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Departments of Obstetrics and Gynecology/Physiology (J.F.M., A.S., H.Y., V.S., C.A.W.), St. George’s Hospital Medical School, London SW17 0RE, United Kingdom; Department of Metabolic Medicine (C.J.S., A.R.K., S.R.B.), Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom; and Center for Neuroscience Research (M.-L.G., S.E.G., T.K., C.W.C.), King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom
Address all correspondence and requests for reprints to: Prof. C. A. Wilson, Department of Obstetrics and Gynecology/Physiology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom. E-mail: cwilson{at}sghms.ac.uk.
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Introduction |
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There are two orexin peptides derived from prepro-orexin, designated as A and B and sharing 46% homology (7). Orexin A appears to be the more potent on physiological actions tested in vivo (6, 7, 8). This may be due to its greater resistance to degradation because orexin A possesses two interchain disulfide bonds, the B peptide being linear (7). Their effects are mediated by receptors, orexin 1 and 2. Orexin A has a similar affinity for both receptors, whereas orexin B has a higher affinity for the orexin 2 receptor subtype (7, 9). The receptors are widely distributed in the brain; within the hypothalamus, expression of orexin 1 receptors is greatest in the anterior hypothalamus and ventromedial nucleus, whereas orexin 2 receptors are expressed more strongly in the paraventricular nucleus, dorsomedial nucleus, arcuate (ARC) nucleus, and lateral hypothalamus. There is moderate expression of orexin 1 and 2 receptors in the rostral, medial, and lateral preoptic area (POA; Refs. 9 and 10). Orexin-immunoreactive (IR) fibers are present in the hypothalamus at the sites of orexin receptor mRNA expression (11, 12). Orexin neurons are found in high concentrations in hypothalamic areas considered to be important in the regulation of food intake, and intracerebroventricular (icv) administration of orexin A or B stimulates food intake (7, 8). However, after those initial investigations, orexins have been found to be involved in a variety of other actions. The orexin peptides have marked effects on arousal and sleep (13, 14) and are involved in the regulation of body temperature, the immune system, and stress responses (15). The orexins also have neuroendocrine effects, and they stimulate ACTH release, inhibit GH release, and have a dual effect on prolactin release (13, 16, 17). The effects of orexin on LH release appear to be complex. In ovariectomized rats exhibiting pulsatile release of LH, icv administration of orexin A or B inhibits LH mean levels and pulse frequency (6, 18, 19). Orexin A also inhibits GnRH-induced LH release from dispersed pituitary cells (20). In contrast, orexin A stimulates GnRH from rat hypothalamic explants of intact males and females in vitro (20). Furthermore, in ovarian steroid-treated ovariectomized or intact female rats, orexin A and B have been reported to stimulate LH release (6, 17); this may be of physiological importance because icv administration of an orexin antiserum inhibits the preovulatory LH surge in intact rats (17). A recent report indicates that orexin-IR fibers form close associations with GnRH neurons in sheep (21).
In this report, the effect of orexin A on GnRH release was investigated in vitro using hypothalamic explants. Its effect on LH release was studied in vivo at three possible sites of action within the hypothalamus. These sites were the medial POA (mPOA), in the region of the sexually dimorphic nucleus (22) and have been implicated in the control of LH release (23); the rostral POA (rPOA) at the level of the organum vasculosum of the lamina terminalis (OVLT), where the GnRH neurons are concentrated; and the ARC/median eminence (ME), the region of GnRH nerve terminals (24). These are all areas innervated by orexin originating from the ZI and/or lateral hypothalamus (11, 12). We have confined our studies to orexin A because it has been found to be the more potent of the two peptides in various contexts (6, 7, 8).
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Materials and Methods |
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In vivo experiments
Hormone treatments.
