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Endocrinology Vol. 140, No. 12 5922-5928
Copyright © 1999 by The Endocrine Society

ARTICLES

Effects of N-Methyl-D-Aspartate on Luteinizing Hormone Release and Fos-Like Immunoreactivity in the Male White-Crowned Sparrow (Zonotrichia leucophrys gambelii)1

Simone L. Meddle2, Donna L. Maney3 and John C. Wingfield

Department of Zoology, University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: Dr. Simone L. Meddle, Department of Biomedical Sciences, University of Edinburgh Medical School, Teviot Place, Edinburgh, Scotland EH8 9AG, United Kingdom. E-mail: slmeddle{at}srv4.med.ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Seasonal breeding is terminated in the White-crowned sparrow by the onset of absolute photorefractoriness, a condition in which the reproductive system is switched off indefinitely until it is dissipated by short day lengths. Absolute photorefractoriness is controlled by the central nervous system; however, the mechanisms underlying GnRH quiescence in photorefractory birds have yet to be elucidated. Using the excitatory amino acid glutamate agonist N-methyl-D-aspartate (NMDA), plasma LH levels in White-crowned sparrows were significantly elevated regardless of the reproductive or photoperiodic condition. NMDA also significantly induced Fos-like immunoreactivity (FLI) within the infundibular nucleus and median eminence, regions previously shown to express FLI after a photoperiodically driven LH rise. NMDA did not induce FLI within GnRH I neurons; instead, it significantly activated cells within the organum vasculosum of the lamina terminalis in close proximity to GnRH I perikarya.

These findings provide the first evidence that photorefractoriness is not due to depletion of GnRH stores, as LH and presumably GnRH were secreted in response to excitatory amino acid stimulation. NMDA activation of FLI in the region of the organum vasculosum of the lamina terminalis and the basal tuberal hypothalamus suggests that seasonal reproductive neuroendocrine control may be mediated via cells in the region of the GnRH I perikarya and terminals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE WHITE-CROWNED sparrow (Zonotrichia leucophrys gambelii) seasonal breeding is essentially determined by changes in day length, although supplementary cues, such as temperature, food, and mate availability, can accelerate or perturb this photoperiodic response (see Ref. 1 for review). Photostimulation triggers gonadal maturation via GnRH secretion, but it also initiates absolute photorefractoriness, an important condition that spontaneously shuts down the reproductive system and overrides the stimulatory actions of a long photoperiod. Termination of the breeding stage is critical, as the sparrows must undergo a molt before migrating out of high latitudes ahead of winter storms. The gonads will remain in a regressed condition as long as the birds experience long days, and photosensitivity only returns after exposure to short days (1, 2). This is in contrast to a relatively refractory condition expressed by some birds (e.g. Japanese quail, Coturnix japonica), which is characterized by the lack of spontaneous gonadal regression by long photoperiod exposure. The reproductive neuroendocrine system in this case will only become inactive when day length is shortened.

Two forms of GnRH exist in birds; both are potent releasers of LH and FSH in vivo. GnRH I perikarya are detected within the hypothalamus, and their fibers terminate in the external zone of the median eminence. GnRH II cells, on the other hand, are located within the mesencephalon, and they have distinctly fewer fibers projecting to the median eminence (see Ref. 3 for review). It has been established, after numerous anatomical and physiological studies, that it is GnRH I, rather than GnRH II, that directly controls pituitary function in birds (see Ref. 3 for review). The adenohypophysis of male photorefractory White-crowned sparrows contains LH ß-subunit messenger RNA (mRNA) (4) and has been shown to be sensitive to GnRH, as a single iv injection results in a surge of circulating LH (5). This suggests that reproductive shutdown during photorefractoriness must involve changes in the hypothalamic control of GnRH I secretion. Studies in the photorefractory White-crowned sparrow suggest that it is the rate of GnRH secretion as opposed to synthesis that determines the reproductive state, as hypothalamic reserves of GnRH I (this study) and its precursor, prepro-GnRH (Meddle, S. L., manuscript in preparation) are not depleted at this time.

