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Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology and Center for Reproductive Biology (H.T.J.), Washington State University College of Veterinary Medicine, Pullman, Washington 99164-6520; Department of Cell Biology, Neurobiology, and Anatomy (C.C., M.N.L.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521; and Department of Physiology (S.H., R.L.G.), West Virginia University Health Sciences Center, Morgantown, West Virginia 26506-9229
Address all correspondence and requests for reprints to: Heiko T. Jansen, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Programs in Neuroscience and Center for Reproductive Biology, Washington State University, 205 Wegner Hall, Pullman, Washington 99164-6520. E-mail: heiko{at}vetmed.wsu.edu.
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Abstract |
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-aminobutyric acid, catecholamine, neuropeptide Y, or ß-endorphin) inputs to GnRH neurons change seasonally, and concomitant with any changes in neural inputs is a change in glial apposition. Using triple-label immunofluorescent visualization of GnRH, glial acidic fibrillary protein and neuromodulator/neural terminal markers combined with confocal microscopy and optical sectioning techniques, we confirmed that total numbers of neural inputs to GnRH neurons vary with season and demonstrated that specific inputs contribute to these overall changes. Specifically, neuropeptide Y and -aminobutyric acid inputs to GnRH neurons increased during BS and ß-endorphin inputs were greater during either anestrus (GnRH somas) or BS (GnRH dendrites). Associated with the changes in GnRH inputs were seasonal changes in glial apposition, glial acidic fibrillary protein density, and the thickness of glial fibrils. These findings are interpreted to suggest an increase in net stimulatory inputs to GnRH neurons during the BS contributes to the seasonal changes in GnRH neurosecretion and that this increased innervation is perhaps stabilized by glial processes. |
Introduction |
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Perhaps the best-studied example of seasonal plasticity is that expressed in the songbird brain. For example, annual song learning is associated with recurring changes in both the size of specific nuclear groups and neural connectivity (5, 6, 7, 8). Both types of changes are dependent on gonadal steroids and can be driven by changing the steroid milieu (9, 10). Within the GnRH system of some bird species, changes in neuronal morphology and gene expression have also been reported (11, 12, 13, 14, 15). However, whether these changes in the avian GnRH system also reflect an underlying change in neuronal connectivity, glial associations, or other mechanisms is not clear.
In mammals, examples of short-term (hours to days) neural plasticity are abundant. Dehydration and lactation both result in profound increases in synaptic inputs onto hypothalamic magnocellular neurons and changes in glial association (16, 17). During the estrous cycle, changes in neural connectivity (18, 19) and glial associations (20) have been documented. Examples of long-term (months) seasonal neural plasticity in mammals are less abundant, despite the fact that many physiological parameters (e.g. behavior, endocrine status, reproduction) in temperate zone species exhibit robust seasonal variation. At best, our knowledge of the neural processes underlying these events is incomplete; however, a recent report demonstrated that the number of synaptic inputs onto GnRH neurons increase significantly during the breeding season (BS), compared with anestrus (AN) in sheep (21).
A variety of different types of neural inputs to GnRH neurons have been identified and together with a large body of experimental evidence suggest they may serve important physiological functions (for reviews, see Refs.22, 23, 24, 25, 26). For seasonal breeders, such as sheep, these inputs may be necessary to convey steroid feedback information to the GnRH neuron. Indeed, the sheep has become a model species in which to examine the neural mechanisms underlying seasonal changes in steroid feedback within the reproductive neuroendocrine axis. For example, sheep exhibit seasonal changes in GnRH neurosecretion (3, 27), total numbers of GnRH inputs (21), and activation of potential GnRH afferents (28, 29) associated with gonadal steroid feedback. Anatomical and physiological studies in this species have implicated catecholaminergic, neuropeptide Y (NPY)ergic, -aminobutyric acid (GABA)ergic and opioidergic systems, among others, as potential transducers of steroid feedback onto the GnRH system. Specifically, dopamine (DA) is suggested to mediate estrogen-negative feedback via an inhibitory influence on the GnRH system during AN (29, 30, 31, 32), whereas the role of norepinephrine may vary depending on the stage of the annual reproductive cycle (30, 33, 34). An inhibitory influence of GABA on the GnRH system is suggested and may be expressed during negative feedback in AN and during negative feedback throughout the estrous cycle (35, 36). Opioid peptides, such as ß-endorphin, also appear to exert an inhibitory influence on GnRH during progesterone-mediated and estradiol-mediated negative feedback (37, 38, 39). For NPY, both stimulatory and inhibitory influences on the GnRH system have been reported (40, 41, 42, 43). Despite the evidence in support of a role for various neurochemical systems in regulating GnRH at different times of the year and under different reproductive conditions, it has not been established, in any seasonal-breeding mammal, whether this regulation occurs because of changes in inputs to GnRH neurons directly.