Three hormonal models were used in these experiments: 1) ovariectomized rats untreated with estrogen and thus, in the absence of negative feedback, possessing high plasma levels of LH. This model is suitable for testing any putative inhibitory effects of orexin (n = 69 animals investigated in this model); 2) ovariectomized rats treated with estradiol benzoate (EB) at 10 µg/rat in 0.1 ml corn oil sc on d 1 at noon (50–51 h before the central injection). The low levels of LH in this model, due to the negative feedback of estradiol, make it suitable for testing putative stimulatory effects on LH release (n = 136); and 3) ovariectomized rats treated with 10 µg EB followed by 0.5 mg progesterone in 0.1 ml corn oil sc on d 3 at noon (i.e. 48 h after EB); this treatment results in an LH surge approximately 4 h (range, 3.5–5 h) after the progesterone injection, an event that is potentially subject to blockade or attenuation (n = 140). The possible inhibitory effect of orexin in this model was noted by injecting orexin centrally approximately 2 h after the progesterone and thus approximately 2 h before the expected time of the LH surge. These experimental models are well established and have been previously used to investigate the role of melanin-concentrating hormone, dopamine, and 5-hydroxytryptamine (5HT) on LH release (4, 5, 25).
Peptide and antagonist treatments.
Orexin A was dissolved in saline and bilaterally injected intrahypothalamically at doses of 0.04, 0.2, or 1 µg/0.5 µl per side. Control rats received 0.5 µl saline bilaterally. The injections were administered stereotaxically under Saffan anesthesia (alphaxalone, 0.9%; alphadone acetate, 0.3%; National Veterinary Supplies, Stoke-on-Trent, UK) at 3 ml/kg ip. Although this anesthetic attenuates the steroid-induced LH surge, it permits investigation of the effect of agents on the occurrence of this surge (24). The injection sites were established according to stereotaxic coordinates (26). Thus, the rPOA at the level of the OVLT was 0.4 mm rostral to bregma, 0.5 mm lateral to midline, and 8.2 mm ventral to the surface of the cortex; the mPOA was 0.8 mm caudal to bregma, 0.5 mm lateral to midline, and 8.0 mm ventral to the surface of the cortex; the ARC/ME was 2.3 mm caudal to bregma, 0.4 mm lateral to midline, and 9.5 mm ventral to the surface of the cortex.
SB 334867-A, a selective orexin 1 receptor antagonist (pKB 7.27; Refs. 27 and 28), was dissolved in 10% encapsin plus 2% dimethoxysulfoxide (DMSO) and given intrahypothalamically at 0.5 µg or 2 µg/side either alone or together with the orexin A in a total volume of 0.5 µl. Controls received 0.5 µl of the encapsin/DMSO vehicle. SB 334867-A was also given by ip injection at 10 mg/kg/ml; this injection was delivered 20–30 min before central administration of orexin A or saline (28).
Procedures.
All rats were anesthetized with Saffan (see above) and placed on a heated electric pad to maintain body temperature (25). A blood sample (0.2 ml) was taken from the tail vein just before the intrahypothalamic treatment, 50–51 h after the EB priming; this means that in groups treated with EB plus progesterone, the intrahypothalamic injection was given 2–3 h after the progesterone and thus approximately 1–2 h before the expected LH surge. The rats were kept on the heated pad thereafter, and blood samples were taken at 10-min intervals for 1 h after the intrahypothalamic treatment in the EB-primed rats and at 30-min intervals for 3 h post injection in the EB plus progesterone-treated groups, to cover the expected period of the LH surge. Anesthesia was maintained throughout the period. There did not appear to be any interaction between the anesthetic effect of Saffan and the arousal effect of orexin, because the depth and duration of anesthesia was similar in the saline- and orexin-treated animals.
At the end of the sampling period, the rats were decapitated, and the brains were removed and fixed in 10% formal saline for histological assessment of the injection sites. The blood samples were centrifuged at 2000 x g, and the plasma was stored at −20 C pending RIA.
