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Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912
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Abstract |
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 Top Abstract I. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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II. The EEA System: Overview, Description, and Localization in Neuroendocrine Tissues |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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illustrates the various
components that comprise the EAA neurotransmission system. The three
major components of the system (endogenous ligands, receptor proteins,
and transporter proteins) will be discussed individually below.
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Glutamate exists as four different pools in the CNS: a transmitter
pool, a metabolic pool, a glial pool, and a 
-aminobutyric acid
(GABA) precursor pool (21, 22, 23). Biochemical and immunocytochemical
approaches have demonstrated that synaptic vesicle concentrations of
glutamate (transmitter pool) are very high, approaching approximately
100 mM (21, 23) (Fig. 1
). Average glial concentrations of
glutamate, on the other hand, are much less (4–5 mM),
while glutamate concentrations in a GABAergic terminal are estimated to
be from 9–11 mM (21, 23). As also illustrated in Fig. 1, glutamate is synthesized by several enzymes. The mitochondrial enzyme,
phosphate-activated glutaminase (PAG), is thought to be an important
glutamate biosynthetic enzyme in that it catalyzes the production of
glutamate from glutamine and is present in both neurons and glia (21, 24). Phosphate and calcium activate PAG, while glutamate is inhibitory
(24). Thus, in states of neuronal activation, which involves hydrolysis
of ATP and increases of intracellular calcium, PAG activity would be
expected to be up-regulated in the presynaptic neuron. The source of
glutamine for neuronal production of glutamate appears to be glial
cells since the biosynthetic enzyme for glutamine (glutamine
synthetase) is found only in glial cells (25) (Fig. 1). Glutamine
synthetase serves to convert glutamate transported into glial cells
into glutamine, which is then released to the extracellular space for
uptake by neurons for use as a substrate for the PAG-driven production
of glutamate (25). The enzymes aspartate aminotransferase and glutamate
dehydrogenase have also been implicated to play a role in the synthesis
of glutamate (26, 27). Aspartate aminotransferase catalyzes the
reversible reaction: aspartate + 2-oxoglutarate 
glutamate +
oxaloacetate, while glutamate dehydrogenase catalyzes the reversible
reaction: glutamate + NADP 2-oxoglutarate + NH3 + NADPH
+ H+ (22, 26, 27). The aspartate aminotransferase- and glutamate
dehydrogenase-driven reactions are reversible, with the direction of
the reactions determined by the concentration of substrates. Hence,
aspartate aminotransferase and glutamate dehydrogenase can function
either to synthesize or to metabolize glutamate.
2. Localization of EAAs in the hypothalamus.
A number of
investigators have examined the distribution of glutamate in the
hypothalamus of various species. These studies have revealed that
within the hypothalamus of the rat and monkey, dense glutamate
immunostaining is found in the magnocellular and parvocellular
paraventricular nucleus (PVN), ventromedial nucleus (VMN), supraoptic
nucleus (SON), lateral hypothalamic area, suprachiasmatic nucleus
(SCN), arcuate nucleus (ARC), infundibular stalk, and median eminence
(ME) (3, 17, 18, 28, 29, 30). The highest level of glutamate
immunoreactivity was observed in presynaptic axon terminals, while
astrocytic processes showed significantly less immunostaining (17). As
a whole, these immunolocalization studies demonstrated that glutamate
is extensively localized in presynaptic terminals throughout the vast
majority of the nuclei of the hypothalamus, and hence strategically
positioned to control many different functions of the hypothalamus.
Glutamate release from neuron terminals is through exocytosis, which is
an ATP- and calcium-dependent event (31, 32). Current data suggest that
glutamate exocytosis is due to calcium entry through calcium channels
(Ref. 33, for review). Glutamate exocytosis can be modified by
activation of facilitatory or inhibitory receptors on presynaptic
neurons. Glutamate can enhance its own release by activation of the
facilitatory metabotropic glutamate receptor on presynaptic neurons,
which leads to phosphorylation and inactivation of presynaptic delayed
rectifier K+ channels, enhanced calcium influx, and thus
enhanced glutamate release (34). Agents that can inhibit glutamate
exocytosis by acting at inhibitory presynaptic receptors include
adenosine (35, 36), GABA-B agonists (37), and opioids (38). Once
released into the synaptic cleft, glutamate can bind to specific EAA
receptors on the pre- or postsynaptic neuron or it can be cleared from
the synaptic cleft by either glial (GLT-1 or GLAST) or neuronal (EAAC1
or EAAT4) transporter proteins (Fig. 1), each of which will be
discussed in sections below.
3. Regulation of EAA concentrations in the hypothalamus by steroid
hormones and developmental changes.
Despite intense investigation,
the specific factors that control EAA release at synaptic terminals
remain poorly understood. Several laboratories have investigated the
possibility that endocrine signals, such as steroid hormones, may be
important regulators of EAA release in the hypothalamus. Regulation of
hypothalamic EAA release by ovarian steroid hormones is of considerable
interest to neuroendocrinologists as it could provide a potential
mechanism for steroid hormone induction of hypothalamic GnRH release at
midcycle, which produces the preovulatory LH surge. Microdialysis
studies by Ping et al. (39) appear to support ovarian
steroid hormone control of hypothalamic EAA release, as estrogen plus
progesterone treatment in ovariectomized adult rats was found to
significantly enhance the release rates of glutamate in the preoptic
area (POA) of the hypothalamus 3–5 h after its administration (Fig. 2). Aspartate release rates in the POA
were also significantly enhanced by estrogen plus progesterone
treatment. The release rates of the neurally inactive amino acid,
serine, were not significantly affected by progesterone treatment,
indicating that the changes in glutamate and aspartate were due to
changes in neurotransmitter release and not to changes in metabolism.
Since the increase in POA release rates of glutamate and aspartate
occurred immediately before peak serum LH levels, it was proposed that
the increased release of EAAs in the POA was necessary for activation
of GnRH neurons. Work by Jarry et al. (40) also showed that
the POA release rates of aspartate and glutamate are increased during
the estrogen-induced LH surge. The steroid-induced increase in
aspartate and glutamate release appears to occur only in the POA, as a
recent study by Jarry et al. (41) demonstrated no change in
aspartate or glutamate release in the mediobasal hypothalamus during
the estrogen-induced LH surge. EAA levels in the hypothalamus have also
been reported to be increased during puberty. Along these lines, Goroll
et al. (42) recently demonstrated that the release rates of
glutamate and aspartate are increased in the POA at the time of puberty
in the female rat. Similarly, POA/mediobasal hypothalamus (MBH)
concentrations of glutamate and aspartate have been reported to be
higher in 30-day-old as compared with 16-day-old female rats (43). It
is tempting to speculate that the elevations in hypothalamic EAA
release rates observed during puberty are due to changes in circulating
levels of ovarian steroids that are known to occur at the time of
puberty. However, before definitive conclusions can be reached, further
investigations are needed to establish whether the observed changes are
actually due to fluctuations in steroids or to other puberty-related
factors.
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B. Types of EAA receptors and their localization in the
CNS and hypothalamus
1. Types of EAA receptors.
EAA receptors are the
most abundant excitatory neurotransmitter receptors in the CNS (1, 4, 6, 13). They are also known as "glutamate receptors" since
glutamate is believed to be the major endogenous ligand, although there
may be other endogenous ligands as discussed above. Two principal
groups of EAA receptors have been identified to date: 1) ionotropic and
2) metabotropic (Table 1). Ionotropic
receptors contain integral, cation-specific ion channels, whereas
metabotropic receptors are coupled to G proteins and modulate the
production of second messengers such as inositol phosphates and/or
adenylate cyclase. Ionotropic receptors can be subdivided into 1)
N-methyl-D-aspartate (NMDA), 2) kainate, and 3)
DL-
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) receptors according to their selective agonists. Activation of
ionotropic EAA receptors leads directly to the opening of a group of
ion channels that are typified by their different permeabilities to
Na+, K+, and Ca2+ ions. Stimulation
of these "ionotropic" receptors underlies rapid glutamate-mediated
excitatory synaptic transmission in the CNS. Multiple subunits exist
for each class of ionotropic EAA receptors as described in Table 1, and
it is thought that these subunits combine together to form a functional
EAA receptor.