The neuroexcitatory amino acid glutamate analog N-methyl-D-aspartate (NMDA) can induce acute elevations in circulating LH levels when administrated to rodents (6, 7, 8, 9, 10, 11, 12), primates (13, 14), estrogen-primed ovariectomized sheep (15), and the domestic chicken (Gallus domesticus) (16). Indeed, NMDA overrides the suppressive effect of short days on LH secretion in hamsters, thereby ascertaining that GnRH neurons in reproductively quiescent animals can respond to the stimulatory action of excitatory amino acids (9, 17). As pituitary sensitivity to GnRH does not change seasonally (5), NMDA does not act directly on pituitary gonadotrophs (18), and GnRH antagonists block the stimulatory action of NMDA on LH secretion (7, 14), NMDA administration provides a direct method to quantify the action of a glutamatergic stimulus on GnRH cells throughout a seasonal cycle.

The expression of the immediate early gene c-fos has been successfully employed as a means to demonstrate GnRH activation at the time of a steroid-induced LH surge (19) or at proestrus (20) in the rat. The rationale for this activation is thought to be a compensatory mechanism by which depleted GnRH reserves are replenished or a mechanism by which the LH surge is terminated (21). As GnRH neurons in the rodent and chicken brain contain copious amounts of the neuropeptide, the finding that NMDA receptor activation does not induce Fos (c-fos gene product) expression within GnRH neurons is not unexpected (8, 10, 16, 22). Instead, an increase in Fos expression is observed in various regions associated with the control of LH release, such as the preoptic region, arcurate nucleus, and locus coeruleus. NMDA receptors are widespread throughout the hypothalamus (16, 22, 23, 24), but the literature contains controversies on whether glutamate acts directly on GnRH neurons. Evidence in vivo suggests that GnRH perikarya express ionotropic glutamate receptors (23, 24, 25). However, other studies have reported no evidence of colocalization (26, 27), and recently Ebling et al.. (27) demonstrated GnRH neurons to be resistant to glutamate toxicity in vivo. In contrast, immortalized GT1 cells appear to express glutamate receptor mRNA, although this expression varies from extensive (28) to very low levels (29). Nonetheless, consistent with the theory that GnRH cells contain no or extremely low concentrations of glutamate receptors, GT1–7 cells appeared to show no toxicity to glutamate (29). It does, of course, remain questionable whether GT1–7 cells are a true representation of the mature in vivo GnRH neuron population, especially as immortalized GnRH neurons are grown in monoculture and are the descendants of a single GnRH neuron that was transformed in early development (26).

The primary aim of this study was to examine the effects of NMDA on GnRH release via indirect evaluation (by measuring LH levels) during absolute photorefractoriness and throughout the seasonal cycle. In addition, using Fos as a marker of cell activation, we sought to determine brain regions activated after NMDA administration and establish whether GnRH I neurons express Fos at this time. The study was designed to test the hypothesis that suppression of the reproductive axis during photorefractoriness in the White-crowned sparrow is not due to GnRH store depletion and in doing so provide valuable insights into the brain circuits involved in the control of avian seasonal reproduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
White-crowned sparrows (Zonotrichia leucophrys gambelii) were captured during autumnal migration in mist nets in Eastern Washington State and held in outdoor aviaries at the Department of Zoology, University of Washington (Seattle, WA). Before the winter solstice they were anesthetized with methoxyflurane vapor (Metofane, Mallinckrodt, Inc., Chicago, IL) and laparotomized via a small incision in the body cavity, just behind the ribs, to determine sex. All males had regressed testis, were housed singly in wire cages (41 x 39 x 35 cm), and were maintained on short days of 8 h of light/day (8L:16D; at a controlled temperature of 20 C). Water (drinking water and birdbaths) and bird seed were freely available. The animal care and use committee at the University of Washington approved all procedures used in these experiments.