Although GnRH neurons receive direct neural innervation, albeit, less than their non-GnRH-expressing neighbors within the POA in sheep (21, 44, 45, 46, 47), they are also extensively associated with nonneuronal elements, specifically glial cells (45, 46, 47). Therefore, glial cells may provide an important method for modulating the number of inputs onto the GnRH neuron, as has been suggested in the monkey (45, 46) and sheep (25). Additional evidence supporting a role for glia in modulating GnRH function comes from our own studies demonstrating that the association between GnRH neurons and the cell adhesion molecule, polysialylated form of neural cell adhesion molecule (PSA-NCAM), varies seasonally (48). PSA-NCAM is expressed in both glia and neurons (49, 50, 51) and studies in the rat demonstrate that removal of PSA from the NCAM molecule in vivo blocks both the glial retraction and increase in synaptic inputs onto magnocellular neuroendocrine neurons during lactation and dehydration or after estradiol administration (49, 52).
Given the observation that a seasonal change in total innervation of GnRH neurons is associated with seasonal changes in reproductive activity in the ewe, we asked whether this reflected changes in specific neural afferents, changes in glial associations, or both.
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Materials and Methods |
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Before euthanasia, jugular blood samples were collected by venipuncture for 4 h from all ewes at either 10-min (BS) or 20-min (AN) intervals. Serum was subsequently assayed for LH concentrations as previously described (54). Following blood collection, animals were heparinized and then killed with an overdose of sodium pentobarbital during AN (August, n = 6) or BS (December, n = 6). The heads were immediately perfused with 6 liters 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) containing 0.1% sodium nitrite as a vasodilator (26). The forebrain and hypothalamus were removed, blocked, and then placed in fixative for an additional 24 h followed by cryoprotectant (20% sucrose in PB) until infiltrated (2–3 d). Frozen sections (55 µm) extending from the diagonal band of Broca rostrally to the mammillary nuclei, caudally, were then collected in six series (distance between sections within a series = 330 µm) and subsequently stored in cryopreservative (55). Each series of sections was then processed for immunocytochemical identification of GnRH, glial processes, and either total neural inputs or specific neuromodulatory afferents as described below. Postmortem examination revealed that two BS ewes had lost their estrogen implants at some time before being euthanized.
Western blotting
Sheep brains (n = 3) were obtained from the Meat Science Laboratory at Washington State University. After slaughter, the POA/hypothalamus was dissected out and immediately frozen at -80 C. Total protein was extracted from each of the three brains as described previously (56) and then subjected to SDS-PAGE (7.5% resolving + 4% stacking gel) using a Criterion apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins were resolved at 50 mA for 60 min and then transferred to polyvinyl difluoride membranes (12 V for 2 h, Idea Scientific Co., Minneapolis, MN). Proteins on gels were visualized with Coomassie blue staining. After transfer, the membranes were incubated overnight in blocking buffer (Bio-Rad Laboratories, Inc.) containing 3% BSA and on the following day were transferred to primary antiserum (anti-Synapsin I, Molecular Probes, Inc., Eugene OR; diluted 1:200) containing blocking buffer, 3% BSA, and 5% normal donkey serum. Membranes were incubated overnight for 16 h at room temperature with shaking. On the following day, the membranes were washed in PBS and transferred to horseradish peroxidase-conjugated donkey antirabbit IgG (diluted 1:10,000 in blocking buffer) and incubated for 1 h at room temperature with shaking. Immunoreactive bands were visualized using chemiluminescence methods (ECL, Amersham, Piscataway, NJ) and subsequently recorded on x-ray film.
