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Division of Paediatric Nephrology, University Children's Hospital, University of Heidelberg, Heidelberg, Germany.
Correspondence to Dr. Franz Schaefer, University Children's Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany. Phone: +49-6221-568377; Fax: +49-6221-564203; E-mail: franz_schaefer{at}med.uni-heidelberg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid [GABA],
taurine) amino acid transmitter outflow in the MPOA was measured by
microdialysis via stereotactically implanted cannulae in the awake, freely
moving rats. In addition to basal extracellular concentrations, the
neurosecretory capacity was assessed by the addition of 100 mM KCl to the
dialysis fluid. The mechanisms of neurosecretion were evaluated further by
inhibition of vesicular release with the use of Ca2+-free,
Mg2+-enriched dialysis fluid and by local perfusion with inhibitors
of voltage-dependent synaptic release (1 µM tetrodotoxin) and of GABA
reuptake (0.5 mM nipecotic acid). In the uremic rats, basal outflow of GABA,
glutamate and aspartate, and K+-stimulated aspartate outflow were
increased. K+-stimulated GABA and glutamate release was less
sensitive to Ca2+ depletion in the uremic than in the control rats.
The elevated basal GABA and essential amino acid outflow in the uremic rats
was due to a voltage- and Ca2+-independent mechanism. GABA reuptake
was inhibited proportionately by nipecotic acid in uremic and pair-fed control
rats. Testosterone supplementation had no independent effects on
neurotransmitter outflow. In summary, the amino acid neurotransmitter milieu
is altered in the MPOA of uremic rats by a nonsynaptic, nonvesicular
mechanism. These abnormalities may contribute to the impaired function of the
GnRH pulse generator. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid), noradrenergic, NOergic
(nitric oxide), and opioidergic inputs
(11). Moreover, the activity
of the GnRH pulse generator is influenced by the sex steroid milieu
(12). In this work, we tested
the hypothesis that the reduced activity of the GnRH pulse generator in uremia
may be caused by an imbalanced local amino acid neurotransmitter milieu. We
applied the intracerebral microdialysis technique
(13) to evaluate in
vivo the extracellular concentrations of the excitatory amino acids (EAA)
glutamate and aspartate and of the mainly inhibitory GABA and taurine in the
MPOA of unanesthetized, freely moving, experimentally uremic and controls
rats. The secretory capacity (neuronal versus glial) and origin of
these amino acids was assessed by a series of experimental protocols. To rule
out secondary effects of uremic anorexia and gonadal dysfunction, we conducted
the studies not only in uremic and ad libitum-fed rats but also in
pair-fed controls and generally in castrated male rats with or without
testosterone substitution. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abdominal Surgery
All surgical interventions were performed under anesthesia, induced by
intraperitoneal administration of chloral hydrate (400 mg/kg). Experimental
uremia was induced by removal of the upper and the lower pole of the left
kidney, followed by complete removal of the right kidney 7 d later. In the
control rats, sham procedures involving kidney decapsulation were performed.
Simultaneously with the initial partial nephrectomy/sham surgery, all rats
were bilaterally orchidectomized. A tablet (diameter, 0.5 cm), containing
either 15 mg of testosterone (Innovative Research of America, Toledo, OH) or
placebo, was inserted subcutaneously through a tunnel extending to
approximately 2 cm lateral of the nephrectomy incision site. Gradual
resorption of the tablets guaranteed stable plasma testosterone concentrations
in the low normal range of adult male rats for approximately 3 wk.
Microdialysis Experiments
Four to 6 d after the second abdominal surgical intervention, a guide
cannula was implanted immediately dorsal to the right hypothalamic MPOA in
each rat. After the head was placed in a stereotaxic frame (Kopf Instruments,
Thujunga, CA), the skull was exposed and the midline between the bregma and
lambda points was positioned in a horizontal plane at a precision of 
0.1
mm. A small hole was drilled 2.0 mm cranial to the bregma and 0.5 mm lateral
to the midline, through which a guide cannula (CMA/12 Guide Cannula,
CMA/Microdialysis AB, Stockholm, Sweden) with a dummy in place was advanced
6.5 mm below the dura (14).
Two stainless steel screws were anchored into the skull, to which the guide
cannula was fixed by dental acrylic cement.
