% inhibition was obtained using the highest dose
The effects of GABA on growth hormone release from
dispersed pituitary cells in vitro
Detection of a GABAergic innervation and speciﬁc
GABA-transaminase activity in the pituitary suggested that
GABA may directly regulate growth hormone release in
goldﬁsh. In vitro and in vivo approaches were used to address
this possibility. Incubation of dispersed pituitary cells with
GABA had no effect on in vitro growth
hormone release (Fig. 3A). The viability and responsiveness
of the cultured growth hormone cells were conﬁrmed by high
levels of basal growth hormone release, and growth-
hormone-release responses to dopamine and 4-β-
tetradecanoyl phorbol acetate (data not shown), consistent
with our extensive data on growth hormone release in vitro
(Chang et al., 1994). In this experiment, the presence of
penicillin and streptomycin in the culture medium was
potentially a concern since penicillin may act as a GABA
antagonist in some circumstances (Macdonald and Olsen,
1994). However, a second experiment using dispersed cells
cultured in the complete absence of penicillin and
streptomycin also indicated that GABA does not directly
affect growth hormone release (Fig. 3B). In this experiment,
high basal levels of release under control conditions were also
noted. Incubation of cells with 1nmoll
GABA did not affect growth hormone release (P>0.05). In
another experiment (Fig. 3C), the GABA
receptor antagonists bicuculline and saclofen, respectively,
did not affect growth hormone release either alone or in
combination with GABA (P>0.05). Cell viability and
secretory responses were conﬁrmed by growth-hormone-
release responses to 100nmoll
chicken GnRH-II (control
100±3.5%; chicken GnRH-II, 113.2±3.9%, P<0.05; not
The effects of injection of GABA into the third brain ventricle
on growth hormone release in vivo
Fig. 4 shows the effect of injection of GABA into the third
brain ventricle on serum growth hormone levels in ﬁsh in post-
spawning condition (at the beginning of gonadal regression).
At 30min following central injection, GABA (50µg)
suppressed serum growth hormone levels by approximately
23% (Fig. 4). In contrast, taurine did not affect growth
V. L. TRUDEAU AND OTHERS
Fig. 1. (A) Electron micrograph at the
level of the proximal pars distalis of the
pituitary showing the presence of γ-
aminobutyric acid (GABA)-positive
proﬁles (*) located either in digitations of
the neurohypophysis (N) or in direct
contact with growth-hormone-positive
cells (arrowheads). Scale bar, 0.5µm.
(B) High-power view of a GABA-
positive nerve ending (*) in close
association with a growth-hormone-
positive cell. Note the strong labelling of
the secretory vesicles (arrowhead) by
gold particles. Scale bar, 0.5µm.
Fig. 2. Speciﬁc γ-aminobutyric acid (GABA)-transaminase activity
in the goldﬁsh pituitary. Increasing concentrations of γ-vinyl-GABA
(GVG) inhibit GABA-transaminase activity. Values are presented as
0 0.01 0.1 1 10 100
Specific activity (µmol mg
= 1.4 µmol l
[GVG] (µmol l
1481GABA inhibits growth hormone secretion
The effects of GABA and sex steroids on growth hormone
In sexually regressed goldﬁsh implanted with control
silastic pellets, GABA injected intraperitoneally
bodymass) did not affect serum growth hormone
levels (Fig. 5). We reasoned that this lack of effect of GABA
might be indicative of the decreased growth hormone
secretory response associated with sexual regression and low
sex steroid levels in the summer (Trudeau et al., 1992).
Therefore, sexually regressed goldﬁsh were treated for 5 days
with silastic implants containing testosterone or oestradiol.
Testosterone alone had no effects on serum growth hormone
levels, and GABA had no effect in the testosterone-treated
group. In contrast, oestradiol treatment caused an
approximately ﬁvefold increase in serum growth hormone
levels (Fig. 5). In the oestradiol-implanted group,
intraperitoneal injection of GABA suppressed growth
hormone release by 50%.
The effects of intraperitoneal injection of GABA in ﬁsh in
the early stages of seasonal gonadal redevelopment
(recrudescence) were also tested. In these animals, GABA
suppressed growth hormone release by 50% within 30min of
injection (Fig. 6). Treatment with progesterone or testosterone
alone for 10 days had no effects on basal serum growth
hormone levels compared with ﬁsh implanted with silastic
pellets without steroid. However, the inhibitory effects of
injected GABA on serum growth hormone levels found in
control implanted (no steroid) recrudescent ﬁsh were absent in
the progesterone- and testosterone-treated ﬁsh. Oestradiol
treatment again increased serum growth hormone levels
ﬁvefold, and GABA injection suppressed this stimulated
release by approximately 70% (Fig. 6).