Acute effects of systemic NMDA on LH secretion throughout the seasonal cycle
Male White-crowned sparrows were randomly assigned to one of three photoperiodic groups (n = 12 in each group). The first group, termed photosensitive, was maintained on 8L:16D. The second, photostimulated, group experienced 2 weeks of long days (20L:4D). The third group was comprised of photorefractory birds that were subjected to 10 weeks of long days (20L:4D). These groups were chosen to generate birds in different stages of the reproductive cycle, and birds were transferred to 20L:4D at different times so that all groups could be sampled on the same calendar day. Each group was divided into two treatments (n = 6 in each treatment), where they were given either vehicle (0.9% sodium chloride) or NMDA (Sigma, St. Louis, MO) at a dose of 20 mg/kg BW, sc. Injections were administered between 1–4 h after lights on. Blood samples were collected by wing venipuncture (~100 µl whole blood) before and 2, 8, and 20 min after injection. Blood was centrifuged, and the plasma samples were stored at -20 C until processing for LH measurement in a single assay to eliminate interassay variation using a micromodification of the RIA originally devised by Follett et al. (30) outlined for use in the White-crowned sparrow by Follett et al. (31). The intraassay coefficient of variation was 3% at 1.71 ng/ml, and the level of assay sensitivity or the minimum detection limit was 0.15 ng/20 µl plasma sample. Results are expressed in terms of nanograms per ml against a chicken LH standard (fraction IRC2).

Acute effects of systemic NMDA on the expression of Fos-like immunoreactivity (FLI) in the forebrain throughout the seasonal cycle
Male White-crowned sparrows were divided into three experimental groups (photosensitive, photostimulated, and photorefractory; n = 12 in each group). Birds in each group were subjected to either vehicle (0.9% sodium chloride) or 20 mg/kg NMDA (Sigma), sc, between 1–4 h after lights on, as described in the above experiment. One hour after treatment, the sparrows were deeply anesthetized with an overdose of 250 mg/kg pentobarbital sodium (Nembutal, Abbott Laboratories, North Chicago, IL), im, and perfused intracardially with 10 ml heparinized 0.9% saline followed by 150 ml modified Zamboni fixative (1.8% paraformaldehyde and 7.5 ml saturated picric acid in 0.01 M PBS, pH 7.4). The experimental design allowed birds from all groups and treatments to be killed on the same calendar day. After perfusion, the testes were removed and weighed. Brains were dissected out of the skull, postfixed at 4 C overnight, washed in PBS, and embedded in gelatin. Immunocytochemistry for FLI was performed as described previously (32, 33). Coronal 50-µm sections were cut on a Vibratome from the preoptic region to the median eminence (ME). Free floating sections were labeled for FLI, and sections from the preoptic region were double labeled for both FLI and GnRH I. In brief, sections were rinsed for 2 h in PBS (six changes) containing 0.2% Triton X-100 (PBST). Endogenous peroxidase was blocked by incubation in 0.3% hydrogen peroxide in PBS (10 min), followed by washing in PBST (three times, 10 min each time).

The sections were then incubated for 70 h in primary antiserum [rabbit polyclonal antichicken Fos (code 9/3), gift from Dr. P. J. Sharp, Roslin Institute, Midlothian, UK] diluted 1:5000 in 2% normal goat serum in PBST at 4 C (see Refs. 32, 34 for details and validation of this antibody). The antibody:antigen complex was localized using the avidin-biotin complex method using a Vector Elite kit (Vector Laboratories, Inc., Burlingame, CA) and PBST as the wash buffer. Sections only to be labeled for FLI were then rinsed in 0.05 M Tris buffer, pH 7.4 (10 min), and the peroxidase was visualized with a solution of 0.025% diaminobenzidine tetrahydrochloride (DAB) containing 0.03% hydrogen peroxide in Tris buffer. The reaction was terminated by several washes in PBS. In sections to be additionally labeled for GnRH I, FLI was located using the above protocol, except that visualization was performed in a solution of DAB nickel sulfate in 0.0175 M sodium acetate. After visualization, sections were incubated in a polyclonal rabbit antichicken GnRH I (code 3/3, gift from Dr. P. J. Sharp, Roslin Institute) (see Ref. 35 for validation of this antibody) diluted 1:10,000 in 2% normal goat serum for 70 h at 4 C. GnRH I was visualized using the peroxidase-antiperoxidase technique. Sections were rinsed in PBST and incubated in goat antirabbit IgG diluted at 1:100 in PBST for 2 h at room temperature. Sections were washed in PBST and then transferred to the peroxidase-antiperoxidase complex diluted at 1:100 in PBST for 2 h at room temperature. After rinses in PBST and Tris buffer, the peroxidase was visualized with DAB containing 0.03% hydrogen peroxide in Tris with the reaction terminated by several washes in PBS. Serial sections were mounted on gelatin-coated slides and left to dry overnight. Slides were dehydrated and mounted in DPX mountant (Aldrich Chemical Co., Inc., Milwaukee, WI).