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In contrast to the EAA ionotropic receptors described above,
metabotropic receptors, on the other hand, do not activate ion
channels. Instead, their response is characterized by a G
protein-stimulated release of Ca2+ from intracellular
stores mediated by ITPs and modulation of adenylate cyclase activity
(6, 14, 55). Hence, metabotropic receptors exert prolonged synaptic
modulation via modulation of second messenger systems. Metabotropic
receptors have been divided into three groups based on agonist
interactions and second messenger activation (Refs. 55 and 56, for
review) (Table 1). Group 1 is comprised of mGluR1 and mGluR5 subunits
due to the fact that they respond best to the ligand quisqualate, and
their activation leads to enhanced phosphatidylinositol hydrolysis.
Group 2 is comprised of mGluR2 and mGluR3 subunits based on the fact
that they respond best to the ligand t-ACPD, and they inhibit cAMP
formation. Finally, the mGluR4, mGluR6, mGluR7, and mGluR8 subunits
comprise group 3 based on their responding best to the ligand L-AP4 and
their inhibition of cAMP formation. Phenylglycine derivatives appear to
selectively inhibit metabotropic receptors and may be useful in
studying specific mGluR subunit effects and regulatory actions (Ref.
56, for review).
2. Localization of EAA receptors in neuroendocrine tissues.
For glutamate to be an important transmitter in neuroendocrine
regulation, it would be essential for glutamate receptors to be
localized in key neuroendocrine regulatory sites such as the
hypothalamus and pituitary. As indicated in Table 1, a significant
number of EAA receptor subunits have been demonstrated to be localized
in the hypothalamus and pituitary of the rat. Immunostaining studies by
Bhat et al. (74) demonstrated moderate to dense
immunostaining for the NMDA-R1 receptor in many hypothalamic nuclei of
the female rat, including the organum vasculosum of the lamina
terminalis (OVLT), VMN, SON, ARC, ME, medial preoptic area (MPOA), and
the PVN. Similarly, moderate to dense NMDA-R1 receptor mRNA and
immunostaining have been reported by Kus et al. (75) and
Petralia et al. (69) in almost all hypothalamic nuclei of
the male rat. With respect to the pituitary, Petralia et al.
(69) have reported moderate NMDA-R1 immunostaining in the anterior
pituitary of the male rat, and Bhat et al. (68) found that
approximately 9–11% of LH and FSH cells in the female rat anterior
pituitary colocalize the NMDA-R1 receptor subunit. GH and PRL cells in
the female rat anterior pituitary were also demonstrated to colocalize
the NMDA-R1 receptor subunit (68).
Using polyclonal antibodies to AMPA type glutamate receptor subunits, Brann et al. (60) found that the AMPA GluR1 receptor subunit was moderately dense in the OVLT, ARC, SON, PVN, and SCN with lighter staining observed in the MPOA of the female rat. GluR2–3 antibody staining yielded a similar pattern to that of GluR1, while immunostaining for the GluR4 receptor showed only very light staining in the ARC and MPOA. AMPA GluR subunits, particularly GluR1 and GluR2, also appear to be widely distributed throughout the male rat hypothalamus (59). Kainate receptor localization has been less intensely studied in the hypothalamus, but kainate binding has been reported to be high in the ARC and ME of the monkey (76). The in situ distribution of kainate GluR6 mRNA is similar to the autoradiographic localization of kainate binding described above (62, 63), and kainate GluR5 subunit mRNA is also expressed in the rat hypothalamus with high levels reported in the SCN and moderate to low levels in the rest of the hypothalamus (62, 63).
With respect to metabotropic receptors, Van den Pol and co-workers (71, 72) have recently reported that the mGluR1 and mGluR5 subunits are strongly expressed in the rat POA, SCN, and lateral hypothalamus, while Petralia et al. (73) reported moderately dense mGluR2–3 immunostaining in the SON, and light to moderate immunostaining in other regions of the hypothalamus and the anterior pituitary.
In agreement with the rather wide distribution of EAA receptor subtypes observed throughout the hypothalamus using immunocytochemical and in situ hybridization studies, Meeker et al. (77) found that each major glutamate receptor class is present in all hypothalamic regions of the rat as determined using receptor-binding studies. The approximate relative densities for glutamate receptor localization in the hypothalamus was NMDA > metabotropic glutamate receptor > kainate > AMPA. Thus, the above-described studies clearly demonstrate that all classes of EAA receptors are present in the hypothalamus and are widely distributed and strategically localized in many different nuclei of the hypothalamus so as to regulate a diverse number of hypothalamic functions.
3. Regulation of EAA receptors in the hypothalamus by steroid
hormones and developmental changes.
It has been postulated that
steroid hormones may up-regulate EAA receptor concentrations in the
hypothalamus as a potential mechanism for enhancing glutamate
neurotransmission. Such a mechanism would possibly explain the enhanced
activation of GnRH neurons noted at midcycle when circulating steroid
hormone levels are very high. Brann et al. (66, 67) recently
examined this issue in the rat and found that neither acute nor chronic
treatment with estradiol or progesterone affected NMDA receptor binding
or NMDA-R1 mRNA levels in the hypothalamus of female rats. Testosterone
treatment likewise had no effect on NMDA receptor binding and NMDA-R1
mRNA levels in the hypothalamus of the male rat. Similarly, Kus
et al. (75), using the in situ hybridization
technique that allows detection of mRNA transcripts in specific
hypothalamic nuclei, found that castration or dihydrotestosterone
treatment had no effect on NMDA-R1 mRNA levels in the ARC and MPOA of
the hypothalamus of adult male rats. Weiland (78), on the other hand,
found that estradiol plus progesterone caused a significant increase in
[3H]glutamate binding in the POA of the ovariectomized
rat. The increase in [3H]glutamate binding was not
displaced by NMDA, suggesting that the increased binding was most
likely due to an elevation of non-NMDA receptor sites. In support of
this possibility, Brann and Mahesh (79) found that estradiol plus
progesterone treatment increases AMPA receptor GluR1 subunit
immunoreactive levels in the POA and ARC of immature female rats. Diano
et al. (80) also recently reported an up-regulation of GluR1
and GluR2–3 immunoprotein levels in the hypothalamus of the adult
female rat after estradiol treatment. Thus, while hypothalamic NMDA
receptors do not appear to be regulated by steroids, hypothalamic AMPA
receptor levels, on the other hand, appear to be regulated by both
estradiol and progesterone. To our knowledge, comparative studies
examining whether steroids exert regulatory effects on kainate or
metabotropic receptors in the hypothalamus have not been performed.
Likewise, little is known concerning the specific cell types in the
hypothalamus that possess EAA receptors, although Bhat et
al. (74) recently reported that the NMDA-R1 receptor is
colocalized in nitric oxide neurons in many hypothalamic nuclei of the
female rat.
With respect to developmental changes in EAA receptors in the hypothalamus, Dees and co-workers (81) recently reported that NMDA-R1 mRNA levels increased at 20 days in the POA and at 25 days in the ARC/ME. Also, NMDA-R1 mRNA levels were reported to be increased in the POA during first proestrus in the rat. In contrast, Zamorano et al. (82), using quantitative RT-PCR, showed only an increase in NMDA-R1 mRNA levels in the female rat POA at day 20, and no increase thereafter. Likewise, Gore et al. (83) found an increase in NMDA-R1 mRNA levels in the POA only on day 20. With respect to the MBH, Zamorano et al. (82) found that NMDA-R1 mRNA levels were increased at days 15, 20, and 25 in the female rat, with no increase observed thereafter. With respect to the AMPA GluR1 receptor, Zamorano et al. (82) found an increase in GluR1 mRNA levels in the MBH at day 15 in the female rat, followed by an increase in the POA at day 20, with no changes thereafter. Finally, EAA receptor binding studies by Brann et al. (67) found that neither NMDA nor kainate receptor binding changed in the hypothalamus of the female rat during the time of puberty. However, since these studies used a hypothalamic block, it is possible that changes in discrete nuclei could have been undetected. Obviously, further studies on developmental regulation of hypothalamic EAA receptors are needed.