C Bic GABA
+ Bic + Sac
C 1 100
[GABA] (µmol l
Growth hormone release (% control)
[GABA] (µmol l
Fig. 3. The effect of γ-aminobutyric acid (GABA) on the in vitro
release of growth hormone (GH) from dispersed goldﬁsh pituitary
cells. (A) Values are means +
S.E.M. (N=8–9) and are expressed as a
percentage of control (C) basal GH levels (1179±61ngml
derived from pituitaries of sexually mature ﬁsh were incubated in the
presence of penicillin and streptomycin. (B) Values are +
(N=12) and are expressed as a percentage of control (C) basal GH
). Cells derived from pituitaries of sexually
regressed ﬁsh were incubated in the absence of penicillin and
streptomycin. (C) Values are +
S.E.M. (N=12) and are expressed as a
percentage of control (C) basal GH levels (1143±24ngml
derived from pituitaries of sexually regressed ﬁsh were incubated in
the absence of penicillin and streptomycin. Drug concentrations
used were 10µmoll
Bicuculline (Bic) and
erum [GH] (ng ml
Fig. 4. The effects of injection of γ-aminobutyric acid (GABA)
(50µg in 2µl of saline) and taurine (50µg in 2µl of saline) into the
third brain ventricle on serum growth hormone (GH) levels in
sexually regressed female goldﬁsh. Values are +
Means with different superscripts are signiﬁcantly different
The presence of GABA and the GABA-metabolising enzyme
GABA-T in the pituitary
The goldﬁsh hypothalamus and pituitary contain high levels
of GABA as determined by HPLC analysis (Sloley et al.,
1991). Double-labelling electron microscopic studies
demonstrated that GABA-producing neurons innervate that
part of the anterior pituitary where somatotroph cells are
located (Fig. 1). The origin of this GABAergic innervation is
unknown. However, retrograde tracing studies have shown that
the preoptic region and the mediobasal hypothalamus, both of
which exhibit high densities of GABA-immunoreactive cell
bodies (Martinoli et al., 1990), are the main hypophysiotrophic
regions in the goldﬁsh brain (Anglade et al., 1993). We have
previously demonstrated that these GABA neurons are
important for the neuroendocrine control of pituitary function
because injection of the GABA-transaminase inhibitor GVG
raises GABA levels by approximately threefold in the preoptic
region, hypothalamus and pituitary, leading to the upregulation
of pituitary GTH-II β-subunit mRNA levels (Trudeau et al.,
2000) and release of GTH-II in vivo (Trudeau et al., 1993b).
We also show that the pituitary contains GABA-transaminase
(Fig. 2) activity, indicating that the pituitary is also an active
site for the metabolism of GABA. The cellular localization of
GABA-T in the pituitary remains to be determined.
GABA inhibits growth hormone release in vivo in goldﬁsh
treated with oestradiol
A few studies in rats have demonstrated that GABA can act
directly at the anterior pituitary (Fiók et al., 1984; Ács et al.,
1990, 1993), but also centrally (McCann and Rettori, 1988;
Ács et al., 1993), to regulate growth hormone release. Despite
the detection of GABA-immunoreactive nerve terminals
apposed to growth-hormone-secreting cells, we were not able
to show that GABA directly affected in vitro release of growth
hormone from dispersed pituitary cells obtained from mature
or regressed goldﬁsh (Fig. 3). However, injection of GABA
into the brain ventricle inhibited release of growth hormone in
post-spawning goldﬁsh (Fig. 4), implicating GABA in the
control of growth hormone secretion in this species. An
inhibitory effect was not seen when GABA was injected
intraperitoneally in sexually regressed ﬁsh (Fig. 5). This effect
could have been dose-related, although 100µgg
effective in stimulating GTH-II release in sexually regressed
goldﬁsh (Trudeau et al., 1993b). In contrast, the 100µgg
dose of GABA inhibited growth hormone release when
injected intraperitoneally into ﬁsh in the early stages of gonadal
recrudescence (Fig. 6), suggesting that gonadal steroids may
modulate the action of GABA.