FLI staining was prevented by incubation of the sections in nonimmune rabbit serum instead of the antibody or the Fos antiserum preabsorbed with 1 µg/ml synthetic chicken c-Fos peptide. Similarly, incubation of the GnRH I antibody preabsorbed with 1 µg/ml synthetic chicken GnRH I abolished GnRH I staining.

Analysis of data
Plasma LH measurements were normalized by log transformations before conducting data analysis and are plotted graphically as the mean ± SEM. Differences between photoperiodic groups and treatments were compared using a multivariate repeated measures ANOVA, with photoperiodic group and treatment as the two factors and LH levels across time as the repeated measure. Any differences were considered significant at an {alpha} level of P < 0.05.

High power oil immersion on a brightfield microscope was used to describe the relationship between FLI and GnRH I neurons in the preoptic region. GnRH I perikarya are located within the organum vasculosum of the lamina terminalis (OVLT), a region located between the preoptic recess and the anterior commissure, extending in a continuum from the preoptic area to the habenula (36). The numbers of GnRH cells were counted single blind in every second section in the NMDA-treated birds from all three photoperiodic groups. Sections were aligned using the septomesencephalic tract and the anterior commissure as neuroanatomical landmarks. Cell counts were determined with multivariate repeated measures ANOVA followed by polynomial contrasts.

The number of labeled FLI cells was directly quantified by single blind analysis. Counts were taken from every second section within the region of the OVLT and paraventricular nucleus (PVN), with the sections again aligned using neuroanatomical landmarks. GnRH I staining provided a useful guide to the boundaries of the OVLT. The numbers of FLI cells in every 50-µm section within the infundibular nucleus (IN), ME, and the bed nucleus of the commissura pallii (BPC) were quantified (see Ref. 37 for definition of these nuclei). Cell counts within the OVLT were analyzed by a multivariate repeated measures ANOVA, with photoperiodic group and treatment as the two factors and FLI counts per section as the repeated measure, followed by polynomial contrasts to describe the distribution. Testicular mass and FLI cell counts taken from other brain regions were normalized by log transformations and analyzed using a one-way ANOVA followed, when appropriate, by post-hoc Tukey’s all pairwise multiple comparison procedures test. Any differences were considered significant at an {alpha} level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Acute effects of NMDA on LH secretion throughout the seasonal cycle
Photoperiodic treatment significantly altered plasma LH levels [F(2,30) = 58.01; P < 0.001]. In agreement with previous findings (1), the photostimulated group had LH levels at least 3 times as high as those of photosensitive and photorefractory birds (see Fig. 1Go). Photorefractoriness was confirmed by the fact that the birds were molting, a typical feature of this condition.



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  		Figure 1. Acute effects of a single sc injection of NMDA (20 mg/kg) or vehicle (injection time indicated by arrows) on plasma LH concentrations in White-crowned sparrows in three different reproductive conditions. Values are the group mean ± SEM (n = 6 for each group). *, P < 0.05 vs. control.

 
Administration of NMDA significantly increased LH in all photoperiodic groups compared with that in vehicle-treated controls [F(3,90) = 4.88; P < 0.001; Fig. 1Go], and the LH response to NMDA did not differ significantly between groups [F(6,90) = 1.65; P > 0.05]. Twenty minutes after sc NMDA administration, plasma LH levels rose in absolutely photorefractory sparrows from 0.5 ± 0.12 to 0.92 ± 0.18 ng/ml, an 84% increase; photosensitive birds showed a 163% increase from 0.46 ± 0.15 to 1.21 ± 0.2 ng/ml. Neither of these groups achieved baseline LH levels observed in photostimulated birds, which exhibited a 51% increase in LH from 1.76 ± 0.2 to 2.66 ± 0.35 ng/ml.