4. Evidence that glutamate functions as a major excitatory
transmitter in the hypothalamus.
Application of glutamate,
aspartate, and L-homocysteic acid has been shown to excite
neurons from virtually all areas tested in the hypothalamus including
the ARC, ME, and other medial hypothalamic locations (84). Similarly,
work by van den Pol and co-workers (3) demonstrated that hypothalamic
neurons responded to glutamate, aspartate, kainate, and NMDA with an
increase in intracellular Ca2+ that mediates a wide variety
of biochemical events in the cell. Furthermore, spontaneous excitatory
postsynaptic potentials (EPSPs) in the ARC, PVN, and mPOA of the rat
were found to be markedly attenuated by application of the non-NMDA
receptor antagonist, cyano-3,3-dihydro-7-nitroquinoxaline (CNQX) (3).
NMDA antagonists were found to only slightly suppress induced EPSPs;
however, the test conditions were not suitable for the testing of NMDA
since the membrane potential was maintained at hyperpolarized ranges
that cause the NMDA current to be almost completely blocked. Thus,
these studies demonstrated that EAA receptors in hypothalamic nuclei
are functional and that glutamate is a major regulator of synaptic
activation in the hypothalamus.
C. EAA transporters
Glutamate released into the synaptic cleft can be bound to
receptors, or it can be removed by high-affinity glutamate transporters
localized on neurons and glia. The removal of glutamate by transporter
proteins prevents toxicity by removal of excess glutamate and allows
for replenishment of glutamate transmitter stores through its recapture
(Fig. 1). To date, four glutamate transporter proteins have been
isolated from the brain. Two of the glutamate transporter proteins
(GLAST and GLT-1) were isolated from glia (85, 86) while a third
(EAAC1) was isolated from neurons (87). The fourth (EAAT4) appears also
to be a neuronal transporter and is predominantly expressed in the
cerebellum (88). For each glutamate transported into the cell by a
transporter, two Na+ are cotransported into the cell while a
K+ and a OH- ion are transported out of the
cell. Work from our laboratory has shown that the mRNA and protein for
the GLT-1 and GLAST transporters are expressed in the cycling adult rat
hypothalamus (R. Unda, V. B. Mahesh, and D. W. Brann,
unpublished findings). EAAC1 has also been reported to be expressed in
the hypothalamus, although only lightly (87).
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III. EAAs and Reproduction |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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A. Effect of EAA agonists on LH secretion
1. Glutamate.
The first report demonstrating that glutamate
can stimulate LH secretion came from the laboratories of Olney (89) and
Ondo (90) in 1976. Using systemic (subcutaneous) and third
intracerebroventricular administration routes, Olney et al.
(89) and Ondo et al. (90) demonstrated that glutamate
treatment markedly increased LH release in adult male rats without
affecting FSH release. The effect of glutamate on LH secretion was
suggested to be due to a hypothalamic site of action since direct
pituitary injection of glutamate was found to have no effect on LH or
FSH plasma levels (91). Testosterone levels were also elevated in the
male rats after subcutaneous glutamate treatment, presumably due to the
elevations in LH (89). Subsequent studies demonstrated that glutamate
also was capable of stimulating LH secretion in the prepubertal male
monkey (92) and the immature female rat (93).
2. NMDA.
The first report that selective agonists for the NMDA
receptor (NMDA or NMA) could stimulate LH secretion originated from the
laboratory of Cicero in 1978 (19), in which NMA
(N-methyl-D,L-aspartate) was
demonstrated to stimulate LH secretion in immature male rats. The
ability of NMDA to increase LH in male animals has been confirmed in a
variety of species including rats (91, 94, 95, 96, 97, 98), monkeys (92, 99), and
hamsters (100, 101). In female animals, numerous other studies have
demonstrated that NMDA also stimulates LH secretion, and less
consistently FSH secretion, in the intact immature female rat (43, 102, 103), the estrogen-primed ovariectomized rat and ewe (43, 93, 104, 105, 106), as well as in the cycling female rat (107, 108) and monkey
(109) (see also Fig. 3). Steroid hormones
also appear to modulate NMDA effects on LH secretion, and this is
discussed in detail in Section III A.6. below.
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, central administration of kainate into the third cerebroventricle
also enhances LH secretion in the ovariectomized estrogen-primed adult
rat, suggesting that kainate probably acts at the level of the
hypothalamus to control LH secretion. The effect of AMPA on LH release
has not been studied extensively; however, there are recent reports
demonstrating that AMPA increases LH release in the hamster (110), ram
(111), and ovariectomized estrogen-primed adult female rat (Fig. 3).
Thus, activation of non-NMDA receptors also leads to enhanced LH
secretion in a variety of species.
4. Metabotropic receptors.
To our knowledge, there are no
published reports describing activation of metabotropic receptors in
the regulation of LH secretion. Since various metabotropic receptor
subtypes have been demonstrated to be localized in important
reproductive neuroendocrine nuclei of the hypothalamus (71, 72, 73), it is
hoped that examination of this issue will be forthcoming in the near
future.
5. Temporal and dose-dependent effects of EAAs on LH
secretion.
NMDA, kainate, and AMPA regulation of LH secretion is
rapid, with peak LH levels occurring 10 min after third
intracerebroventricle (icv) injection in adult estrogen-primed
ovariectomized rats (Fig. 3). The effect is transient as LH levels
typically return to baseline by 30–60 min postinjection. The effect of
EAAs on LH secretion is dose-dependent with effective icv doses ranging
from 0.5–5 nmol for AMPA, kainate, and NMDA. Systemic injection
(subcutaneous or intravenous) of EAA receptor agonists is also
effective in eliciting a rapid release of LH (and in some cases of
FSH), with effective doses ranging from 1–2.5 mg/kg body wt for
kainate and AMPA and from 20–40 mg/kg body wt for NMDA (19, 43, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111).
6. Steroid dependency of EAA effects on LH secretion.
Recent
work by several laboratories has shown that the effect of NMDA on LH
secretion is steroid-dependent. Comparable studies examining the
steroid dependency of AMPA and kainate effects on LH secretion have not
been performed to our knowledge. Concerning the steroid dependency of
NMDA effects, NMDA has been shown to stimulate LH secretion in intact
or steroid-primed ovariectomized animals, but it has no effect or is
actually inhibitory to LH secretion in non-steroid-primed
ovariectomized animals (93, 95, 106, 108, 112, 113, 114). Subsequent studies
by Arias et al. (113) found that NMDA stimulated GnRH
release equally well from hypothalamic fragments obtained from either
steroid-primed or non-steroid-primed ovariectomized rats. This suggests
that the difference in LH response to NMDA may be due to different
sensitivities of the anterior pituitary to released GnRH in the
steroid-primed vs. the non-steroid-primed ovariectomized
rat. In apparent support of this possibility, Kalra and co-workers (95)
found that if one suppresses the hypersecretion of LH in the
non-steroid-primed ovariectomized rat by giving an opioid agonist, then
a stimulatory effect of NMDA on LH secretion could then be observed in
the ovariectomized non-steroid-primed rat.
Progesterone also appears to modulate EAA effects, as several groups have demonstrated that progesterone treatment significantly enhances the effect of NMDA on stimulating LH release in both the rat and the monkey (43, 112). Along these lines, Reyes et al. (112) found that the LH response to NMA was highest during the luteal phase of the monkey when serum progesterone levels were the highest. Likewise, Carbone et al. (43) found that NMDA administration induced plasma elevations of LH of 4.9-fold and 94.5-fold, respectively, in 30-day-old female rats and 30-day-old female rats treated with estrogen plus progesterone. How estradiol and progesterone may enhance LH response to EAAs is not entirely clear, but it could involve either an enhancement of pituitary sensitivity to GnRH, an enhancement of releasable GnRH stores in the hypothalamus, or an enhancement of EAA receptors in the hypothalamus (79, 80).