Immunocytochemistry
Tissue sections were removed from cryopreservative and washed four times, 5 min each, in PB containing 0.1% Triton X-100 with shaking followed by a 1-h incubation in a cocktail of blocking sera (1% each) specific to the species in which the secondary antibodies were raised. Next the sections were incubated in a cocktail of primary antibodies against GnRH, glial fibrillary acidic protein (GFAP) and one of the following: Synapsin-I (Syn-I), tyrosine hydroxylase (TH), PY, ß-endorphin (ß-EN), or glutamate decarboxylase (GAD). The antibody cocktail was made in PB containing 0.1% Triton X-100 containing 3% blocking sera plus 0.01% sodium azide; sections were incubated for 48 h at 4 C with shaking. On the third day, sections were rinsed in PB four times, 5 min each, and then incubated in fluorescent secondary antibodies (Table 1
; diluted 1:100) appropriate for GnRH (antirabbit or mouse Cy3-conjugated), GFAP (antiguinea pig Cy5-conjugated) for 1 h at room temperature with shaking. Sections were washed four times, 5 min each. To enhance the visualization of Syn-I, GAD, ß-EN, and TH immunoreactivity (ir), the fluorescent signal (Alexa 488) was intensified using a multiple-bridging amplification method as follows: Sections were transferred to fluorescent secondary antibody (diluted 1:100) for 30 min and then washed. This was followed by incubation in unlabeled bridging IgG (10 µg/ml) specific to the species in which the primary antibody was raised (e.g. rabbit, mouse) for 30 min. After additional washes, the entire process (primary-secondary) was repeated twice more. Because of the intensity of NPY fluorescence, amplification was not required. All sections were processed through a final series of washes and then mounted on SuperFrost-plus slides (Fisher Scientific, Pittsburgh, PA), dried in the dark overnight, and coverslipped with Gelvatol (57).
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. The characteristics of GnRH antibodies have been previously described (58, 59). For the other antibodies, controls consisted of either preincubating the antibody cocktail with 1–10 µg purified antigen (anti-NPY, -Syn-I, -ß-EN) or omission of the primary antibody (anti-GAD, -GFAP, -TH); in each case, specific staining was lost. Additional controls consisted of omission of one of the three secondary antibodies; again, this resulted in the complete loss of signal for the particular antigen (species) against which the antibody was raised (not shown).
Imaging and analysis
Slides were viewed and the confocal images obtained using a LSM-510 laser-scanning microscope (Zeiss, Thornwood, NY) equipped with Ar, and He/Ne lasers capable of producing monochromatic excitation at 458/488/514, and 543/643-nm wavelengths, respectively. The pinhole diameter was optimized to 1.0 Airy disk. Image size was set at 512 x 512 pixels. Scans at each wavelength were performed independently (multitracking) to eliminate bleed-through between individual channels.
Individual GnRH neurons were visualized at x20 magnification under epifluorescent (mercury vapor) illumination. Then the illumination was switched to confocal mode (laser) and optical sectioning (Z-thickness, 0.45 µm) of a selected GnRH neuron was performed using a x 63 C-Apochromat water immersion objective (numerical aperture 1.2). For each GnRH neuron, 20 optical sections, each composed of scans through three separate channels (488, 543, and 643 nm excitation) were captured and saved (Fig. 1). Composite images, as well as individual images from each channel, were then converted to TIF format, saved, and subsequently analyzed using NIH Image software (Object IMAGE, version 2.07) as described below. GnRH neurons were grouped, post hoc, into either rostral or caudal populations based on their location rostral to, or caudal to, the midline crossing of the anterior commissure, respectively. The plane of sectioning was adjusted for each brain such that similar landmarks were present within a section between animals.
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Inputs to GnRH neurons were identified based on the proximity of terminal immunoreactivity to the presumptive GnRH cell membrane. Although the limit of resolution with the light microscope does not allow identification of synaptic specializations, and thus bona fide synapses, additional criteria were developed to maximize the identification of axon terminals (i.e. GnRH inputs). Previous studies using confocal microscopy to examine close appositions between axon terminals and GnRH neurons (60) were limited in their ability to resolve close associations, and thus we incorporated GFAP immunoreactivity as an additional means to eliminate false positives. Thus, only those terminals (green pixels) apposed to the GnRH cell membrane (red pixels) and not having any GFAP-ir elements (blue pixels) interposed between them were considered (Fig. 1). The sum of all Syn-I-ir GnRH inputs (soma and dendrite) was then used to provide an estimate of the total number of afferents per GnRH neuron. Based on these results, the percentage of the total number of somatic and dendritic GnRH inputs represented by each type of neurotransmitter/neuropeptide input was derived.