Acute effects of systemic NMDA on the expression of Fos-like immunoreactivity in the forebrain throughout the seasonal cycle
The effect of photoperiodic treatment on the reproductive physiology of the birds was confirmed by measurement of paired testicular mass. Short day photosensitive birds and photorefractory birds had significantly smaller testes compared with photostimulated birds ([0.01 ± 0.00 vs. 0.03 ± 0.01 vs. 0.14 ± 0.02 g; F(2,35) = 30.21; P < 0.001]. In addition, photorefractoriness was verified by feather molt.

Subcutaneous administration of NMDA did not induce FLI within GnRH I cell bodies regardless of photoperiodic treatment; examples from photosensitive and photorefractory birds are illustrated in Fig. 2Go, A–D. Nonetheless within the OVLT, NMDA elicited significantly more FLI than did vehicle [F(11,330) = 6.15; P < 0.001]. There was no significant difference among the photoperiodic groups in the response to either NMDA [F(15,165) = 0.61; P > 0.05; Fig. 3Go] or vehicle [F(15,165) = 0.65; P > 0.05] as measured by FLI cell number. After NMDA treatment FLI cells appeared to be in close proximity to GnRH perikarya and fibers (see Fig. 2Go, A and C). Polynomial tests described both the pattern of the GnRH I-labeled neurons and the population of FLI-labeled cells through the OVLT as quadratic (P = 0.00 in each case; see Fig. 3Go). This implies that both types of labeled cell share the same pattern, with the highest labeling within the central part of the OVLT. The number of positively stained GnRH I perikarya quantified for every other section was not significantly different between photoperiodic groups [F(11,165) = 0.71; P > 0.05; see Fig. 2Go, A–D, and 3].



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  		Figure 2. Photomicrographs A–F illustrate double labeling of GnRH I (A–D, brown cell bodies and fibers; E and F, brown fibers only) and FLI (black nuclei) within the OVLT (A–D) and BPC (E and F). Higher levels of FLI are observed in these regions after sc administration of NMDA (20 mg/kg; A, C, and E) compared with vehicle (B, D, and F). A and B are taken from photostimulated birds; photomicrographs C–F are from photorefractory birds. Note the lack of colocalization but the close proximity of FLI expression to GnRH I cells in the OVLT in response to NMDA. Also note that GnRH I immunoreactivity is similar in photostimulated and photorefractory birds. Photomicrographs G–L demonstrate higher FLI activation within the IN and ME of NMDA-treated birds (G, I, and K) compared with controls (H, J, and L). Birds in three different reproductive states are represented: photosensitive (G and H), photostimulated (I and J), and photorefractory (K and L). Scale bars: A–F, 20 µm; G–L, 200 µm.

 


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  		Figure 3. Distribution and number of GnRH I-labeled cells (gray symbols) and FLI-positive cells (black symbols) in the OVLT of vehicle-treated NMDA (20 mg/kg) White-crowned sparrows in three reproductive conditions: photosensitive (circles), photostimulated (triangles), and photorefractory (squares). The mean numbers (±SEM; n = 6 for each group) of cells per 50-µm section are arranged in rostral to caudal order after alignment using the anterior commissure (AC) as a landmark. The numbers of GnRH I-labeled cells within the OVLT did not significantly differ between photoperiodic treatments [F(11,165) = 0.71; P > 0.05]. NMDA injection resulted in highly significant activation of cells throughout the OVLT [F(11,330) = 6.15; P < 0.001] compared with controls (data not shown). Note the pattern of FLI through the OVLT is similar to that of GnRH I-labeled cells.