Four to 6 d after insertion of the intracerebral guide cannula, i.e., 19 d after orchidectomy and 14 d after completion of the 5/6 nephrectomy (or second sham surgery), the intracerebral microdialysis studies were performed. To this end, the rats were connected to the collection assembly under light ether anesthesia. The inlet tubing of the probe was connected to a 1-ml Hamilton gastight syringe (Ziemer, Mannheim, Germany) in a perfusion pump (Precidor; INFORS AG, Bottmingen, Switzerland) and was perfused continuously with an artificial cerebrospinal fluid containing 130 mM NaCl, 3.8 mM KCl, 1.15 mM CaCl2, 1.2 mM KH2PO4, 0.75 mM MgSO4, and 1.6 mM Na2HPO4, buffered to a pH of 7.4, at a flow rate of 2 µl/min. The dummy was removed from the guide cannula, and the microdialysis probe was inserted with the tip extending 2.5 mm into the MPOA. Concentric double-lumen microdialysis probes with polycarbonate hollow-fiber dialysis membranes (length, 2 mm; outer diameter, 500 µm; cut-off, 20 kD) were used in all experiments (CMA/12, CMA/Microdialysis AB). The total dead space of the outlet part of the probe and the outlet tubing was 6.5 µl. After an equilibration period of at least 2 h, samples were collected in 10-min fractions.
The four experimental protocols performed are illustrated in Figure 1. Five to seven experiments with confirmed correct probe location per treatment group (uremic, pair-fed, ad libitum-fed) and testosterone status (replacement, placebo) were performed with each protocol. The first four fractions obtained in each protocol were collected to determine baseline neurotransmitter concentrations. In protocols A through C, the perfusion fluid was switched after 40 min for three consecutive 10-min periods to a medium containing 100 mM KCl (with NaCl content reduced to 24 mM to maintain constant osmolarity), followed by a second 30-min baseline period. Protocol A was concluded by a second 30-min depolarizing period with high K+ content. In protocol B, an additional 30-min baseline period and a second 30-min high K+ period followed during perfusion with a medium in which CaCl2 was replaced by 12.5 mM MgCl2. Mg2+ acts as a Ca2+ antagonist by blocking Ca2+ channels and has been established to enhance the effects of Ca2+ omission from the dialysis fluid (15). In protocol C, a second baseline and high K+ perfusion sequence were performed in the presence of the sodium channel blocker 10-6 M tetrodotoxin (TTX; Sigma, St. Louis, MO) to the dialysate. In protocol D, nipecotic acid (Sigma), a GABA reuptake inhibitor, was added to the dialysate after the initial baseline period in a final concentration of 0.5 mM for 30 min, followed by a 30-min washout period (16). A gastight liquid switch (CMA/110, CMA/Microdialysis AB) was used to change between the different perfusion media.
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The samples were snap-frozen immediately after collection and stored at -70°C. Immediately after the end of the study, the rats were killed by aortic puncture. To verify the location of the probe, we injected eosin through a used, membraneless probe. The brains were conserved in 10% formaldehyde and embedded in paraffin. The probe track was located by inspection of sequential 50-µm-thick transversal brain slices counterstained with hematoxylin. Correct probe location was confirmed in 85% of the experiments. Experiments with incorrect probe location were excluded from further analysis.
The microdialysis probes were used up to three times. After each use, the probes were flushed with water and stored for 12 h in a 0.5-mM HCl solution to remove any matrix layers from the membranes. The mean in vitro recovery rates, determined for each probe before each experiment by perfusion at 2 µl/min in a fluid containing a known amount of the amino acids, were 30% for glutamate, 18% for aspartate, 20% for taurine, and 16% for GABA. The recovery rates decreased by an average of 3% per use. Because the relationship between in vitro and in vivo solute recovery is complex, the measured dialysate concentrations were not corrected for in vitro recovery.