B. Effect of blockade of EAA neurotransmission on the
steroid-induced and preovulatory LH surge and pulsatile LH secretion
1. Steroid-induced and preovulatory LH surge.
To study the
physiological role of EAA neurotransmission in endocrine signaling to
produce the LH surge, several groups have employed specific EAA
receptor antagonists in vivo to block EAA neurotransmission
and determine the effect on the steroid-induced LH surge. A role for
EAA neurotransmission in the estradiol-induced LH surge was
demonstrated by Urbanski and Ojeda (102, 115) and Lopez et
al. (116), who demonstrated that administration of NMDA and
non-NMDA antagonists blocked the estradiol-induced LH surge in immature
and adult ovariectomized rats, respectively. Likewise, work by our
laboratory (104, 117) demonstrated that the administration of a NMDA
receptor antagonist completely blocked the progesterone-induced LH and
FSH surge in the estrogen-primed ovariectomized immature rat. Work by
our laboratory has also demonstrated that administration of the
non-NMDA receptor antagonist DNQX (20 nmol) at 0800 h and
1030 h via icv injections (10 µl) also attenuates the
progesterone-induced LH surge (118). Since DNQX blocks both AMPA and
kainate receptors, it was unclear which specific non-NMDA receptor
(AMPA or kainate) was involved in the progesterone-induced LH surge. A
recent follow-up study by our group provided evidence that AMPA
receptors are critical mediators of the LH surge as icv injection of
the selective AMPA receptor antagonist, NBQX, was found to
significantly attenuate the steroid-induced LH surge in the adult
ovariectomized rat (L. Ping, G. Bhat, V. B. Mahesh, and D. W.
Brann, unpublished findings). Additional studies using specific kainate
receptor antagonists (when they become available) are needed to further
establish the physiological role and contribution of kainate receptors
in the production of the LH surge.
Since the above studies were performed in discrete steroid-replaced ovariectomized animal models and one cannot be absolutely sure that the natural steroid fluctuations and patterns of the intact animal are exactly reproduced, it was important to confirm the above findings in the intact natural cycling adult rat. Toward this end, our laboratory examined the effect of an NMDA antagonist (MK-801) on the proestrous LH surge in the adult cycling rat. These studies showed that blockade of NMDA receptor-mediated neurotransmission via MK-801 treatment resulted in abolishment of the proestrous LH surge (104). Mean FSH levels, on the other hand, were not statistically different in MK-801 vs. vehicle-treated proestrous rats. Non-NMDA receptors also appear to be important for production of the preovulatory LH surge as treatment of the PMSG-primed immature rat with the non-NMDA receptor antagonist DNQX (15 nmol at 1100 h and 1500 h on the day of the surge) significantly attenuated the preovulatory LH surge while having no effect on the FSH surge (118). In agreement with our findings, Schwartz and co-workers (108) also found that the NMDA receptor antagonist MK-801 completely blocked the proestrous LH surge in the adult female rat. To our knowledge, there have been no studies reported in the literature concerning the role of metabotropic EAA receptors in LH surge production. This issue awaits further study. Nevertheless, taken as a whole, the above studies demonstrate that EAA neurotransmission through NMDA and non-NMDA receptors is an important component of the neurotransmission line mediating the natural preovulatory LH surge.
2. Pulsatile LH secretion.
In addition to a role in LH surge
expression, evidence has accumulated that EAAs have a regulatory role
in the natural pulsatile pattern of LH secretion. Along these lines,
repetitive NMDA stimulation has been demonstrated to produce GnRH
pulses in vitro and LH pulses in vivo upon each
stimulation (92, 107). Additional support for a role of NMDA receptors
in the control of pulsatile LH secretion has come from Arslan et
al. (119) who found that systemic administration of a competitive
NMDA receptor antagonist, AP5, suppresses pulsatile LH release in the
castrate male rat. Similarly, Bourguignon et al. (120) found
that pulsatile GnRH release from male hypothalami in vitro
is abolished by MK-801 treatment. Work from our laboratory has
demonstrated that a single icv administration of AP5 decreased LH and
FSH mean and trough levels and LH and FSH pulse frequency in the
castrate adult male rat (121). In contrast, a single icv injection of
the non-NMDA receptor antagonist, DNQX, did not have any effects on
pulsatile LH or FSH secretion in the castrate adult male rat. However,
if multiple injections of DNQX were used, then LH pulses were
suppressed. In the ovariectomized female rat, icv administration of the
NMDA receptor antagonist, AP5, or the non-NMDA receptor antagonist,
DNQX, resulted in a significant decrease in mean and trough LH levels
as well as LH pulse frequency and pulse amplitude (122). Since DNQX
blocks both kainate and AMPA receptors, the question arises which class
of non-NMDA receptors is the more important in regulating pulsatile LH
secretion. While this question awaits further experiments for its
resolution, it should be pointed out that while kainate does stimulate
LH secretion in male and female animals (19, 91, 92, 107), its effect
on LH release has been shown to rapidly desensitize, such that kainate
stimulates LH secretion on the first application, but subsequent
applications fail to cause elevations of LH secretion (92, 107). Thus,
attempts to show that kainate can induce pulsatile LH secretion have
not been successful. To our knowledge, there are no published studies
that have tested whether AMPA can induce pulsatile LH secretion.
Likewise, the role of metabotropic EAA receptors in the control of
pulsatile GnRH or LH secretion has not been thoroughly addressed. While
further work in this area is clearly needed, the above studies do
provide evidence that glutamate (acting through both NMDA and non-NMDA
receptors) is able to modulate the natural pulse generator in the
hypothalamus and thereby control pulsatile LH secretion in male and
female animals. Thus, glutamate neurotransmission appears to be
important for both surge and pulsatile LH secretion.
C. Site of action of EAAs in the control of LH secretion
1. Hypothalamic vs. pituitary site of action.
A number of
studies have focused on elucidating the precise site of action of
glutamate in the control of LH secretion. The majority of the evidence
accumulated to date suggests that glutamate acts primarily at the
hypothalamus to control LH secretion. Evidence supporting this
contention has derived from studies in which glutamate agonists were
administered centrally, a route that would appear to preclude an action
directly at the pituitary. For instance, central administration of
glutamate agonists through either direct injection into the
hypothalamus (90) or injection into the third cerebroventricle (Refs.
74, 105; see also Fig. 3) was demonstrated to induce a significant
elevation of serum LH levels in male and female animals within 10–15
min. A hypothalamic site of action is also supported by studies that
demonstrated that GnRH antagonists block the ability of EAAs to
stimulate LH release (96, 123, 124). Conversely, direct injection of
glutamate into the anterior pituitary was found to have no effect on LH
secretion (90). Likewise, NMDA was found to have no effect on LH
release from rat anterior pituitary cells in static culture (125).
Conversely, it should be pointed out that a few studies have reported
an enhancement of LH and other pituitary hormones by glutamate agonists
when perifused anterior pituitary cell cultures were used (126, 127, 128),
and ionotropic glutamate receptors have been reported in anterior
pituitary cells of the rat by several investigators (52, 59, 61, 68, 69, 129). Nevertheless, these studies require further confirmation, and
the general consensus remains that glutamate controls LH secretion
through actions exerted primarily at the hypothalamus of male and
female animals.