 
Within the basal tuberal hypothalamus, NMDA treatment significantly induced FLI within the IN [F(5,35) = 16.42; P < 0.001] and ME [F(5,35) = 11.06; P < 0.001] compared with animals injected with vehicle (see Fig. 4Go and photomicrographs in Fig. 2Go, G–L). There was no significant difference in the numbers of FLI among the different reproductive conditions in either the IN (P > 0.05 for both treatment groups) or the ME (P > 0.05 for both treatment groups) or in its distribution (Fig. 2Go, G–L). Within the BPC, a region rich in GnRH fibers, NMDA significantly increased FLI compared with controls [F(5,35) = 17.69; P < 0.001], and again there was no significant difference in the amount of activation among the three photoperiodic groups (P > 0.05 for both treatment groups; see Fig. 4Go). Examples of FLI and GnRH fiber staining in photorefractory birds given either NMDA or vehicle is shown in photomicrographs E and F in Fig. 2Go. The PVN was not significantly activated by NMDA treatment, nor was there any effect of photoperiodic condition [F(5,35) = 1.47; P > 0.05; see Fig. 4Go].



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  		Figure 4. Induction of FLI activity (mean numbers of cells per 50-µm section ± SEM; n = 6 for each group) within four brain regions of the White-crowned sparrow after sc administration of NMDA (20 mg/kg) or vehicle. Birds in three different reproductive conditions are compared: photosensitive, photostimulated, and photorefractory. NMDA significantly increased numbers of FLI within the ME, IN, and BPC (***, P < 0.001 vs. control), but not in the PVN (P > 0.05 vs. control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since avian photorefractoriness was first described by Bissonnette in the 1930s (38), the mechanism underlying the condition has remained elusive. Previous studies have suggested that the decline in releasable GnRH I underlies photorefractoriness, as levels of GnRH I are correlated with this condition (1, 5). However, this theory does not account for why European starlings (Sturnus vulgaris) become absolutely photorefractory before detectable levels of GnRH I or its precursor decline in the ME (39, 40). This present study demonstrates for the first time that photorefractory birds have the capacity to increase their plasma LH levels, presumably by stimulation of GnRH I release. These findings strongly support the hypothesis that quiescence of the reproductive axis is not a consequence of GnRH store depletion or reduced releasability of GnRH (41), particularly as all NMDA-treated birds (regardless of their photoperiodic condition) responded with a rapid LH rise reflecting prompt GnRH release. This is complemented by the discovery that the numbers of GnRH I-immunoreactive perikarya throughout the OVLT are not significantly different among photoperiodic groups. This finding reinforces the view that changes in GnRH neuron number are not central to seasonal changes in reproductive function. Studies in the hamster also show no relationship between photoperiodic condition and the number of immunocytochemically labeled GnRH cells (42, 43) or cells containing GnRH mRNA (44).

However, these studies contrast with those in the European starling (36) and dark-eyed junco (Junco hyemalis) (45), in which numbers of GnRH neurons were lower in photorefractory individuals. Further research is warranted to investigate whether the actual hypothalamic content of GnRH I differs seasonally in the White-crowned sparrow. Nonetheless, direct comparisons among photoperiodic groups reveal that LH levels attained in photosensitive and photorefractory birds do not reach those in photostimulated birds. One explanation for this difference could be the differing pituitary sensitivities to GnRH as a consequence of varying levels of endogenous GnRH. In the rhesus monkey, priming of the pituitary gland with GnRH was required before LH release could be effectively stimulated (14). Secondly, the discrepancy in LH levels may be attributed to a single dose (as in this experiment) as apposed to pulsatile administration of NMDA. Pulsatile NMDA infusion either increases or decreases the LH response in hamsters depending upon the reproductive state (7), so that experiments involving pulsatile NMDA may result in higher circulating LH levels in short day birds. Thirdly, the results may be explained by higher circulating sex steroids in photostimulated birds compared with those in the other groups. Sex steroids are required for the excitatory action of NMDA in the monkey (46) and sheep (15), although the LH response to NMDA is attenuated in castrated rats (12). Alternatively, GnRH neurons may be inhibited by an unknown endogenous mechanism that is not completely overridden by the action of NMDA.