Those GnRH neurons processed for determination of total numbers of inputs (i.e. were Syn-I-ir), were also used to estimate the amount of glial association with the soma and proximal dendrites. Data from the individual channels in three optical sections were used to calculate the percentage of GnRH-ir membrane (red pixels; both soma and proximal dendrite) in direct contact with GFAP-ir elements (blue pixels) according to the formula: (LGFAP/LGnRH) x 100, where LGFAP = length of GFAP-ir pixels in contact with GnRH-ir membrane and LGnRH = length of GnRH-ir pixels associated with either the soma or dendrite(s). The overall density of GFAP-ir was also estimated in 100 randomly selected fields of GnRH neurons using the method described previously (48) with a minor modification to increase the size of the area analyzed from 150 pixels to 260 pixels to incorporate more of the GnRH proximal dendrite(s). First, for each 512 x 512 pixel field, GFAP density was estimated within an area containing the GnRH neuron and proximal dendrite(s) and then in an area of the same field but devoid of GnRH-ir (usually the lower right quadrant). Finally, within the area lacking GnRH-ir, the diameter of individual glial filaments was quantified by drawing a line (15 µm in length) bisecting the region of interest and oriented perpendicular to the majority of glial filaments in the field. The distribution of gray-scale values along the line was then plotted and for each plot, a threshold value 2 SD above the minimum was computed. The diameter of each glial fiber was estimated from the points at which the peak crossed the threshold value. This procedure was repeated in three adjacent optical sections centered about the middle of each z-series.
Statistical analysis
For each parameter, the mean ± SEM was determined for all rostral or caudal neurons in each animal. Statistical comparisons were made by three-way ANOVA (main effects: season, cell location, dendrite vs. soma, and interactions). LH pulse parameters were identified using the Pulsar algorithm as described previously (61, 62) and compared between seasons by Mann-Whitney U test. Post hoc comparisons of group means were performed by Fisher’s least significant difference analysis. Differences were considered statistically significant if P was less than 0.05.
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Results |
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). As expected, for the two BS ewes that lost their estradiol implants, pulse frequency was similar to that of estrogen-implanted ewes (6 pulses/4 h). This similarity reflects the low sensitivity of the GnRH system to estradiol-negative feedback at this time of the year (2, 3). Similar to LH pulse frequency, mean LH concentrations were also significantly greater during BS than during AN (P < 0.001; Fig. 2).
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). However, the number of neural inputs to GnRH neurons did vary by season, and the results of analysis of total and specific inputs are summarized in Figs. 4 and 5.
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). Immunocytochemical analysis of Syn-I-ir terminals revealed that immunoreactivity was limited to small puncta (
0.25 µm in diameter, Table 2) and these were densely scattered throughout the neuropil (Fig. 1C). Syn-I-ir terminals were present on GnRH neurons (Fig. 3). Quantitation of input numbers revealed that GnRH somas of BS ewes received greater numbers of inputs than somas of AN ewes (effect of season, P < 0.01; Fig. 4). A similar picture emerged for GnRH dendrites (Fig. 5), i.e. greater numbers of inputs were present during the BS than during AN (P < 0.01). There was no difference in total numbers of inputs between rostral and caudal populations or when comparing soma and dendrites (effect of region, P > 0.05). Overall, the number of inputs on GnRH dendrites (per micrometer cell membrane) within rostral and caudal regions was greater than inputs to GnRH somas (0.06 ± 0.013 vs. 0.03 ± 0.02; P < 0.05, respectively).