Assays
The hypothalamic perfusates were analyzed for glutamate, aspartate,
taurine, and GABA content with the use of an isocratic HPLC system with
fluorometric detection after precolumn derivatization
(17). The system was composed
of a degasser (CMA/252, CMA/Microdialysis AB), an isocratic pump (CMA/250,
CMA/Microdialysis AB), a refrigerated autoinjector (CMA/200, CMA/Microdialysis
AB), a C18 column (100*3.1 mm Zorbax; Phenomenex, Aschaffenburg,
Germany; 3 µm), and a fluorescence detector (CMA/280, CMA/Microdialysis
AB). The mobile phase consisted of 0.1 mM acetate (pH 5.4) with 17.5%
acetonitrile and 2.5% 1-propanol. The 20-µl microdialysate sample was
derivatized with 2 µl of ophthaldehyde/mercaptoethanol for 120 s, separated
in the column at a flow rate of 0.5 ml/min, and analyzed in the fluorescence
detector at 340/440 nm excitation/emission wavelengths. The lower limit of
detection was 100 fmol. The dose-signal relationship was linear between 100
fmol and 200 pmol for each amino acid. The coefficients of variation at
concentrations of 0.5 pmol/20 µl and 50 pmol/20 µl were 8.5% and 4.9%
for glutamate, 7% and 12% for aspartate, 7.5% and 4.1% for taurine, and 3% and
11% for GABA, respectively. Serum testosterone was measured by RIA after
ethanol extraction. The detection limit of the assay was 10 mg/dl.
Statistical Analyses
Time-averaged mean values of amino acid outflow were calculated both during
the baseline periods and during the pharmacologic interventions in each rat.
These intraindividual mean values were used for subsequent statistical
comparisons. Data are given as means ± SEM.
After we evaluated the mode of distribution using the Shapiro-Wilk statistic, variables that did not exhibit a Gaussian distribution were log-transformed. Subsequently, data were analyzed with the use of parametric techniques, i.e., two-way ANOVA with treatment (ad libitum-fed, pair-fed, uremic) and gonadal state (testosterone-replaced versus nonreplaced) as covariates, followed by Duncan's test for multiple comparisons, for evaluation of between-group differences, and two-tailed paired t tests for assessment of longitudinal (within-group) differences. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Basal Amino Acid Outflow
The baseline periods of all experiments (protocols A through D) were
combined to evaluate the effects of uremia, nutrition, and testosterone
substitution on basal outflow of neuroactive amino acids
(Table 2). Testosterone
substitution did not affect basal amino acid concentrations in any of the
treatment groups. When the groups with and without testosterone
supplementation were considered together, basal glutamate was significantly
higher in the uremic rats (43.9 ± 6.9 pmol/20 µl) than in the
pair-fed controls (21.7 ± 3.5; P < 0.05), and aspartate was
increased in the uremic (7.9 ± 0.8), compared with both the pair-fed
(4.9 ± 0.4; P < 0.01) and the ad libitum-fed
control groups (5.5 ± 0.7; P < 0.05). Taurine levels were
similar in all groups. Basal GABA outflow was significantly elevated in the
uremic (1.93 ± 0.28 pmol/20 µl) compared with the pair-fed (1.18
± 0.12; P < 0.05) and the ad libitum-fed controls
(1.05 ± 0.13; P < 0.01).
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K+-Stimulated Amino Acid Outflow
To test the integrity and overall neurosecretory capacity of the neuronal
tissue, the maximal neurotransmitter release was assessed by membrane
depolarization. The depolarizing stimulus of 100 mM KCl in the dialysate led
to immediate increases in the outflow concentrations of each of the four amino
acids studied. The analysis of the first baseline and KCl periods in the rats
of protocols A, B, and C showed maximal increases to 365 ± 29% of
baseline for glutamate, 504 ± 80% for aspartate, 437 ± 27% for
taurine, and 7126 ± 800% for GABA, respectively
(Figure 2). The
K+-stimulated time-averaged outflow of aspartate was increased in
uremic (19.8 ± 2.8 pmol/20 µl), compared with ad
libitum-fed (12.7 ± 1.38; P < 0.01) and pair-fed rats
(14.1 ± 1.8; P < 0.05). No significant treatment-related
differences were observed for glutamate, taurine, and GABA. Testosterone
supplementation had no effect on stimulated time-averaged amino acid
neurotransmitter release in any of the treatment groups. In protocol A, a
second period of K+ stimulation was performed after another 40-min
baseline period. During the second baseline period, the dialysate
concentrations of all amino acids returned to the previous baseline levels.