In an independent experiment, ovariectomized rats not treated with ovarian steroids were fitted with a permanent bilateral cannula in the mPOA (29). After a recovery period of 1 wk, the animals were anesthetized with ketamine at a dose (100 mg/kg ip) that does not affect the pulsatile release of LH (30). Blood samples were taken from the tail vein (as described above) at 5-min intervals. Sampling took place for 110 min before and 110 min after administration of either saline (0.5 µl/side) or orexin A (1.0 µg/side) into the mPOA.
Histological examination of the brains.
Serial sections of 60 µm were cut from the fixed brains over the area of the needle tract using a freezing sledge microtome and stained with thionine blue. The lowest point of the tract was taken as the site of injection. Results were not included when the bilateral injections were outside the desired area (number of bilateral injections made inside the rPOA, 101; outside the rPOA, 36; inside the mPOA, 125; outside the mPOA, 17; inside the ARC, 27; outside the ARC, 9).
RIA for LH.
Plasma samples of 20 µl were assayed in duplicate using reagents kindly supplied by the National Hormone and Pituitary Program (Baltimore, MD). The standard was NIDDK-LH-RP3, and the antibody was NIDDK-anti-rLH-S10. The inter- and intraassay coefficients of variation were 8.0 and 9.0%, respectively, and the sensitivity was 25 pg/tube (2.5 µg/ml). Detection of LH pulses was established by use of the algorithm ULTRA (31).
In vitro experiments
Effect of orexin on GnRH release from hypothalamic explants.
A static incubation system was used as previously described (20). Groups of ovariectomized rats, either left untreated or primed 48 h before with 10 µg EB, were decapitated, and their brains were removed. The brain was mounted with the ventral surface uppermost and placed in a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A 1.7-mm slice was taken from the base of the brain, and the hypothalamus block was dissected and incubated in individual tubes (Sarstedt, Leicester, UK) containing 1 ml of artificial cerebrospinal fluid (aCSF; 20 mM NaHCO3, 126 mM NaCl, 0.09 mM Na2HPO4, 6 mM KCl, 1.4 mM CaC12, 0.09 mM MgSO4, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 µg/ml aprotinin), equilibrated with 95% O2 and 5% CO2. These hypothalamic explants contain multiple hypothalamic nuclei, including the ARC, dorsomedial, periventricular, and paraventricular nuclei, lateral hypothalamic area, and the whole POA. The tubes were placed in a water bath maintained at 37 C. After an initial 2-h equilibration period, the hypothalami were incubated for 45 min in 500 µl aCSF (basal period) before being challenged for 45 min with orexin A (1, 10, 100, or 1000 nM) in 500 µl aCSF. Finally, the viability of the tissue was verified by a 45-min exposure to 56 mM KCl. Isotonicity was maintained by substituting K+ for Na+. At the end of each period, the aCSF was removed and frozen at −20 C until measurement of GnRH by RIA as previously described (32). In the first experiment (Fig. 1A
), 14 hypothalamic explants were harvested from ovariectomized animals that had not been treated with EB; after the preincubation period, each of the 14 explants was exposed to a 45-min basal period, a 45-min period of orexin A 1000 nM, and finally a 45-min potassium period (56 mM). In the second part of this experiment (Fig. 1B), 14 hypothalamic explants were harvested from ovariectomized animals that had been primed with EB and then treated as described above. Both parts of experiment 1 were performed on the same day. The second experiment was designed to investigate the dose response of the effect of orexin on GnRH release from hypothalamic explants. Hypothalamic explants (n = 64) were harvested from ovariectomized animals that had been primed with EB. After the preincubation period, all of the 64 explants were exposed to a 45-min basal period. In the second 45-min incubation period, 14 explants were exposed to orexin A 1 nM, 10 explants were exposed to orexin A 10 nM, 17 explants were exposed to orexin A 100 nM, and 23 explants were exposed to orexin A 1000 nM. In a final incubation period, all 64 were exposed to 56 mM potassium. All 64 explants received only one dose of orexin A. Experiment 2 was spread over 3 d due to the number of explants investigated. All of the orexin doses were studied on each of the days. Explants were considered viable if the release of GnRH in the potassium period was greater than that in the basal period. The n values quoted above represent the explants that were considered viable. In both experiments, some explants were excluded as nonviable (i.e. the potassium GnRH release was less than the basal period); this was approximately 10% in both experiments. The stated P value represents the difference between the basal incubation period and the orexin A period (paired t test).