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and 6) were present on GnRH neurons located in both rostral and caudal regions of their distribution. Overall, GnRH somas received a significantly greater total number of NPY inputs during the BS than AN (BS: 5.5 ± 0.08 vs. AN: 1.7 ± 0.1, P < 0.001; Figs. 4 and 5). This difference was significant for the caudal population of GnRH neurons (BS: 5.1 ± 0.1 vs. AN: 1.0 ± 0.08, P < 0.001; Fig. 4). For the rostral population, the numbers of NPY inputs were not statistically different with respect to season (BS: 6.7 ± 3.9 vs. AN: 2.5 ± 1.0, P = 0.20). Comparison of NPY inputs onto GnRH dendrites followed the same trend as for the cell soma (Fig. 5). Thus, the dendrites of caudal GnRH neurons received significantly greater numbers of inputs during the BS than AN (P < 0.01; Fig. 5). In addition, for the caudal GnRH population, the numbers of NPY inputs onto GnRH dendrites was greater than onto somas (P < 0.02).
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). In addition to terminals, occasional GAD-ir cell bodies were evident (not shown). Quantitative analysis revealed that inputs onto GnRH somas did not change seasonally (BS: 5.2 ± 0.3 vs. AN: 6.3 ± 1.5; P = 0.3) or with region (Fig. 4). However, GAD-ir inputs onto GnRH dendrites were greater during the BS than during AN (P < 0.01; Fig. 5). This change was specific to the rostral GnRH population (Fig. 5). No significant seasonal changes in GAD-ir inputs onto GnRH dendrites were observed for the caudal population.
Catecholaminergic (TH-ir).
A few widely scattered TH-ir fibers and terminals were present in the sections examined, and a few were found in close association with GnRH neurons (Fig. 6). However, neither TH-ir inputs onto GnRH somas nor onto dendrites exhibited any significant differences with respect to season or region (Figs. 4 and 5). In general, the total number of inputs was extremely low for both somas (1.02 ± 0.09 inputs/soma) and dendrites (0.003 ± 0.002/µm) and overall for somas between regions (rostral: 0.9 ± 0.4 inputs vs. caudal: 1.3 ± 0.8 inputs).
ß-EN.
Fibers and terminals immunoreactive for ß-EN (Fig. 6) were present in both rostral and caudal regions with a slighter greater density in caudal regions (not shown). ß-EN inputs onto GnRH somas were significantly greater during AN in both the rostral and caudal population (main effect of season, P < 0.01; Fig. 4). Overall, somas of caudal GnRH neurons received significantly more inputs than rostral GnRH neurons (P < 0.05). For GnRH dendrites, the picture was more complex. Within the caudal population of GnRH neurons, ß-EN inputs onto GnRH dendrites were significantly greater (P < 0.01) during BS (Fig. 5). No significant differences in ß-EN innervation were observed within the rostral GnRH population, (P = 0.17).
Glial association
The plasma membrane of all GnRH neurons was closely associated with glial processes, as evidenced by more than 90% of GnRH immunoreactivity being directly in contact with GFAP-ir fibrils (Fig. 7). In general, dendrites were less heavily associated with glial processes than were somas (P < 0.02). However, dendrites exhibited the most significant seasonal changes (main effect of season, P < 0.001). Dendrites of both rostral and caudal GnRH neurons exhibited a greater degree of glial association during BS, compared with AN (main effect of season, P < 0.01; Fig. 7). By contrast, somas of rostral GnRH neurons exhibited significantly more glial association during AN (P < 0.05); this difference was no longer evident in the caudal population (Fig. 7).
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). This was characterized by a 153% increase in the ratio of GFAP-ir to GnRH-ir (main effect of season, P < 0.01) and a 133% increase in GFAP-ir during BS, compared with AN (main effect of season, P < 0.01; Fig. 8). Neither the size of GnRH somas (Table 2) nor the density of GFAP immunoreactivity between regions (rostral vs. caudal) varied with season (not shown). The density of GFAP-ir in areas immediately adjacent to the GnRH neurons, but devoid of GnRH-ir, also did not exhibit a seasonal variation (Fig. 8); thus, the increase in GFAP-ir was specific to GnRH neurons and their proximal dendrites. Within the areas adjacent to the GnRH-ir, a significant increase in glial filament diameter (53%) was observed in AN ewes, compared with BS ewes (P < 0.01; Fig. 8).