The subsequent repeated K+ stimulation generally raised dialysate
amino acid levels to a similar degree as the first stimulation. The only
treatment-related difference in restimulation was a higher second aspartate
peak (time-averaged mean, +24%) in the uremic rats, contrasting significantly
(P < 0.05) with a slightly diminished restimulation (-20%) in the
pair-fed rats.
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Effects of Ca2+ Depletion on Amino Acid Outflow
The exocytotic release of neurotransmitter vesicles into the synaptic cleft
is Ca2+ dependent. Moreover, Ca2+ regulates presynaptic
neurotransmitter reuptake via specific transporter proteins. Switching to a
Ca2+-free, Mg2+-enriched dialysis fluid increased
glutamate (+44 ± 10%; P < 0.0005) and decreased taurine
levels significantly (-49 ± 2.7%; P < 0.0001) but did not
affect the basal dialysate concentrations of aspartate and GABA
(Figure 3A). The change in
basal outflow concentrations of the individual amino acids did not differ
between the treatment groups. During K+ stimulation, amino acid
concentrations were reduced by Ca2+-free dialysis fluid, compared
with the preceding K+ stimulation with normal Ca2+
content, by 32 ± 5% for aspartate (P < 0.0001), 17 ±
7% for glutamate (P < 0.05), 27 ± 3% for taurine
(P < 0.0001), and 56 ± 4% for GABA (P < 0.0001)
(Figure 3B). The suppression of
K+-stimulated glutamate release was evident in the ad
libitum-fed (-36 ± 9%) and the pair-fed control groups (-13.5
± 11%) but not in the uremic rats (-2.5 ± 11%; P <
0.05 versus ad libitum-fed controls). Similarly, K+
-stimulated GABA effluent concentrations were diminished by
Ca2+-free dialysis fluid more markedly in the ad
libitum-fed (-62 ± 5%) and pair-fed controls (-66 ± 10%)
than in the uremic rats (-42 ± 5%; P < 0.05 versus ad
libitum-fed and pair-fed controls). Neither the basal nor the
K+-stimulated amino acid concentrations during Ca2+
depletion were affected significantly by testosterone substitution.
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Effects of TTX on Amino Acid Outflow
TTX inhibits the neuron-specific release of neurotransmitters via
voltage-dependent Na+ channels. In the rats that were treated
according to protocol C, addition of 1 µM TTX did not change basal outflow
of aspartate, taurine, and GABA. Basal glutamate concentrations were increased
significantly (+48 ± 11%; P < 0.001), without any
differences between the treatment groups
(Figure 4A).
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Stimulation of amino acid release by K+ during TTX perfusion yielded similar time-averaged peak concentrations as the preceding stimulation without TTX for glutamate, aspartate, and taurine, whereas K+-stimulated GABA levels were reduced by 30 ± 12% (P = 0.01) without a difference between the treatment groups (Figure 4B). Testosterone replacement had no effect on either basal or K+-stimulated amino acid concentrations.
Effect of Local GABA Uptake Inhibition on GABA Outflow
The addition of the GABA uptake inhibitor, nipecotic acid, to the dialysis
fluid resulted in a severalfold increase in GABA outflow, which was rapidly
reversible after discontinuation of the drug
(Figure 5). Whereas higher
absolute GABA concentrations were recovered in the uremic rats (6.33 ±
1.45 pmol/20 µl) compared with the ad libitum-fed (3.93 ±
0.5; P < 0.05) and pair-fed controls (3.73 ± 0.62;
P = 0.07), the percentage increase relative to baseline was
significantly smaller in the uremic (357 ± 47%; P < 0.05)
and pair-fed groups (265 ± 34%; P < 0.01) than in the
ad libitum—fed control group (601 ± 90%). The effect of
nipecotate was independent of testosterone substitution. The concentrations of
the glutamate, aspartate, and taurine were not altered by nipecotate.