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Immunohistochemistry
Double immunohistochemistry for orexin A and GnRH was performed on free-floating sections using a polyclonal rabbit antiserum (OXA11-S, 1:2000; Autogen Bioclear, Wiltshire, UK) and a GnRH mouse monoclonal antibody (A4E1, 1:3000; Dr. D. W. Silversides, University of Saskatchewan, Saskatoon, Canada). Twelve days after ovariectomy, adult female Wistar rats (n = 4) were perfused via the transcardiac route with 4% paraformaldehyde under pentobarbital anesthesia. Vibratome sections (30 µm) were pretreated at room temperature (RT) with 0.1% Triton X-100 in PBS (20 min), 0.5% hydrogen peroxide in PBS (10 min), and 2% normal donkey serum (25 min); after thorough washing, they were incubated with the primary antibodies (40 h at 4 C). The sections were then placed in biotinylated goat antirabbit Ig (Vector Laboratories, Peterborough, UK; 1:1000, 60 min at RT), followed by the Vectastain streptavidin-biotin-peroxidase complex (Vector Laboratories; 1:3000, 60 min at RT); the signal for orexin A was then amplified using the biotinylated-tyramide technique (TSA Biotin System, NEN Life Science Products, Boston, MA; 1:1000, 30 min at RT). Sections were then processed for either brightfield or fluorescence microscopy.
Double-label brightfield immunoreactivity was revealed using nickel-enhanced diaminobenzidine for orexin A and diaminobenzidine alone for GnRH. Double-label immunofluorescence was obtained with fluorescein isothiocyanate-Avidin (Vector Laboratories, 1:400) for orexin A and with Rhodamine Red X conjugated donkey antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:400) for GnRH (overnight incubation at 4 C).
The relationship between orexin A-IR fibers and GnRH perikarya was evaluated in hypothalamic sections extending from the diagonal band of Broca to the region of the suprachiasmatic nucleus. At least nine sections were analyzed for each animal. In brightfield microscopy, GnRH-IR cell bodies (brown) were considered to be in close proximity to an orexin A-IR fiber if black IR varicosities or fibers were observed to be adjacent to the GnRH perikarya in the same plane of focus using a x40 objective. Immunofluorescent staining of GnRH (red) and orexin A (green) was visualized by switching between red and green filters. GnRH cells in close proximity with orexin A fibers were subsequently examined by confocal microscopy (Bio-Rad MRC-600 Laser Scanning Confocal System, Bio-Rad Laboratories, Hercules, CA) using an inverted microscope (x60 oil objective).
Statistical analysis
The Student’s t test was used for comparisons between two groups, and the paired t test was used for comparisons from the same hypothalamic explants. Comparisons between more than two groups were assessed by ANOVA, followed by the multiple comparison Gabriel’s test for unequal group numbers. When sequential concentrations were measured, the area under the curve (AUC; nanograms per milliliter per 40 min) was calculated and used for comparisons. This is considered a more stringent method of comparison over a time course because it includes all of the results (33).
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). Application of a range of concentrations of orexin A (1, 10, 100, and 1000 nM) in the latter model revealed that only the highest concentration (1000 nM) had a significant effect on GnRH release (Fig. 2).