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Discussion |
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Some caution is required in our interpretation of the cause of these changes because animals were not held under constant conditions of photoperiod or temperature; thus, additional factors such as seasonal changes in photoperiod or food consumption and body composition (63) could have contributed to the present endocrine and anatomical findings. However, we feel the latter are unlikely for two reasons. First, our animals were well fed. Second, significant feed restriction is necessary to alter LH secretion in the adult ewe (64). It is also important to keep in mind that ultimately any modulator of GnRH secretion must do so by directly influencing the GnRH neuron at the level of its cell body or terminals in the median eminence. Thus, the rearrangements in neural inputs and glial association described in the present study could reflect one of several processes ultimately involved in the mechanism(s) whereby the long-term seasonal change in GnRH neurosecretion is achieved.
Seasonal variation in GnRH inputs
Changes in total GnRH inputs.
The present study confirms at the light microscopic level what had previously been demonstrated using the electron microscope (21), namely that a seasonal change in total number of inputs to ovine GnRH neurons occurs in the absence of changing gonadal steroid concentrations. Although we cannot confirm the synaptic nature of these changes because the resolution of the system does not allow identification of synaptic specializations, the usefulness of Syn-I immunoreactivity as an indicator of underlying changes in synaptic numbers is not unwarranted. For example, Syn-I is localized to synaptic vesicles within axon terminals (65, 66) and levels of Syn-I immunoreactivity correlate highly with changes in synaptic density within the hypothalamus (67). Despite the limitation in visualizing synaptic specializations, changes in axon terminal numbers were observed in the present study. Similar changes have been shown at the ultrastructural level in sheep; these paralleled those occurring in synaptic input numbers (21). Indeed, the numbers of inputs we report are similar to those made by Xiong et al. (21) previously. Thus, our methods are capable, within the limits of optical resolution, to identify underlying anatomical changes heretofore only described at the electron microscopic level and should make this method useful in a wide range of anatomical studies. in specific GnRH inputs. A substantial literature has elucidated specific roles for various neuromodulators in the control of GnRH output. For example, NPY is well recognized as an important neuropeptide mediating the effects of food intake and metabolic signaling on the reproductive neuroendocrine axis (68, 69), so the changes in NPY innervation may reflect seasonal changes in food consumption or body composition (63). However, the precise effect of NPY on GnRH neurons remains controversial. Based on the current findings that the number of NPY inputs and LH secretion both increase during BS, a stimulatory role is suggested. This is consistent with evidence that NPY is stimulatory to LH secretion in ewes (41, 70), but there are also compelling data supporting an inhibitory role for this neuropeptide (42). It is interesting to note that the numbers of NPY-immunoreactive perikarya also increases in BS ewes (43) so that the increase in NPY-positive inputs may reflect an overall change in NPY-producing neurons.
For GABAergic afferents, only the rostral population of GnRH neurons exhibited a seasonal change. This was also specific to dendrites and was in a direction opposite to that expected; in the BS when estrogen-negative feedback is least, GABAergic innervation is greatest. There are a few possible explanations for this seemingly paradoxical finding. First, we are visualizing changes in only one of the two biosynthetic enzymes that could reflect an inverse relationship to activity. Selective changes in GAD65 and GAD 67 have been reported in sheep (71) and rodents (72). Alternatively, GABA may be serving an excitatory function. Stimulation by GABA in vitro has been demonstrated on numerous occasions (73, 74, 75), and in vivo GABA agonists have been shown to increase GnRH neurosecretion (76). A final possibility is that because of the large percentage of the total GnRH afferents represented by GABA (up to 40%) during BS and AN, GABA input is always sufficient to influence the GnRH system. This would go along well with the multiplicity of roles GABA is thought to play during various portions of the reproductive cycle (36, 76).
The lack of seasonal changes in catecholaminergic inputs to GnRH neurons could be interpreted to indicate an absence of seasonal changes in either DA or noradrenergic (NE) afferents, the latter not being identified specifically in the present study. Moreover, the low number of catecholaminergic inputs raises the possibility that these systems do not directly modulate GnRH significantly. For DA, the present findings fit well with evidence that the activation of A14/A15 DA neurons, rather than a direct response to this catecholamine, increases in AN (29), a finding further strengthened by observations that POA GnRH neurons do not receive direct innervation from A15 neurons (our unpublished observations) (77). Thus, DA neurons appear to act indirectly to modulate GnRH, perhaps via GABAergic neurons (which do change seasonally) as has been shown in the rat (78) and in the present study. A final possibility is that DA projections could be acting on GnRH terminals in the median eminence (79, 80) to affect GnRH release in a seasonal fashion; however, this possibility awaits confirmation.
