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Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine (J.-J.X., M.N.L.), Cincinnati, Ohio 45267-0521; and the Reproductive Sciences Program, University of Michigan (F.J.K.), Ann Arbor, Michigan 49109
Address all correspondence and requests for reprints to: Dr. Michael N. Lehman, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ultimately, the central mechanism upon which the circannual system acts consists of neurons that synthesize and secrete the decapeptide, GnRH. GnRH cells and their projections to the median eminence comprise a final common pathway controlling the secretion of pituitary gonadotropins (15, 16) and play a pivotal role not only in seasonal breeding, but also in neuroendocrine mechanisms underlying puberty and the estrous cycle (1, 17). The major factor responsible for seasonal anestrus in sheep is a striking increase in the ability of estradiol to inhibit pulsatile GnRH and LH secretion (18, 19). In view of these seasonal changes in the responsiveness of GnRH neurons to estradiol negative feedback, it is important to note that the overwhelming majority of GnRH neurons in the sheep (20, 21), like those in other mammals (22, 23), appear to lack estrogen receptors. Therefore, the heightened response to estradiol negative feedback that leads to anestrus is probably conveyed to GnRH neurons via afferents from other neurons. The view that seasonality is not due to fundamental changes in GnRH neurons, but, rather, to changes in afferent neurons that regulate GnRH neurosecretion, is supported by studies in seasonally breeding rodents that have failed to reveal any striking seasonal changes in GnRH peptide or messenger RNA content (24, 25, 26).
In preliminary studies, we found evidence suggesting that structural plasticity may underlie seasonal reproductive transitions in the sheep (27). This hypothesis was initially suggested to us by elegant studies documenting seasonal rearrangements in the songbird brain (28, 29) as well as by evidence of steroid-induced plasticity in the mammalian preoptic area and hypothalamus (30, 31). Our initial seasonal comparisons of GnRH cells in the ewe, at the light microscopic level, provided evidence that immunostained GnRH neurons have longer and more numerous dendrites during anestrous than during the breeding season (32). However, it was unclear whether these changes reflected alterations in the content or localization of immunodetectable GnRH, rather than actual morphological rearrangements. In fact, our preliminary electron microscopic observations revealed that despite the shorter length of their dendrites, GnRH neurons during the breeding season receive approximately twice the mean number of synaptic input per unit of plasma membrane as do GnRH cells during anestrus (27). Because our preliminary observations in ewes were made in animals with intact gonads, it was not certain whether these seasonal alterations were due to changing levels of endogenous gonadal hormones or might reflect the influence of photoperiod or the intrinsic seasonality of the brain. In the present study, we distinguished between these possibilities by analyzing the synaptic inputs on GnRH cells in both intact ewes and ovariectomized ewes bearing implants that maintain fixed steroid levels (OVX+E). The use of OVX+E animals allowed us to eliminate a changing hormonal milieu as a factor as well as to monitor changes in the responsiveness of LH secretion to estradiol negative feedback as an indicator of seasonal reproductive transitions (33). As in our previous study, we focused on GnRH cells in the preoptic area, the site of the majority of GnRH neurons in this species (32, 34). In addition to examining preoptic GnRH neurons, we quantified the synaptic inputs to neighboring unidentified neurons in the same sections.
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Materials and Methods |
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Ewes were anesthetized with sodium pentobarbital and perfused bilaterally via the carotid arteries with 6 liters 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, with 0.1% sodium nitrite added to this fixative as a vasodilator. The first 300–400 ml fixative perfused through the carotids also contained a higher concentrated heparin solution (1000 U/ml); heparin was added to the rest of the fixative in a lesser concentration (100 U/ml). After perfusion, the brain and attached pituitary were carefully removed from the cranium. The preoptic area and hypothalamus were dissected out, placed in the same fixative for an additional 24 h, and thereafter stored in 0.1 M phosphate buffer, pH 7.3, at 4 C. The ovaries of all intact animals were carefully examined at the time of perfusion for the presence of corpora lutea and/or corpora albicantia to confirm their reproductive status.
Immunocytochemistry
The brains were blocked, and the preoptic area and hypothalamus
were embedded in an egg yolk-gelatin matrix to facilitate Vibratome
(Ted Pella, Inc., Reading, CA) sectioning. Egg-gelatin-embedded blocks
were cut coronally on a Vibratome at 60–80 µm. Sections were
collected and washed several times in phosphate buffer, then treated
with 0.5% hydrogen peroxide to reduce endogenous peroxidase activity.
The primary antiserum against GnRH used in this study (LR-1, generously
provided by Dr. Robert Benoit) recognizes amino acids 3, 4, 7, 8, 9,
and 10 of the decapeptide and no other identified neuropeptide.
Sections were incubated for 48–72 h at 4 C in primary antiserum
diluted 1:10,000 in phosphate buffer with 0.02% saponin and 1% normal
goal serum. Sections were then treated sequentially with a biotinylated
goat antirabbit IgG diluted 1:200 in phosphate buffer and an
avidin-biotin-horseradish peroxidase complex diluted 1:1:250 (Elite
Kit, Vector Laboratories, Burlingame, CA). Each step was applied for
1 h at room temperature. The horseradish peroxidase was
demonstrated using diaminobenzidine, and the glucose oxidase method was
used for generating the hydrogen peroxide substrate (37).