Indeed, GABA clearly inhibited growth hormone release in
both regressed and recrudescent animals treated with oestradiol
to increase basal levels of growth hormone secretion (Figs 5,
6). This action of GABA in inhibiting growth hormone release
within 30min is very robust considering that oestradiol
increases pituitary growth hormone content and enhances
V. L. TRUDEAU AND OTHERS
Serum [GH] (ng ml
Fig. 5. The effects of γ-aminobutyric acid (GABA)
bodymass) on growth hormone (GH) release in
sexually regressed female goldﬁsh implanted for 5 days with
testosterone (Testo) or oestradiol (E2). *P<0.01, oestradiol
stimulates GH release. ‡P<0.05, GABA inhibits GH release in
oestradiol-treated ﬁsh. Values are means +
S.E.M. (N=10–12). Note
that the error for the group treated with GABA alone is too small to
Serum [GH] (ng ml
Fig. 6. The effect of γ-aminobutyric acid (GABA)
bodymass) on growth hormone (GH) release in
sexually recrudescent female goldﬁsh implanted for 10 days
with testosterone (Testo), progesterone (P4) or oestradiol (E2).
*P<0.01, oestradiol stimulates GH release. ‡P<0.05, GABA
inhibits GH release in control and oestradiol-treated ﬁsh. Values
are means +
S.E.M. (N=10–12). Note that the errors for the groups
treated with P4 plus GABA or testosterone alone are too small to
1483GABA inhibits growth hormone secretion
growth hormone secretion for at least 10 days (Zou et al.,
1997). The effects of oestradiol on serum growth hormone
levels were different from those of the other steroids tested.
Progesterone and testosterone did not affect basal serum
growth hormone levels. However, recrudescent animals treated
with progesterone or testosterone did not respond to the
inhibitory effects of GABA on serum growth hormone levels
(Fig. 6), indicating a differential role for these steroids on
growth hormone release. Contrasting effects of the sex steroids
on GABA synthesis have also been observed in the goldﬁsh
hypophysiotropic system. For example, oestradiol increased,
whereas both testosterone and progesterone decreased, GABA
synthesis in the pituitary (Trudeau et al., 1993a).
The site of GABA action for inhibiting growth hormone
release in vivo
GABA could be inhibiting growth hormone release either
by direct actions on the somatotroph or indirectly via the
release of other neurotransmitters or neuropeptides. GABA or
GABA antagonists did not affect in vitro growth hormone
release from dispersed pituitary cells that were responsive to
GnRH. Lesioning of the preoptic region and basal
hypothalamus has shown that, in vivo, growth hormone
release in the goldﬁsh is under tonic inhibition by somatostatin
(Marchant et al., 1989b). In contrast, for pituitary cells in
vitro, these predominant inhibitory inﬂuences have been
removed. Therefore, dispersed pituitary cells of the goldﬁsh
typically secrete high levels of growth hormone in vitro. In
this situation of high basal release, GABA did not inhibit
growth hormone release over a wide range of doses
administered in vitro to goldﬁsh pituitary cells in static
culture. In contrast, intracerebroventricular injection of
GABA reduced serum growth hormone levels. In Atlantic
salmon, at least, GABA
receptor subunits have been
demonstrated immunohistochemically throughout the
preoptic–hypophysiotropic system (Anzelius et al., 1995).
Using patch-clamp electrophysiology, we have demonstrated
functional inhibitory GABA
receptors in the ventral preoptic
area of goldﬁsh (Trudeau et al., 2000). In goldﬁsh, GABA
injected into the third brain ventricle could activate preoptic
GABA receptors to decrease serum growth hormone levels,
suggesting a central site of action. However, the modest
reduction of growth hormone levels following
intracerebroventricular injection compared with the more
obvious effects of intraperitoneally injected GABA suggest
that GABA may be acting predominantly on
hypophysiotrophic nerve terminals rather than centrally.
Alternatively, intracerebroventricularly injected GABA could
be rapidly degraded by GABA-T or removed from interstitial
spaces in the brain by GABA transporters, thus attenuating the
growth-hormone-release response. Although we cannot yet
entirely rule out a direct action of GABA on somatotrophs
under some other physiological conditions that we have not
yet tested, the currently available data suggest that GABA
probably acts indirectly, through unidentiﬁed neuronal
systems, to inhibit growth hormone release. Studies in the
chicken also demonstrate that the inhibitory effects of GABA
on growth hormone release are indirect (Hall et al., 1984).
Possible mechanisms for GABAergic inhibition of growth
Studies in the rat indicate that GABA can stimulate growth
hormone release by inhibiting somatostatin-secreting neurons
(McCann and Rettori, 1988). In goldﬁsh, a similar effect of
GABA on somatostatin release is not a likely mechanism for
the GABAergic inhibition of growth hormone release observed
here. An inhibition of somatostatin release would lead to
stimulation rather than inhibition of growth hormone release
in the goldﬁsh model. In goldﬁsh, GABA has been shown
to stimulate GnRH release (Kah et al., 1992), and GnRH
stimulates growth hormone release (Marchant et al., 1989a),
especially in oestradiol-treated animals (Trudeau et al., 1992).
These observations do not support the involvement of GnRH
in the GABAergic inhibition of growth hormone release.