2. Site of action of EAAs in the hypothalamus: POA vs. ARC/ME.
To pinpoint the sites in the brain where EAAs could act, several
investigators have used c-Fos immunoreactivity as a marker of neuronal
activation after EAA treatment. Along these lines, results from our
laboratory demonstrated that administration of AMPA or kainate into the
third ventricle of estrogen-primed ovariectomized adult rats induced
immunoreactive c-Fos in the ARC (129), suggesting that the ARC may be a
major site of action for AMPA and kainate in the induction of LH
release. In support of this suggestion, we found that AMPA and kainate
are more potent than NMDA in stimulating GnRH release in
vitro from proestrous rat ARC/ME fragments (130). Likewise,
Negro-Vilar and co-workers (131, 132) found that AMPA and kainate were
much more potent than NMDA in stimulating GnRH release from male rat
ARC/ME fragments. Finally, glutamate-induced GnRH release from male rat
ARC/ME fragments in vitro was found to be blocked by the
AMPA/kainate receptor antagonist, DNQX, but not by the NMDA receptor
antagonist, AP-7 (131). Thus, non-NMDA receptors appear to be important
in controlling LH secretion through actions at the level of the ARC/ME;
however, it should be pointed out that kainate has been shown to
stimulate LH release when injected into the POA (91), and kainate has
also been demonstrated to stimulate GnRH mRNA levels in the POA of male
rats (133). Hence, one cannot rule out that non-NMDA receptor
activation in the POA does not contribute to regulation of GnRH and LH
secretion.
With respect to NMDA induction of c-Fos in the brain, several groups have shown that NMDA induces c-Fos immunoreactivity in the ARC and ME, the OVLT, the periventricular hypothalamus, the PVN, the cerebral cortex, and in noradrenergic cells of the locus ceruleus of immature and adult rats (129, 134, 135, 136). These studies suggest that NMDA could act at multiple sites in or outside the hypothalamus to influence LH release. Considerable evidence has appeared in the literature suggesting that a major site of action of NMDA in the control of LH secretion is the POA of the hypothalamus. For instance, NMDA has been shown to stimulate LH secretion when injected into the MPOA, but not when injected into the anterior hypothalamic nucleus, ventromedial nucleus, or ARC (91), and NMDA is still capable of stimulating LH release even after destruction of the ARC by monosodium glutamate treatment (103, 137). In addition, NMDA has been demonstrated to stimulate POA GnRH synthesis and secretion (120, 138), and the elevation of GnRH mRNA levels in the POA corresponded with the NMDA-induced rise in plasma LH levels at 15 and 60 min (139). Liaw and Barraclough (140) confirmed and extended these findings to the female by demonstrating that NMDA administration increased OVLT/rostral POA GnRH mRNA levels at 1 h after injection in estrogen-primed ovariectomized rats. Finally, recent work by Bourguignon et al. (141) demonstrated that treatment with antisense oligonucleotides to the NMDA-R2A subunit inhibits pulsatile GnRH release from the complete POA/MBH fragment but not when the MBH fragment alone is used.
Based on the above findings, it is suggested that the POA constitutes a
major site of action of NMDA in the regulation of GnRH and LH release;
however, other sites, such as the locus ceruleus and ARC, may also be
important contributing sites in mediating the regulatory actions of
NMDA on GnRH and LH secretion. Figure 4
illustrates the potential different sites of action on which NMDA and
non-NMDA receptors could be activated by glutamate to exert regulatory
control over LH secretion. Based on the literature cited above, NMDA
receptor-mediated actions are proposed to dominate in the POA, while
non-NMDA receptor-mediated actions are proposed to dominate in the
ARC/ME. The locus ceruleus may also be an additional important site of
action for EAAs, since it provides a major catecholaminergic imput to
the neuroendocrine hypothalamus, and NMDA has been shown to induce
c-Fos in noradrenergic cell bodies in the locus ceruleus (136). Recent
studies have also demonstrated that NMDA and non-NMDA receptors are
localized in the locus ceruleus (142), and although there are
conflicting reports in the literature, there is some evidence
supporting a role for catecholamines in mediating NMDA effects on GnRH
secretion, as will be discussed in detail in a subsequent section (20, 98, 103, 140, 143). Finally, the anterior pituitary may also be a
secondary site of action for EAAs, although this possibility requires
further confirmation.
|
Zuo et al. (130) recently reported that middle-aged rats display an attenuated GnRH neurosecretory response to glutamate agonists on proestrus afternoon as compared with young proestrous rats (130). This has led to the suggestion that a defect in hypothalamic glutamate neurosignaling may be an important contributing factor in reproductive aging in the female. The defect in hypothalamic glutamate neurosignaling may be due to a decrease in hypothalamic glutamate receptors as quantitative RT-PCR revealed significantly lower NMDA-R1 mRNA levels in the POA and ARC/ME of the middle-aged proestrous rat vs. the young proestrous rat (130).
2. EAA regulation of GnRH neurons: direct or indirect?
A major
question concerning EAA regulation of GnRH secretion is whether the
effect is exerted directly on the GnRH neuron or indirectly through
interneurons. The majority of evidence supporting a direct effect of
EAAs on GnRH neurons has come from studies utilizing the immortalized
GnRH neuronal cell line (GT1–7 cell line). For instance, Spergel
et al. (145) found that glutamate, NMDA, AMPA, kainate,
and the metabotropic receptor agonist, t-ACPD, all were able to induce
Ca2+ influx and subsequent GnRH release in GT1–7 cells.
The effects were reportedly specific as they were each blocked by
specific EAA receptor antagonists. Similarly, Grumbach and co-workers
(146) reported that NMDA stimulates GnRH release from GT1–7 cells
in vitro, and they and others have reported that GT1–7
cells strongly express the NMDA-R1 mRNA transcript (146, 147, 148). However,
in situ hybridization studies have revealed that less
than 5% of GnRH neurons in adult male and female rats express NMDA-R1
mRNA (149). Likewise, GnRH neurons in adult male Syrian hamsters have
been reported not to colocalize NMDA-R1 receptor mRNA or protein or
AMPA receptor subunit mRNA (Ref. 150, for review). Furthermore, two
different laboratories (135, 136) have reported that GnRH neurons do
not express c-Fos after NMDA treatment, but neurons that surround GnRH
neurons did. Thus, these findings suggest that the effect of NMDA on
GnRH neurons may be an indirect one that involves mediation by other
"interneurons." In agreement with the reported lack of EAA
receptors in GnRH neurons in vivo, a recent study
demonstrated that GnRH neurons in the hypothalamus appear to be immune
to the neurotoxic effects of high doses of glutamate, kainate, and NMDA
(151). Taken as a whole, the available in vivo data
appear to support an indirect mechanism of action for EAAs in the
control of GnRH secretion, although a direct effect on the GnRH neuron
cannot be ruled out.
3. Potential mediators of EAA effects on GnRH neurons: a role for
catecholamines?
Since the majority of the reports in the
literature appear to support an indirect effect of EAAs to control GnRH
neurosecretion, there has been intense work to identify
"interneurons" that mediate EAA effects on GnRH neurons. Along
these lines, a number of studies have demonstrated that EAAs can
stimulate hypothalamic norepinephrine release in the rat (152, 153, 154),
leading to the suggestion that catecholamines may mediate the effects
of EAAs on GnRH release. As stated previously, NMDA and non-NMDA
receptors have been demonstrated in the locus ceruleus where many
hypothalamic-projecting catecholamine cell bodies are located (142),
and Saitoh et al. (136) reported that NMDA induces c-Fos
in noradrenergic neurons of the locus ceruleus and in dopaminergic
neurons in the MBH in the rat. In contrast, no c-Fos immunoreactivity
was observed in neuropeptide Y-immunoreactive cells in the POA, septum,
or locus ceruleus area after NMDA administration. Hence, either
dopaminergic or noradrenergic neurons could play a role in mediating
EAA effect on GnRH release. Liaw and Barraclough (140) reported that
pretreatment with the 1-adrenergic antagonist, prazosin, blocked
NMDA-induced LH release in four of seven rats. The remaining animals
responded to NMDA with a significant increase in plasma LH. Studies
utilizing norepinephrine synthesis inhibitors have also produced
somewhat contradictory results, with a block of NMDA-induced LH release
observed in one study (103) and no effect observed in a second study
(20). In vitro studies, on the other hand, have been
more straightforward and less ambiguous as McCann and co-workers (143)
found that phentolamine, a noradrenergic -receptor antagonist,
clearly and effectively blocked the ability of glutamate to stimulate
GnRH release from male rat ARC/ME fragments in vitro.