As noted above, we did not identify NE inputs specifically. It is possible that a portion of the TH-ir inputs to GnRH could have originated from NE neurons in the brain stem. Noradrenergic and DA terminals appear to contact GnRH neurons in sheep (26, 81), and both catecholamines appear to be important modulators of GnRH neurosecretion in this species (82, 83). Further evidence to support a role for noradrenergic modulation of GnRH comes from the demonstration of adrenergic receptors being expressed on GnRH neurons in the rat (84). Yet despite their possible presence, our findings would suggest that noradrenergic systems are not able to influence GnRH in a fashion that involves altering their numbers of inputs onto GnRH neurons. However, as for DA, a possible change in activation of these afferents by estradiol cannot be discounted and indeed may be an important mechanism for regulating GnRH, especially during the estrous cycle (34). A final possibility is that NE could modulate GnRH via corelease at terminals other than those expressing TH-ir. Indeed, considerable evidence suggests overlap or colocalization between catecholamines and NPY in the brain (81, 85). Because the number of NPY inputs onto GnRH neurons exhibited clear seasonal differences, caution is warranted when interpreting the absence of gross seasonal changes in TH-ir inputs to GnRH neurons because the overall number of sites for interaction may have been underestimated.
A complex pattern of innervation by ß-EN systems was revealed in the present study. Because the nature of opioid peptide interactions with the GnRH system appears equally complex in sheep (38, 39, 86, 87), a single explanation for the seasonal anatomical changes is not feasible. For example, during the BS, opioids appear to inhibit GnRH in a progesterone-dependent fashion (38). This system is still responsive during AN because administration of exogenous progesterone is more effective in inhibiting LH secretion at this time of year (88). Thus, an increase in numbers of ß-EN perikarya (43) and numbers of ß-EN inputs onto GnRH somas in AN (present report) may provide a mechanism for the seasonal variation in progesterone negative feedback. However, because progesterone was not present in our ovariectomized ewes, it would not cause these changes. Finally, we also observed seasonal changes in ß-EN input to GnRH dendrites (increase) that did not correlate with this response and thus these changes may reflect other actions of opioids in modulating GnRH secretion (89, 90).
Seasonal variation in glial apposition
We report here that GnRH proximal dendrites were much more heavily associated with glia during BS than AN. This observation is somewhat surprising given their expected role, i.e. to prevent dendritic innervation (18). However, the recent demonstration that astrocytes dramatically increase the number of synapses onto hippocampal neurons and stabilize them (91) suggests a different function for these glial processes. Indeed, a stabilizing role would be ideally suited to the GnRH system in which long-term changes in innervation pattern must be sustained; this would also support our hypothesis that an increase in predominantly stimulatory inputs is present during BS. Why the direction of change in glial association differed between cell bodies and dendrites is not clear, but the former was quite small and only observed in rostral GnRH somas. One possible explanation for this difference is that the glial response varies, depending on the type of input to the GnRH cell. In other words, inputs provided by excitatory or inhibitory transmitters could affect the glial cell’s ability to interact with the GnRH neuron differently. Thus, although GABAergic inputs represent roughly 60% of the inputs to GnRH cell somas, they represent only about 10% of the total inputs onto GnRH dendrites. At least a portion of these unidentified inputs could use glutamate as a neurotransmitter. Given the intimate relationship between glial cells (astrocytes) and glutamatergic terminals, it remains a possibility that the increase in total GnRH inputs we observed reflected an increase in glutamatergic inputs and that the associated increase in glial apposition is necessary as a means of buffering the surrounding neuropil from excess glutamate efflux.