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0.25 nmol) into the rPOA at the level of the OVLT significantly raised plasma LH levels over a 30-min period, resulting in a significantly greater AUC (0–40 min) after orexin A administration compared with the AUC over the same period after saline (AUC ± SEM, saline, 215 ± 28; orexin, 314 ± 32; P < 0.01; Fig. 3A). The lower doses of 0.2 and 0.04 µg were ineffective in the rPOA. When orexin A was injected into the mPOA, it had a transient stimulatory effect on LH release that was only significant at 10 min for 1 µg orexin A and at 10 and 20 min for 0.2 µg orexin A. However, the rise seen after either dose into the mPOA was not significant when calculated as AUC over 0–40 min (AUC ± SEM, saline, 153.5 ± 23; orexin 1 µg, 216 ± 32.4; orexin 0.2 µg, 232 ± 34.8; Fig. 3B). Orexin A (1 µg) had no effect when injected into the ARC/ME area (Fig. 3C). Figure 4 shows the sites of injections of orexin A or saline in this experiment.
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are presented as the difference between the LH concentration 2 h after the progesterone (immediately before the central injection) and the highest concentration observed after the central injection (peak postinjection concentration). Thus, the surge observed when saline was injected into the rPOA, mPOA, or ARC/ME was +6.0 ± 0.9, +6.8 ± 1.2, or +10.6 ± 1.8 ng/ml ± SEM, respectively (Table 1). When 1 µg orexin A per side was injected into the rPOA, it failed to alter the surge, but when injected into the mPOA, it significantly reduced the expected rise in LH (+2.1 ± 0.4 ng/ml); a comparable effect was achieved with 0.2 µg/side (+1.8 ± 0.6 ng/ml; P < 0.001) in the mPOA. The surge was also attenuated when 1 µg orexin A per side was injected into the ARC/ME (+3.1 ± 0.9ng/ml; P < 0.001; Table 1).
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), LH concentrations were measured in samples collected at 10-min intervals from ovariectomized rats without estrogen treatment; intrahypothalamic administration of 1 µg orexin A per side had a slight stimulatory effect on LH levels in these animals when injected into the rPOA, although the AUC was not significantly altered (AUC ± SEM, saline, 305.5 ± 30.7; orexin, 248.5 ± 37.3). However, the same treatment into the mPOA significantly reduced LH concentrations over a 40-min period (AUC ± SEM, saline, 327.0 ± 57.1; orexin, 204.1 ± 20.9; P < 0.05). An additional experiment was performed on untreated ovariectomized rats bearing permanent bilateral cannulae in the mPOA. LH levels in blood samples collected at 5-min intervals revealed the pulsatile pattern of LH release (Fig. 6). Saline administration into the mPOA did not alter LH pulse frequency; in contrast, injection of orexin A at this site led to a small but significant reduction in this parameter (P < 0.05; Table 2). There were no significant changes in LH pulse amplitude.
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). This stimulatory effect of orexin A on LH release was blocked when SB 334867-A, the orexin receptor 1 antagonist, was injected at 10 mg/kg ip 20–30 min before the peptide (AUC ± SEM, saline, 215 ± 28; orexin A, 314 ± 32; orexin A plus antagonist, 188 ± 28; P < 0.05; Fig. 7). LH levels measured after SB 334867-A given before saline in the rPOA were not different from levels observed after saline alone (AUC ± SEM, saline alone, 215 ± 28; saline plus antagonist, 184 ± 20). This indicates that SB 334867-A has no inherent effect in this model.
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). Administration of SB 334867-A either peripherally (10 mg/kg ip) or intrahypothalamically (0.5 µg/side in the mPOA) reversed this inhibitory effect (Table 3). In contrast, administration of SB 334867-A alone (2 µg/side) into the rPOA significantly attenuated the expected LH surge in rats treated with EB plus progesterone (Table 4).
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), with relatively few present more caudally in the mPOA. Sites of orexin A-IR fibers included the rPOA and mPOA. Double immunohistochemistry for GnRH and orexin A, using both brightfield and fluorescence techniques, revealed fibers and varicosities IR for orexin A in close apposition to approximately 10% of GnRH neurons (Fig. 8).