Dopamine appears to be less important than norepinephrine as a mediator
of NMDA effects, as Price et al. (98) found that
pretreatment with dopamine antagonists had no effect on NMA-induced LH
release in the male rat.
With respect to other potential mediators of glutamate action, Rossmanith et al. (155) recently demonstrated that administration of the NMDA receptor antagonist, MK-801, resulted in a significant attenuation of galanin mRNA levels in GnRH neurons during the time it blocked the steroid-induced LH surge in the rat. Since galanin is a potent stimulator of GnRH secretion and also has been implicated in the LH surge apparatus (156, 157, 158), it is possible that galanin neurons may also be a mediator of glutamate effects on GnRH neurons. Recent studies by Voight et al. (159) and Melcangi et al. (160) have also provided evidence that glial factors can facilitate GnRH secretion. This is interesting as glutamate receptors of all classes (excepting NMDA) have been demonstrated to be present on glial cells (161), and glutamate has been shown to induce calcium waves in hypothalamic and hippocampal glial cells (18, 162, 163). Thus, it is possible that in addition to recruiting other neurons into the accelerator mechanism for GnRH surge production, glutamate may also recruit and activate glial cells in the hypothalamus to release active factors to potentiate GnRH neurosecretion. This interesting possibility awaits further study.
4. Potential interaction of glutamate neurons with nitric oxide and
opioid neurons in the hypothalamus: importance to the control of GnRH
secretion.
Recent studies by a number of laboratories has provided
significant evidence that the novel gaseous neurotransmitter, nitric
oxide, plays an important role in mediating glutamate effects upon GnRH
neurons (164, 165). Work from our laboratory and others has
demonstrated that nitric oxide neurons are present in significant
concentrations in key reproductive hypothalamic nuclei including the
OVLT, POA, and ME (74, 166, 167). GnRH neurons in the OVLT and POA of
the male and female rat were found not to possess nitric oxide synthase
(NOS); however, GnRH neurons were often surrounded by nitric oxide
neurons, and potential contacts between the two neuronal types in the
OVLT and POA were observed (74, 166, 167). Furthermore, there was a
significant overlap between en passant GnRH neuronal fibers
and nitric oxide neuronal fibers in the ME (74). Further work by our
group has demonstrated that b-NOS (brain-NOS or neuronal-NOS) is the
major NOS isoform in the hypothalamus with virtually no e-NOS
(endothelial-NOS) or m-NOS (macrophage or inducible-NOS) detected
(168). The above findings provide anatomical evidence that nitric oxide
neurons are in position in the hypothalamus to regulate GnRH secretion
and thus could serve to mediate glutamate effects on GnRH neurons.
That nitric oxide neurons in the hypothalamus could be targets for
glutamate action was demonstrated by Bhat et al. (74) who
found that nitric oxide neurons in many hypothalamic nuclei appear to
colocalize the NMDA-R1 receptor. Functional evidence supporting a role
for nitric oxide in mediating glutamate stimulation of GnRH release has
come from the study of Rettori et al. (164), who
demonstrated that glutamate-induced GnRH release from ARC/ME fragments
in vitro is blocked by coadministration of a competitive
inhibitor of nitric oxide, NG-monomethyl-L-arginine (NMMA)
as well as by hemoglobin, which scavenges nitric oxide. Moreover,
Moretto et al. (169) were the first to demonstrate that
nitric oxide itself stimulates GnRH release from hypothalamic fragments
and from immortalized GnRH neurons in vitro, a finding
subsequently confirmed by McCann and co-workers (164). As shown in Fig. 5, work from our laboratory has also
demonstrated that the NMDA-induced LH increase in vivo is
blocked by prior treatment with a NOS inhibitor (74). A functional role
for nitric oxide in the production of the steroid-induced and
preovulatory LH surge has been demonstrated by Kalra and co-workers
(170, 171), who showed that administration of NOS inhibitors blocks the
steroid-induced and proestrous LH surge in the female rat. Likewise,
Aguan et al. (172) found that central administration of
b-NOS antisense oligonucleotides significantly attenuated the
steroid-induced LH surge in the adult ovariectomized rat.
|
) (168),
and cGMP analogs have been shown to stimulate GnRH release, while a
cGMP-dependent protein kinase inhibitor conversely blocks nitric
oxide-induced GnRH release (169). Thus, nitric oxide may regulate the
heme-containing signaling enzyme, guanylate cyclase, and thereby
elevate the second messenger cGMP and facilitate GnRH secretion.
Additionally, one cannot rule out an effect of nitric oxide on the
heme-containing enzyme, cyclooxygenase, which controls production of
prostaglandins, a known stimulator of GnRH secretion (173).
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E. The opioid-glutamate-nitric oxide-guanylate cyclase pathway in
the control of GnRH secretion: a proposed model
Based on the findings described above, a working model has been
proposed that suggests a central role for glutamate in endocrine
signaling to produce the preovulatory LH surge in the rat (Fig. 7). As illustrated in the model,
regulation of GnRH neurosecretion involves both inhibitory and
excitatory neurotransmitters, which are regulated by steroid hormones.
The inhibitory transmitters are proposed to function as a "brake"
to restrain neurosecretion, whereas the excitatory transmitters are
proposed to function as an "accelerator" to enhance GnRH
neurosecretion. Opioid neurons are proposed to be the major component
of the inhibitory brake, with GABA and neuropeptide K neurons also
participating (95, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185). Based on the findings presented above,
glutamate is proposed to be a major component of the "accelerator"
mechanism, with nitric oxide, neuropeptide Y, galanin, and
catecholamines also participating (2, 74, 79, 104, 155, 156, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 186, 187). During nonsurge conditions, the opioid inhibitory brake acts
to tonically restrain GnRH secretion by inhibiting excitatory
"accelerator" components such as glutamate (174, 175),
catecholamine (188), and neuropeptide Y (178, 185) neurons. Opioids may
also directly inhibit GnRH neurons. During surge conditions, such as
those observed on the day of the preovulatory LH surge, rising steroid
hormone levels in the blood are proposed to exert actions in the brain
to turn off opioid neurons, thereby allowing activation of excitatory
"accelerator" components (glutamate, nitric oxide, neuropeptide Y,
catecholamines, galanin), which stimulate GnRH neurons to produce the
GnRH surge. As shown in the model, glial cells (astrocytes) may also
participate in facilitating GnRH secretion (159, 160, 189) and may act
to mediate glutamate-excitatory signals to GnRH neurons, although this
requires further validation. As a whole, the evidence cited above
clearly supports a critical and central role for glutamate as an
important neuroendocrine signaler in the hypothalamus to control GnRH
secretion and the preovulatory LH surge.
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IV. Role of EAAs in Puberty and Reproductive Behavior |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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, a remarkable degree of synchronization
of the day of vaginal opening was observed in the NMDA-treated rats
with the majority of the animals displaying vaginal opening on a single
day (day 32). The non-NMDA receptor agonist, kainate (2 mg/kg), did not
significantly affect the timing of puberty in the female rat (Fig. 8),
and likewise treatment with the non-NMDA receptor antagonist, DNQX (15
nmol icv at 1400 and 1600 h, days 29–36), had no significant
effect on the time of vaginal opening in the female rat (67). In
contrast, a physiological role for endogenous glutamate acting at NMDA
receptors in puberty was evidenced by the demonstration that treatment
with the NMDA receptor antagonist MK-801 delayed puberty in female rats
(115). Activation of GnRH neurons appears to underlie the ability of
NMDA to advance puberty as evidenced by the ability of a GnRH
antagonist to block the facilatory effect of NMDA on puberty (99).
Further support for a role for endogenous glutamate in the achievement
of puberty has come from studies that have demonstrated that endogenous
release rates for glutamate and aspartate increase in the POA in female
rats at the time of puberty (42, 43). To our knowledge there have been
no correlative studies performed to address whether AMPA or
metabotropic EAA receptors play a role in puberty. Nevertheless, the
above studies provide substantial evidence that endogenous glutamate
acting through NMDA receptors is an important component in the CNS
mechanism regulating puberty in female animals.