GABA could also act by suppressing dopaminergic activity.
We have previously shown that GABA can inhibit dopamine
turnover in the goldﬁsh hypothalamo–pituitary axis (Trudeau
et al., 1993a), and dopamine, through the activation of a
pituitary D1 receptor, stimulates growth hormone release
(Wong et al., 1993; Chang et al., 1994). Preoptic dopamine
neurones also innervate the anterior pituitary, in which the
somatotrophs are localized (Kah et al., 1987b), and
GABA/dopamine axo-axonal interactions are therefore
possible. In a preliminary study (V. L. Trudeau, unpublished
data), the GABA metabolism inhibitor GVG
bodymass injected intraperitoneally) given alone
had no effect on serum growth hormone levels compared with
saline-injection in sexually regressed control ﬁsh. This is
consistent with the lack of effect of intraperitoneally injected
GABA in regressed ﬁsh documented in the present study.
However, in the preliminary study, when GVG was given in
combination with the tyrosine hydroxylase inhibitor α-methyl-
bodymass injected intraperitoneally) to
inhibit dopamine synthesis, a signiﬁcant inhibitory effect on
serum growth hormone levels was observed. It may be that the
potent stimulatory dopamine input to growth-hormone-
secreting cells overrides the inhibitory effects of GABA,
especially in sexually regressed animals. Further analysis
of GABA/dopamine interactions within the goldﬁsh
hypophysiotrophic system is clearly warranted.
The GABA receptor subtypes mediating inhibition of
growth hormone release were not studied. Our previous work
in goldﬁsh indicated that GABA stimulated GTH-II release by
activating the GABA
-type receptor, and an additional
stimulatory component dependent on the GABA
also evident (Trudeau et al., 1993b). These functional studies
indicate the presence of the two GABA receptor subtypes, and
their respective roles in controlling growth hormone release in
the goldﬁsh remain to be determined.
The present series of experiments suggests that GABA is
involved in the inhibitory control of growth hormone release.
This contrasts with the stimulatory effects of GABA on GTH-
II release in goldﬁsh (Kah et al., 1992; Sloley et al., 1991;
Trudeau et al., 1993a,b) and trout (Mañanos et al., 1999).
GABAergic neurons projecting to somatotrophs and
gonadotrophs (Kah et al., 1987a, 1992), therefore,
differentially regulate the secretory activity of these two
adjacent cell types within the anterior pituitary. Furthermore,
we have previously demonstrated that GVG concomitantly
upregulates the expression of mRNA for the secretory vesicle
protein secretogranin-II (SgII) and decreases the cell content
of GTH-II in gonadotrophs, indicating a GABAergic activation
of a regulated secretory pathway (Blázquez et al., 1998). In
contrast, in the same experiment, GVG did not alter
somatotroph SgII mRNA levels, yet increased cell growth
hormone content. We interpret this as inhibition of growth
hormone secretion by GABA, which supports our results
showing that GABA injections reduce serum growth hormone
levels in maturing goldﬁsh. We have also presented evidence
that oestradiol modulates the GABAergic control of growth
hormone release in goldﬁsh. This latter observation is
especially signiﬁcant because it is known that growth hormone
stimulates ovarian oestradiol production (Van Der Kraak et al.,
1990; Le Gac et al., 1993) and oestradiol, in turn, enhances
growth hormone production and release (Trudeau et al., 1992;
Zou et al., 1997). Oestradiol can increase dopamine turnover
in the goldﬁsh hypothalamus and pituitary (Trudeau et al.,
1993a), which would contribute to a positive feedback
regulation of growth hormone release. Conversely, oestradiol
increases hypothalamic and pituitary GABA synthesis
(Trudeau et al., 1993a), and GABA inhibits growth hormone
release in maturing or oestradiol-treated animals. We
hypothesise that the GABAergic neurons in the preoptic region
and/or hypothalamus are part of a gonadal feedback system
controlling growth hormone secretion. Additional studies are
required to characterize fully the receptor subtypes, site and
mechanism of GABA action involved in the inhibition of
growth hormone release.
The authors acknowledge with appreciation the help of C.
S. Nahorniak and C. K. Murthy (Edmonton) with
radioimmunoassay, J. G. Dulka (Omaha) with brain injections
and A. Nisbet (Aberdeen) with IC
donations of H. Kawauchi (puriﬁed carp growth hormone;
Kitasato University, Japan) and E. Bohme (GVG; Hoechst
Marion Roussel Research Institute, Ohio, USA) for these
studies is greatly appreciated. This work was supported by the
NSERC (V.L.T, R.E.P. and J.P.C), Wellcome Trust U.K.
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