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Discussion |
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A number of other neurotransmitters have dual effects on LH release that appear to be steroid dependent, all of them exhibiting a stimulatory effect in the presence of gonadal steroids and either no effect (galanin) or an inhibitory effect in the absence of these steroids (34). Prime examples are norepinephrine (NE), neuropeptide Y (NPY), neurotensin, angiotensin II, and 5HT (34, 35). In the case of the first two examples, a mechanism has been suggested; in the absence of estrogen or when its levels are low, it has been postulated that NE stimulates a GABAergic system in the mPOA that then exerts a direct inhibitory effect on GnRH neurons (36). In similar models, it is hypothesized that NPY stimulates the release of the proopiomelanocortin derivative ß-endorphin in the ARC/ME area, from which site the latter inhibits GnRH release (37). In the presence of ovarian steroids administered in a sequence that induces an LH surge, these actions of NE and NPY disappear, presumably unmasking their stimulatory effects (36, 37). Pu et al. (6) have suggested that the dual effect of the orexins is also dependent on the presence or absence of gonadal steroids. However, in our in vivo experiments both stimulatory and inhibitory effects of orexin A are noted in the presence of these steroids; furthermore, in vitro, we have shown stimulatory effects in both the absence and presence of estrogen. As suggested by Herbison (38), it may be the ongoing activational state of the GnRH neurons that dictates their response to a neurotransmitter. We therefore suggest that these steroids have an indirect influence on the response to orexin A dependent on whether they are exerting positive or negative feedback on the GnRH system.
We have found that the dual effects of orexin appear to be site-dependent, and again there are several other examples of neurotransmitters that also have dual effects according to site. For instance, the dopaminergic tubero-infundibular tract originating in the ARC with terminals in the ME exerts an inhibitory effect on LH release (39, 40), whereas the dopaminergic incertohypothalamic tract with terminals in the ZI and POA is stimulatory (3, 4, 41, 42). Similarly, the serotonergic tract originating in the median raphe and innervating the mediobasal hypothalamus is inhibitory (43), whereas the serotonergic tract from the dorsal raphe passing to the POA is stimulatory (44, 45), albeit only in the presence of steroids (35). The dual effect of orexin noted here and by others (5, 14, 19) indicates that this peptide joins the ranks of many other neurotransmitters in modulating GnRH activity bidirectionally.
In this report, the stimulatory effect of orexin A was more apparent in the rPOA than in the mPOA. A sustained elevation of LH levels was obtained with administration into the rPOA, whereas only a transient rise occurred with treatment in the mPOA. The stimulatory action of orexin may involve direct effects on GnRH cell bodies; this is supported by our observation of orexin A varicosities in close apposition to GnRH neurons in the rPOA using confocal microscopy. Double label immunohistochemistry at the electron microscopic level would be required to demonstrate synaptic associations. Nevertheless, on the basis of confocal microscopy a similar association between these two systems has been recently reported in sheep (21). It should, however, be noted that the percentage of GnRH neurons found with orexin A IR varicosities in close apposition was smaller in rats (10%) than in sheep (30%).
In contrast, orexin A exhibited an inhibitory effect on LH release after injection into the mPOA in two experimental conditions in which LH levels are relatively high, namely the ovariectomized rat without exogenous ovarian steroids and the ovariectomized rat primed with estrogen followed by progesterone and consequently generating an LH surge. In the latter model, orexin A also exerted an inhibitory effect when administered in the ARC/ME.
Others have shown an inhibitory effect of icv orexins on pulsatile release of LH in untreated ovariectomized rats (6, 18). This inhibitory effect on pulsatile release was enhanced in the presence of very low concentrations of estradiol (19). Orexin A may act to inhibit GnRH release from nerve terminals in the ME, or it may exert indirect effects in both the mPOA and ARC/ME via other systems that inhibit GnRH release. Just as described above for other neurotransmitters, candidates for mediating these inhibitory effects of orexin A might include ß-endorphin or 
-aminobutyric acid. Orexin nerve terminals have been found to impinge on these systems and alter their release (46, 47). van den Pol et al. (47) have claimed that approximately one third of hypothalamic neurons express orexin receptors and reported the presence of orexin axon terminals throughout the hypothalamus. These observations suggest that orexin may influence activity in many hypothalamic systems; for example, the orexin effect on ACTH and prolactin release appears to be mediated via NPY (48, 49) and its effect on grooming behavior via 5HT (50). ß-Endorphin may mediate the inhibitory effect of orexin A on LH release, because naloxone has been shown to antagonize this effect (51).