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V. Role of EAAs in the Secretion of Other Anterior Pituitary Hormones |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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2. Site and mechanism of action of EAAs in the control of ACTH
secretion and physiological role of EAAs in stress.
As shown in
Fig. 9A, the PVN in the hypothalamus
appears to be the major site of action for EAAs in regulating ACTH
release since direct injection of glutamate into the PVN has been shown
to stimulate ACTH release (200). This effect appears to be due to EAA
regulation of CRF release in the PVN since EAAs stimulate CRF release
from neonatal rat hypothalami in vitro (201). However, it
should be pointed out that other studies failed to find any effect of
EAAs on CRF release from adult rat hypothalami in vitro
(202, 203). Instead, EAAs stimulated the release of vasopressin,
another ACTH-secretogue present in the PVN. However,
immunoneutralization studies in rats have shown that CRF antiserum, but
not vasopressin antiserum, blocks NMDA- and kainate-induced ACTH
release in vivo (97, 197). Finally, recent studies by Joanny
et al. (204) revisted whether glutamate could modulate CRF
release from adult rat hypothalami in vitro and found that
glutamate and NMDA were capable of stimulating CRF release. Their study
used hypothalamic slices rather than the fragments used in previous
studies, which may have provided better access of the EAAs to the CRF
neurons to stimulate CRF release. Thus, the available evidence to date
tends to support regulation of CRF by EAAs as the primary mechanism of
EAA stimulation of ACTH release, although other mechanisms such as
modulation of arginine vasopressin release may also play a role (Fig. 9A). Furthermore, glutamate may also exert effects at other CNS sites
to modulate the HPA axis, as Gabr et al. (205) recently
reported that direct injection of glutamate into the amygdala of
stressed anesthetized male rats increased CRF release from the ME and
markedly enhanced plasma corticosterone levels. Likewise, Carlson and
Gann (206) demonstrated that direct injection of glutamate into the
locus ceruleus also stimulates ACTH release. This effect may involve
mediation by norepinephrine since the locus ceruleus is a major site of
norepinephrine cell body localization that projects to the PVN, and
EAAs have been reported to induce c-Fos in noradrenergic neurons in the
locus ceruleus (136). A direct effect of EAAs at the anterior pituitary
seems unlikely since glutamate, NMDA, and kainate have been
reported to have no effect on ACTH release from rat pituitaries
incubated in vitro (197).
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A physiological role for EAAs in stress activation of the HPA axis was suggested from the studies of Jezova et al. (209), who found that ACTH secretion in response to immobilization stress in male rats was significantly inhibited by pretreatment with the NMDA receptor antagonist, MK-801. Plasma epinephrine levels were also found to be inhibited by MK-801 pretreatment. Similarly, Soltis and DiMicco (210) reported that microinjection of either a NMDA or non-NMDA receptor antagonist into the dorsomedial hypothalamus of adult male rats attenuated air stress-induced tachycardia, while Papp and Moryl (211) found that NMDA receptor antagonists reduced stress-induced depression. Thus, EAA neurotransmission appears to be an important activator of the HPA axis and may be a central mediator in stress responses.
B. GH
EAA regulation of GH release was first reported in 1983 by Mason
et al. (212), who demonstrated that subcutaneous injections
of NMA or kainate, but not quisqualate, significantly elevated serum GH
levels 7.5 min after administration in adult male rats. EAA stimulation
of GH release was subsequently confirmed in a variety of species
including sheep, bull calves, and monkeys (124, 213, 214, 215).
Additionally, Nemeroff et al. (216) found that
intrahypothalamic injection of the NMDA receptor agonist, quinolinic
acid, markedly stimulated GH release in male rats. As illustrated in
Fig. 9B, stimulation of GHRF release from the ARC appears to be the
major mechanism whereby NMDA stimulates GH release. Evidence supporting
this contention has come from findings demonstrating that 1)
NMDA-induced GH release can be blocked by administration of GHRF
antibodies (215); 2) destruction of the ARC, where GHRF neuronal cell
bodies reside, completely abolishes the ability of NMDA to induce GH
release (215); 3) treatment of rats with an NMDA receptor antagonist
causes a reduction in hypothalamic GHRF mRNA levels while somatostatin
(SRIF) mRNA levels are unaltered (217); and 4) GHRF immunoreactivity in
the ME is reduced in NMDA receptor antagonist-treated rats (217). Other
sites/mechanisms still may have a regulatory role, however, as some
studies have found a stimulatory effect of NMDA on SRIF release from
hypothalami incubated in vitro (218). Stimulation of SRIF
could enhance GH release by exerting a regulatory effect on GHRF
release in the ARC (218, 219). As also shown in Fig. 9B, in addition to
a hypothalamic site of action, EAAs may also act directly at the
anterior pituitary to regulate GH secretion. In support of this
possibility, NMDA has been shown to dose-dependently stimulate GH
secretion from perifused somatotropes in vitro (127). This
effect was blocked by treatment with NMDA receptor antagonists and
appeared to be mediated by an increase in intracellular calcium. Niimi
et al. (220) also confirmed a dose-dependent stimulatory
effect of NMDA, kainate, and glutamate on GH release from somatotropes
in vitro. Thus, while EAA regulation of GHRF release from
the ARC appears to be the primary mechanism for EAA stimulation of GH
release, other sites and mechanisms may also play a role.
C. PRL
Recent work has shown that NMDA treatment induces c-Fos
immunoreactivity in hypothalamic regions known to be important in the
regulation of PRL secretion (i.e., the PVN and the ARC)
(107, 135). Agonist studies verified that NMDA treatment leads to
enhanced PRL secretion in male and female animals of a variety of
species (96, 107, 108, 109, 124, 221). Kainate also stimulates PRL release
when administered into the third cerebroventricle (107). The mechanism
whereby EAAs stimulate PRL release is presently unclear. As shown in
Fig. 9C, EAAs could act to enhance PRL secretion through effects on
PRL-releasing factors in the hypothalamus, such as TRH and vasoactive
intestinal peptide in the PVN or oxytocin in the ARC. Dopamine neurons
in the ARC may also play a role in EAA stimulation of PRL release, as
NMDA has been reported to induce c-Fos immunoreactivity in dopamine
neurons in the MBH (136). Glutamate and NMDA have also been reported to
enhance PRL release directly from perifused anterior pituitary cells
in vitro (128), suggesting that direct modulation by
glutamate at the anterior pituitary is also possible. Clearly, more
studies are needed to clarify the precise site and mechanism of action
of EAAs in the stimulation of PRL release.
Work by a number of investigators has addressed the question of whether glutamate neurotransmission has a physiological role in the control of PRL secretion (104, 118, 222, 223). Brann and Mahesh (104) demonstrated that administration of the NMDA receptor antagonist, MK-801, blocks the proestrous PRL surge in the female rat as well as the PMSG-induced preovulatory PRL surge in the immature female rat. Non-NMDA receptors also appear to be important for the preovulatory PRL surge, as Brann et al. (118) have shown that injection of the non-NMDA receptor antagonist DNQX into the third cerebroventricle also significantly attenuates the preovulatory PRL surge in the PMSG-primed immature rat. Non-NMDA antagonist treatment was also shown to attenuate suckling-induced PRL release, an effect not shared by NMDA receptor antagonists (223). As a whole, the above studies provide significant evidence that glutamate is an important transmitter in the control of PRL secretion.
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VI. Conclusions |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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Although much has been accomplished, significant gaps still remain in our knowledge. For instance, while there is considerable knowledge and understanding with respect to the role of NMDA receptors in neuroendocrine regulation, considerably less is known concerning the role of AMPA and kainate receptors in these events, and virtually nothing is known concerning the role of metabotropic EAA receptors. Furthermore, the interaction of glutamate with other transmitters and neuropeptides in the hypothalamus and the precise cellular messengers and signaling pathways underlying glutamate effects also require further clarification. Undoubtedly, as the field continues to move forward, these gaps in our knowledge will be closed even further. While considerable work still remains, the impressive body of evidence accumulated to date suggests that glutamate is one of the dominant excitatory transmitters in the hypothalamus and functions as a critical central mediator in endocrine signaling and neuroendocrine regulation.