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The studies were conducted to evaluate the possibility of an altered balance of inhibitory and excitatory neurotransmission in favor of neuroinhibitory activity in the hypothalamic MPOA in chronic renal failure. GABA is considered the major inhibitory neurotransmitter in this area of the hypothalamus (18). The maturation of the gonadotropic axis in puberty, which is driven by increasing activity and functional synchronization of the GnRH neurons in the MPOA, is associated with a reduction of the local GABA tone (19), and local GABA administration directly inhibits pulsatile GnRH release (20). In agreement with the only other microdialysis study of GABA outflow in rat MPOA (21), we observed low basal extracellular levels of GABA, an immediate 50-fold increase by the depolarizing stimulus of 100 mM K+, and a rapid return to baseline concentrations after discontinuation of high K+. Consistent with previous investigations in the MPOA (18) and other brain stem regions (22,23), basal GABA outflow was unaffected by Ca2+ depletion and Na+ channel inhibition but increased eightfold when reuptake was blocked pharmacologically. Hence, basal extracellular GABA concentrations in the MPOA seem to be determined by nonvesicular, voltage-independent release from neurons and/or glia and by the efficacy of the GABA uptake carrier system. Neurosecretion from a nonvesicular, e.g., cytoplasmic, pool via reversal of the sodium-GABA membrane transport carrier has been demonstrated (24,25). In contrast to basal outflow, K+-stimulated GABA levels were sensitive to withdrawal of Ca2+ and addition of TTX, which suppressed maximal release by 65 and 30%, respectively. These findings confirm previous evidence that K+-releasable GABA in the MPOA, in major part, is due to neurogenic, Ca2+-dependent exocytosis of vesicles into synaptic clefts (21). The Ca-sensitive but TTX-insensitive portion of stimulated GABA release may reflect voltage-independent vesicular secretion from perikarya and dendrites (26), and the residual Ca2+ - and TTX-insensitive fraction is probably of glial origin, as demonstrated in several other brain areas (15,25).
In keeping with our hypothesis, we observed a consistent increase in basal, albeit not K+-stimulated, GABA outflow in the uremic rats. When GABA reuptake was blocked, GABA concentrations remained elevated in the uremic rats, but the relative increase was similar in uremic and pair-fed control rats. Although the effect of TTX on depolarization-induced GABA secretion did not differ between the treatment groups, stimulated GABA release was less affected by Ca2+ depletion in the uremic rats. The elevation of the neuroinhibitory GABA tone was specific inasmuch as neither basal nor K+-stimulated dialysate concentrations of the other principal inhibitory neurotransmitter, taurine, differed between the treatment groups. K+ stimulation elicited a four- to fivefold increase, and Ca2+ depletion depressed basal and K+-stimulated taurine outflow to the same extent in all treatment groups. Both basal and stimulated taurine release were Ca2+ dependent but not affected by TTX, indicating nonsynaptic, neurovesicular release of this amino acid in the MPOA.
The EAA are regarded as major stimulatory neurotransmitters that act upon the GnRH pulse generator. Their effects are exerted via activation of NO synthesis in the GnRH neurons and, perhaps more important, by indirect signaling through local NOergic neurons and noradrenergic axon endings (27,28,29). In contrast to the wealth of literature describing the neuroendocrine effects of local hypothalamic or systemic EAA receptor stimulation or inhibition (28,30), little is known about the in vivo EAA turnover in the MPOA (31).
The basal outflow of glutamate was increased both by Ca2+ depletion and by TTX perfusion. This response is in agreement with observations in other brain regions. The effect of Ca2+ depletion has been explained by an inhibition of Na+/K(+)-ATPase, which is required for the uptake of amino acids via Na+-dependent carriers, and Na+ channel blockade by TTX might reduce the Na+ gradient required for this countertransport mechanism (32), resulting in a reversal of glutamate transport. The neuronal origin of EAA release in the MPOA was demonstrated by the four- to fivefold concentration increase in response to K+-induced depolarization. Approximately one third of the stimulated EAA outflow was Ca2+ sensitive, whereas TTX had no effect, suggesting that a major fraction of the releasable EAA pool may not be of synaptosomal but of dendritic origin. Similar observations have been reported for other brain regions (15,23).
Whereas basal glutamate and aspartate outflow rates were elevated almost twofold in the uremic rats compared with the control groups, only the elevated aspartate concentrations persisted during depolarization. K+-stimulated glutamate release was less sensitive to Ca2+ depletion in the uremic than in the control rats.