Previous reports have shown that the orexins can stimulate GnRH release from hypothalamic explants taken from intact males and proestrous female rats (20). We have now shown that orexin A is effective in stimulating GnRH release from hypothalamic explants harvested from ovariectomized rats with or without estrogen priming. This appears to be inconsistent with the inhibitory effects we find in vivo in the absence of estrogen. The basis for this apparent discrepancy remains to be determined; thus far, no site has been found in vivo at which orexin treatment results in increased LH release in the absence of estrogen treatment.
Orexin 1 receptors are expressed in the three areas investigated in the present study, namely, the rPOA, mPOA, and ARC (9, 10). Both the stimulatory and inhibitory effects of exogenous orexin A appear to be mediated, at least in part, by this receptor subtype, because the selective orexin 1 antagonist, SB 334867-A (26, 27), prevents the orexin actions in the relevant models. Administration of this antagonist into the rPOA of ovariectomized rats primed with estrogen plus progesterone attenuated the expected LH surge. This result suggests that the antagonist may block the stimulatory effect of endogenous orexin and is consistent with the discovery that central administration of orexin antiserum inhibits the LH surge in proestrous rats (17).
In summary, orexin A has a stimulatory effect on GnRH neurons in vitro and increases LH release in vivo when administered into the rPOA. Double label immunohistochemistry for GnRH and orexin A, analyzed by confocal microscopy, reveals close appositions between orexin A IR varicosities and a small proportion of GnRH cell bodies. This suggests that the stimulatory effect on LH release of orexin A within the rPOA may involve direct actions on GnRH neurons. The stimulation of LH release by orexin A at this site can be blocked by the orexin 1 receptor antagonist, SB 334867-A; this also blocks the inhibitory effects of orexin A observed in the mPOA and ARC/ME. These inhibitory effects may be mediated by neurotransmitter systems located in or near the mPOA and/or ARC. The present findings extend our understanding of the role of orexin in the regulation of LH release.
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Acknowledgments |
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Footnotes |
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Abbreviations: aCSF, Artificial cerebrospinal fluid; ARC, arcuate; AUC, area under the curve; DMSO, dimethoxysulfoxide; EB, estradiol benzoate; 5HT, 5-hydroxytryptamine; icv, intracerebroventricular; IR, immunoreactive; ME, median eminence; mPOA, medial POA; NE, norepinephrine; NPY, neuropeptide Y; OVLT, organum vasculosum of the lamina terminalis; POA, preoptic area; rPOA, rostral POA; RT, room temperature; ZI, zona incerta.
Received November 14, 2002.
Accepted for publication March 25, 2003.
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References |
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-aminobutyric acid on LH release and dopamine expression and activity in the zona incerta in rats. J Reprod Fertil 117:189–197
-MSH and MCH after central administration in the female rat. Peptides 17:171–177[CrossRef][Medline]
-aminobutyric acid neurones demonstration by microdialysis. Neuroendocrinology 52:161–168[Medline]
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, considered inside the area; 
, considered outside the area. Other outside injections (n = 14) were seen more caudally at the level of the appearance of the anterior commissure. LPO, Lateral preoptic area; MPO, medial preoptic area; MEPO, median preoptic nucleus; AVP, anteroventral preoptic nucleus; AVPV, anteroventral periventricular nucleus; OV, vascular organ of the lamina terminalis; och, optic chiasm; NDB, nucleus of the diagonal band; V3, third ventricle. B, Sites of injections into the mPOA: 