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Footnotes |
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1 Parts of the research summarized in this article were supported by NIH
Grants HD-28964 and HD-16688. 
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References |
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TopAbstractI. IntroductionII. The EEA System:...III. EAAs and ReproductionIV. Role of EAAs...V. Role of EAAs...VI. ConclusionsReferences |
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 Z. Chu and S. M. Moenter Endogenous Activation of Metabotropic Glutamate Receptors Modulates GABAergic Transmission to Gonadotropin-Releasing Hormone Neurons and Alters Their Firing Rate: A Possible Local Feedback Circuit J. Neurosci., June 15, 2005; 25(24): 5740 - 5749. [Abstract] [Full Text] [PDF]
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F. J P Ebling The neuroendocrine timing of puberty Reproduction, June 1, 2005; 129(6): 675 - 683. [Abstract] [Full Text] [PDF]
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V. M. Navarro, J. M. Castellano, R. Fernandez-Fernandez, S. Tovar, J. Roa, A. Mayen, M. L. Barreiro, F. F. Casanueva, E. Aguilar, C. Dieguez, et al. Effects of KiSS-1 Peptide, the Natural Ligand of GPR54, on Follicle-Stimulating Hormone Secretion in the Rat Endocrinology, April 1, 2005; 146(4): 1689 - 1697. [Abstract] [Full Text] [PDF]
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V. M. Navarro, J. M. Castellano, R. Fernandez-Fernandez, S. Tovar, J. Roa, A. Mayen, R. Nogueiras, M. J. Vazquez, M. L. Barreiro, P. Magni, et al. Characterization of the Potent Luteinizing Hormone-Releasing Activity of KiSS-1 Peptide, the Natural Ligand of GPR54 Endocrinology, January 1, 2005; 146(1): 156 - 163. [Abstract] [Full Text] [PDF]
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C. Caruso, M. C. Bottino, M. Pampillo, D. Pisera, G. Jaita, B. Duvilanski, A. Seilicovich, and M. Lasaga Glutamate Induces Apoptosis in Anterior Pituitary Cells through Group II Metabotropic Glutamate Receptor Activation Endocrinology, October 1, 2004; 145(10): 4677 - 4684. [Abstract] [Full Text] [PDF]
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H. T. Jansen, C. Cutter, S. Hardy, M. N. Lehman, and R. L. Goodman Seasonal Plasticity within the Gonadotropin-Releasing Hormone (GnRH) System of the Ewe: Changes in Identified GnRH Inputs and Glial Association Endocrinology, August 1, 2003; 144(8): 3663 - 3676. [Abstract] [Full Text] [PDF]
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S. Heger, M. Seney, E. Bless, G. A. Schwarting, M. Bilger, A. Mungenast, S. R. Ojeda, and S. A. Tobet Overexpression of Glutamic Acid Decarboxylase-67 (GAD-67) in Gonadotropin-Releasing Hormone Neurons Disrupts Migratory Fate and Female Reproductive Function in Mice Endocrinology, June 1, 2003; 144(6): 2566 - 2579. [Abstract] [Full Text] [PDF]
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W. Lin, K. McKinney, L. Liu, S. Lakhlani, and L. Jennes Distribution of Vesicular Glutamate Transporter-2 Messenger Ribonucleic Acid and Protein in the Septum-Hypothalamus of the Rat Endocrinology, February 1, 2003; 144(2): 662 - 670. [Abstract] [Full Text] [PDF]
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A. B. Cashion, M. J. Smith, and P. M. Wise The Morphometry of Astrocytes in the Rostral Preoptic Area Exhibits a Diurnal Rhythm on Proestrus: Relationship to the Luteinizing Hormone Surge and Effects of Age Endocrinology, January 1, 2003; 144(1): 274 - 280. [Abstract] [Full Text] [PDF]
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M. C. Kuehl-Kovarik, W. A. Pouliot, G. L. Halterman, R. J. Handa, F. E. Dudek, and K. M. Partin Episodic Bursting Activity and Response to Excitatory Amino Acids in Acutely Dissociated Gonadotropin-Releasing Hormone Neurons Genetically Targeted with Green Fluorescent Protein J. Neurosci., March 15, 2002; 22(6): 2313 - 2322. [Abstract] [Full Text] [PDF]
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 E. Terasawa and D. L. Fernandez Neurobiological Mechanisms of the Onset of Puberty in Primates Endocr. Rev., February 1, 2001; 22(1): 111 - 151. [Abstract] [Full Text]
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 M. E. Freeman, B. Kanyicska, A. Lerant, and G. Nagy Prolactin: Structure, Function, and Regulation of Secretion Physiol Rev, October 1, 2000; 80(4): 1523 - 1631. [Abstract] [Full Text] [PDF]
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 C. D. Buchanan, V. B. Mahesh, and D. W. Brann Estrogen-Astrocyte-Luteinizing Hormone-Releasing Hormone Signaling: A Rolefor Transforming Growth Factor-{beta}1 Biol Reprod, June 1, 2000; 62(6): 1710 - 1721. [Abstract] [Full Text]
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O. Eyigor and L. Jennes Kainate Receptor Subunit-Positive Gonadotropin-Releasing Hormone Neurons Express c-Fos during the Steroid-Induced Luteinizing Hormone Surge in the Female Rat Endocrinology, February 1, 2000; 141(2): 779 - 786. [Abstract] [Full Text] [PDF]
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L. E. Claypool, E. Kasuya, Y. Saitoh, F. Marzban, and E. Terasawa N-Methyl D,L-Aspartate Induces the Release of Luteinizing Hormone-Releasing Hormone in the Prepubertal and Pubertal Female Rhesus Monkey as Measured by in Vivo Push-Pull Perfusion in the Stalk-Median Eminence Endocrinology, January 1, 2000; 141(1): 219 - 228. [Abstract] [Full Text] [PDF]
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B. Shi, G. Bhat, V. B. Mahesh, M. Brotto, T. M. Nosek, and D. W. Brann Bradykinin Receptor Localization and Cell Signaling Pathways Used by Bradykinin in the Regulation of Gonadotropin-Releasing Hormone Secretion Endocrinology, October 1, 1999; 140(10): 4669 - 4676. [Abstract] [Full Text]
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 P. J Emmerson and R. J Miller Pre- and postsynaptic actions of opioid and orphan opioid agonists in the rat arcuate nucleus and ventromedial hypothalamus in vitro J. Physiol., June 1, 1999; 517(2): 431 - 445. [Abstract] [Full Text] [PDF]
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L. C. González, L. Pinilla, M. Tena-Sempere, and E. Aguilar Regulation of Growth Hormone Secretion by {alpha}-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Receptors in Infantile, Prepubertal, and Adult Male Rats Endocrinology, March 1, 1999; 140(3): 1279 - 1284. [Abstract] [Full Text]
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E. Kasuya, C. L. Nyberg, K. Mogi, and E. Terasawa A Role of {gamma}-Amino Butyric Acid (GABA) and Glutamate in Control of Puberty in Female Rhesus Monkeys: Effect of an Antisense Oligodeoxynucleotide for GAD67 Messenger Ribonucleic Acid and MK801 on Luteinizing Hormone-Releasing Hormone Release Endocrinology, February 1, 1999; 140(2): 705 - 712. [Abstract] [Full Text]
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G. K. Bhat, V. B. Mahesh, L. Ping, L. Chorich, V. T. Wiedmeier, and D. W. Brann Opioid-Glutamate-Nitric Oxide Connection in the Regulation of Luteinizing Hormone Secretion in the Rat Endocrinology, March 1, 1998; 139(3): 955 - 960. [Abstract] [Full Text] [PDF]
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