We recently reported that the amount of PSA-NCAM associated with GnRH neurons also increases in BS ewes (48). This, together with our present findings, provides additional support for a stabilizing role of glia. Specifically, these complementary findings indicate that both the increased PSA-NCAM and increased glial association during the BS helps to ensure the long-term stability of synaptic interactions, much like in the magnocellular system of the rat (49). Surprisingly, the changes for both GFAP density and PSA-NCAM are specific to GnRH neurons because neighboring brain regions devoid of any GnRH immunoreactivity showed no seasonal variation. However, certain glial properties in non-GnRH containing brain regions apparently are affected seasonally as is evidenced by the variation in the thickness of glial fibrils. The significance of these latter changes remains unclear but may reflect changes in brain regions containing neurons presynaptic to GnRH neurons. This is concordant with observations made in the rat (20) and suggests a possible common mechanism for modulating GnRH function, both directly and indirectly, among different species. In all, it seems likely that both long-term and short-term responses to estradiol may involve glial and neuronal remodeling.
Seasonal change in net balance between stimulatory and inhibitory inputs
In assessing the possibility of a shift in balance of inputs, it should be emphasized that the four neuromodulatory systems examined in the present study represented nearly 100% of the total number of inputs onto GnRH somas, yet they only accounted for approximately 40% of the total number of dendritic inputs. It remains to be determined whether these unidentified inputs to GnRH dendrites also undergo seasonal reorganization, but this seems likely given the changes in total number of inputs. Regardless, for both somas and dendrites the increase in total numbers of inputs supports the hypothesis that a net increase in stimulatory inputs corresponds with the increase in GnRH output during BS. As indicated above, these unidentified inputs may contain glutamate as a neurotransmitter. Indeed, the evidence implicating glutamate as a neurotransmitter in the control of GnRH release is extensive (for review see Refs.92, 93, 94). However, much of this evidence is based on indirect measures. Despite the failure by GnRH neurons to respond to the neurotoxic effects of N-methyl-D-aspartate or kainic acid and failure to express glutamate receptors (95), the recent description of numerous glutamatergic appositions onto GnRH neurons in the rat (96) indicates the interactions between these two systems may be more prominent than previously thought. Although we have been unsuccessful in identifying glutamatergic terminals directly onto GnRH neurons using immunocytochemistry, this may change given the availability of antibodies to markers against presynaptic glutamate transporters.
For caudal GnRH somas and dendrites, the BS increase in input density (5-fold) is due primarily to NPY afferents. Together with the decrease (somas) or only modest increase (dendrites) in ß-EN afferents and no overall change in GABAergic or catecholaminergic inputs, this yields a presumptive gain of stimulatory tone during BS. Conversely, for rostral GnRH somas a preponderance of GABAergic afferents might be expected to yield a presumptive gain in inhibitory tone (unless GABA is stimulatory). The possibility thus arises that rostral and caudal populations of GnRH subserve different functions, related to different phases of the annual reproductive cycle or to different stages of the estrous cycle. One such possibility supported by the present anatomical findings and by physiological experiments attributes the generation of the GnRH/LH surge to more rostrally located GnRH neurons (97), whereas those in the caudal hypothalamic regions serve to maintain tonic LH release (86).
In summary, this study provides a dramatic example of seasonal plasticity within the GnRH system of the ewe and presents, for the first time in any seasonally breeding mammal, compelling evidence that specific neural inputs as well as glia both modulate GnRH neurons. This plasticity, whether generated endogenously or influenced by environmental and hormonal signals, may be antecedent to changes in GnRH neurosecretory activity exhibited at different times of the sidereal year.
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All procedures were approved by the West Virginia Institutional Animal Care and Use Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Abbreviations: AN, Anestrus; ß-EN, ß-endorphin; BS, breeding season; DA, dopamine; GABA, -aminobutyric acid; GAD, glutamate decarboxylase; GFAP, glial fibrillary acidic protein; ir, immunoreactivity; NE, noradrenergic; NPY, neuropeptide Y; PB, phosphate buffer; POA, preoptic area; PSA-NCAM, polysialylated form of neural cell adhesion molecule; Syn-I, Synapsin-I; TH, tyrosine hydroxylase.
Received December 31, 2002.
Accepted for publication April 16, 2003.
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-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons. Mol Endocrinol 16:2872–2891-aminobutyric acid B agonist reverses the negative feedback effect of testosterone on gonadotropin-releasing hormone and luteinizing hormone secretion in the male sheep. Endocrinology 141:3940–3945This article has been cited by other articles:
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