Consistent with previous studies (8, 10, 16, 22), NMDA did not induce Fos expression within GnRH perikarya. This suggests that all birds, regardless of photoperiodic history, had sufficient stores of GnRH peptide so that gene expression was not required. This discovery is compatible with studies in the Japanese quail (33) and White-crowned sparrow (Meddle, S. L., manuscript in preparation), demonstrating that GnRH I cells do not express FLI after a photoperiodically driven LH rise. Instead, cells within the basal tuberal hypothalamus show increases in FLI expression analogous to the induction observed in this study. The basal tuberal hypothalamus is a vital component of the avian photoperiodic response, as lesions in this region sparing GnRH I fibers block photoinduced gonadal growth (47). In mammalian models, NMDA also elicits high levels of Fos expression in the arcurate nucleus-median eminence region (8, 9, 48), suggesting that the release of GnRH may be mediated via cells in the mediobasal hypothalamic complex.

It is speculated that NMDA induces Fos activation by Ca2+ influx through the NMDA receptor-coupled ion channel or by postsynaptic stimulation from afferents excited by NMDA (49). NMDA receptors are requisite for Fos induction, as Fos expression in the medial basal tuberal hypothalamus and preoptic region can be abolished by preinjection of the glutamate antagonist MK-801 (50). NMDA receptors are widespread throughout the hypothalamus (16, 22, 23, 24), and the substantial FLI induction in close proximity to GnRH I cell bodies within the OVLT may indicate that GnRH I neurons are regulated by a population of glutamate receptor-containing cells in this region. This is supported by evidence in the literature suggesting that there are no or very low concentrations of glutamate receptors on GnRH neurons (26, 27, 29). However, the possibility of a direct action of NMDA on GnRH neurons cannot be ruled out, as other studies have demonstrated the colocalization of NMDA receptors and GnRH neurons (23, 24, 25, 28).

NMDA may cause an increase in LH by releasing GnRH neurons from tonic inhibition. Evidence of possible inhibitory inputs comes from studies in the photorefractory European starling, in which GnRH I neurons have more synaptic input than those in photosensitive birds (51). The pattern of NMDA-induced FLI throughout the OVLT was similar across all photoperiodic groups, suggesting that the same population of cells was stimulated in each group. The high numbers of cells expressing FLI in the BCP could participate in activation of the reproductive axis, but because the avian BCP contains cells immunoreactive for a variety of neuropeptides and receptors (52), it is possible that the NMDA-activated cells are unrelated to the seasonal control of reproduction. The present study did not address the sites of NMDA action within the hypothalamus. Nonetheless, the increase in FLI expression within the OVLT, BPC, ME, and IN implies that these sites are potential constituents of the reproductive neuroendocrine axis, particularly as all of these regions contain GnRH I-immunoreactive material. We can be confident that the significant increase in FLI is specific and not a reflection of a generalized elevation in brain activation, because cells in the posterior OVLT or PVN did not show any significant activation after NMDA treatment.

In the White-crowned sparrow, seasonal alterations in gonadal function are most likely to be caused by changes in GnRH secretion, especially as the numbers of GnRH I-immunoreactive cells do not appear to show a seasonal change. Clearly, further experiments are required to resolve the complex mechanisms underlying avian photorefractoriness and seasonality. In particular, experiments incorporating NMDA receptor antagonists would assist in resolving whether glutamate has a physiological role in absolute photorefractoriness.


   Acknowledgments
 
We thank Dr. Peter J. Sharp for the gift of the avian Fos and chicken GnRH I antibodies, Dr. Eliot A. Brenowitz for use of the photomicroscopy equipment, and Dr. Alexander Kitaysky for assistance with statistical analysis. Thanks also go to Dr. Catherine J. Black and the anonymous reviewers for their critical appraisal of a previous version of the manuscript.


   Footnotes
 
1 This work was supported by NSF Grant IBN-9631350 (to J.C.W.). Back

2 Present address: Department of Biomedical Sciences, University of Edinburgh Medical School, Teviot Place, Edinburgh, Scotland EH8 9AG. Back

3 Present address: Department of Psychology, Behavioral Neuroendocrinology Group, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218. Back

Received February 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wingfield JC, Farner DS 1990 Endocrinology of reproduction in wild species. In: Farner DS, King JR, Parkes KC (eds) Avian Endocrinology. Academic Press, New York, vol 9:164–293
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