These abnormalities of glutamate release were qualitatively and quantitatively similar to those observed for GABA outflow. Possible explanations for the elevated basal outflow of both the EAA and GABA in the uremic rats may be given by an impairment of axonal Na+/K(+)-ATPase and by an increased intracellular Ca2+ content. Na+/K(+)-ATPase activity is diminished in chronic renal failure in various tissues, including brain synaptosomes (33,34). Moreover, intracellular Ca2+ content is increased in the nerve endings of chronically uremic rats (34), probably as a result of a stimulatory effect of hyperparathyroidism on ATP-dependent Na+/Ca2+ exchange (35,36). Both mechanisms have been shown to impair the uptake of monoaminergic neurotransmitters by uremic rat synaptosomes (34,35). These abnormalities also may affect the function of the specific neuronal GABA, glutamate and aspartate uptake carriers, and result in an increased reversed transport of these amino acid neurotransmitters in uremia. Moreover, increased intracellular Ca2+ stimulates the activity of glutamate decarboxylase, the rate-limiting step in GABA synthesis (37). Finally, an increased intracellular Ca2+ content also has been demonstrated to impair the effect of K+ depolarization on neurovesicular release (38), offering a potential explanation for the relative resistance of stimulated GABA and glutamate secretion to short-term Ca2+ depletion.
Little is known about the effects of castration on the activity of the GnRH pulse generator. Whereas the GnRH-producing neurons lack steroid receptors (39), a possible feedback disinhibition on these cells could be mediated by suppression of the steroid-receptive, neuroinhibitory GABAergic neurons surrounding the GnRH cells (40). Decreased GABA turnover was reported in microdissected MPOA of male rats 2 and 6 d after castration (41). We were unable to demonstrate any effect on GABA outflow in the MPOA 2 wk after castration. Previous in vivo studies also failed to demonstrate a marked elevation of hypothalamic GnRH secretion after gonadectomy (42), suggesting that the hypothalamic feedback circuit may be less important for the postcastrational resetting of the gonadotropic axis than the changes that occur at the pituitary level.
We showed previously that the moderate malnutrition associated with experimental uremia may, per se, suppress the GnRH secretion rate by up to 50%. The inclusion of a pair-fed control group in this study rules out alterations of the amino acid transmitter milieu as a causative mechanism for this effect, leaving the increased hypothalamic opioidergic tone induced by food restriction as the most likely cause (43). Although a general increase in endorphin secretion also has been observed in uremia (44), endorphin blockade by naloxone reverses reduced gonadotropin secretion in malnourished (43) but not in uremic rats (45,46). Thus, the observed changes in amino acid transmitter tone may compromise GnRH neuron activity in uremia, independently of the neuroendocrine alterations induced by malnutrition.
Metabolic acidosis may be another factor that independently affects hypothalamic neurotransmitters in chronic renal failure. Acidosis, per se, has been shown to cause peripheral growth hormone (GH) insensitivity but also suppresses GH release from the pituitary (47). However, it is not clear whether reduced GH release is caused by a hypothalamic or a pituitary defect. No published information is available as to potential effects of acidosis on the function of the gonadotropic hormone axis. In one study that evaluated hypothalamic neurotransmitters, even severe acidosis did not alter local monoamine concentrations (48). Although independent effects of acidosis on the central nervous amino acid neurotransmitter milieu cannot be excluded, it should be emphasized that metabolic acidosis was mild in the rats studied here, making a major effect unlikely.
In summary, we observed increased extracellular concentrations of both neuroinhibitory and neuroexcitatory amino acids in the MPOA of rats with chronic renal failure. Irrespective of the mechanism(s) underlying the elevated local GABA and EAA tones, these must be assumed to affect the balance of stimulatory and inhibitory afferents that regulate GnRH neuron activity. In view of the reduced net hypothalamic GnRH release in experimental renal failure (8,9), our findings suggest that the increased inhibitory GABA tone outweighs the concomitant EAA elevation. Of course, this conclusion is still speculative because the design of our studies did not permit direct investigation of the effects of changes in hypothalamic neurotransmitter tone on GnRH release into the pituitary. Moreover, it should be emphasized that the neuronal network that regulates pulsatile GnRH release is far more complex, involving cross talk between EAA and GABA via NOergic neurons (49), indirect effects of GABA and EAA through noradrenergic axons synapsing with NOergic and GnRH producing neurons (50), and modulatory effects of various other neurotransmitters, such as endorphins, dopamine, neuropeptide Y, and leptin.
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Acknowledgments |
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