Regions containing immunoreactive cells in the medial preoptic area were then dissected out. These tissue pieces were postfixed in 2% osmium tetroxide containing 1.5% potassium ferricyanide (38), dehydrated, and flat embedded in Epon 812. Semithin sections (1 µm) were cut and examined under the light microscope for the presence of immunoreactive cells. GnRH cells, which are dispersed and scattered in the preoptic area (32), appeared as isolated cells in the semithin sections and camera lucida drawings made to record their location before ultrathin sectioning. Ultrathin (70 nm) sections were cut from the remaining block, mounted on formvar-coated grids, and stained with uranyl acetate and lead citrate (39).
Data analysis
Between 8–14 GnRH cells and 4–10 unidentified neurons were
analyzed from each ewe (Table 1
). GnRH cells selected
for analysis were located in the preoptic area at the level of the
organum vasculosum of the lamina terminalis, and unidentified cells
selected for analysis were located within 30–40 µm of individual
GnRH cell bodies. A single thin section was analyzed per cell so that a
given cell was only analyzed once. Sections were examined with a JEOL
100S electron microscope (JEOL, Peabody, MA), and a series of
overlapping, low power electron micrographs (x4,000) was taken to
record each GnRH and unidentified cell and their processes. Synapses
were defined by the presence of synaptic densities and/or a synaptic
cleft. Electron micrographs were taken at higher power (x20,000) of
each synapse to confirm the presence of synaptic modifications.
However, as neuropeptides may be exocytotically released at nonsynaptic
sites (40), we also analyzed vesicle-containing terminals that were in
direct membrane contact with GnRH cells but in which synaptic
modifications were not evident. Low power electron micrographs from
each cell were analyzed using a Jandel Scientific digitizer pad and
SigmaScan software (Jandel Scientific, Sausalito, CA) run on an IBM PC
computer (IBM Corp., Boca Raton, FL). For each cell, we measured 1) the
total length of somatic and dendritic plasma membranes, 2) the total
length of direct contacts between apposed axon terminals and somas or
dendrites, and 3) the total length of synaptic modifications. The
boundary between somas and dendrites, which is often indistinct in GnRH
neurons (41), was arbitrarily defined as a radial distance of 1 µm
from the nuclear envelope. Using this boundary, we also measured the
soma area (square microns) for each GnRH neuron. Portions of GnRH
dendrites not attached to the cell body were also analyzed; because of
the scattered nature of the preoptic GnRH cell population and the
identification of these cells at a light microscopic level before
ultrathin sectioning, dendrites were able to be unambiguously assigned
to a given GnRH cell. The identification of immunoreactive elements,
synaptic specializations, and the defined boundaries between somas and
dendrites, were verified by two independent observers.
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The total number of cells examined and the mean length of plasma
membrane analyzed per cell are presented in Table 1
. Differences among
groups were tested for significance at P < 0.05 using
three-way (GnRH vs. unidentified neurons, intact
vs. OVX+E, breeding season vs. anestrus) ANOVA.
In addition, a two-way ANOVA was used for seasonal and ovarian-based
analyses involving only GnRH or unidentified neurons (Figs. 6 and 7).
Post-hoc analyses were performed using Sheffe’s F test.
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Results |
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GnRH perikarya: ultrastructural features
Immunoreactive perikarya in the medial preoptic area were easily
identified in thick (70-µm) and semithin (1-µm) sections. At an
ultrastructural level, GnRH perikarya and processes were recognized by
diaminobenzidine reaction product, which was unevenly distributed
throughout the cytoplasm of GnRH neurons, primarily associated with the
rough endoplasmic reticulum and neurosecretory granules 80–120 nm in
diameter (
Figs. 1–4). Reaction product was also found
over the nuclei of some, but not all, GnRH neurons (Figs. 2 and 3); there were no
obvious differences among the four groups of animals in the presence of
nuclear reaction product.
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), proximal dendrites
(Fig. 5B), and small dendritic spines and protuberances (Fig. 5C).
These terminals formed synapses that were defined by the presence of
either a synaptic cleft or synaptic density (Fig. 5, arrows). In many instances, axon terminals that at lower
magnification appeared to be in close association with GnRH somas or
dendrites were found at higher power to be separated from
immunoreactive somas and dendrites by glial elements (Fig. 5D).
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, A–C). Synaptic contacts exhibited
either symmetric (Fig. 5B) or asymmetric (Fig. 5C) densities.
Seasonal comparisons
GnRH neurons in both intact and OVX+E ewes showed significant
seasonal differences in their degrees of innervation. The mean
percentage of GnRH plasma membrane (both somatic and dendritic) bearing
synaptic modifications was reduced in both intact and OVX+E groups of
anestrous ewes compared to that in animals perfused in the breeding
season (Fig. 6A). This difference was not simply due to
a reduction in the length of individual synaptic modifications, as the
mean length of synaptic modifications (either synaptic clefts or
densities) did not significantly differ between groups (Fig. 6B).
Accordingly, there were also significant seasonal differences in the
mean number of synapses (Fig. 6C) or axon terminals (Fig. 6D) per 10
µm plasma membrane of GnRH neurons. Within season, there was no
significant influence of ovarian status on any of the aforementioned
measures.
Although nonidentified preoptic neurons received significantly greater
numbers of synapses per 10 µm plasma membrane than adjacent GnRH
neurons (F = 82.18; P < 0.0001, by three-way
ANOVA), there were no significant differences among unidentified
neurons based on season or ovarian status in either the mean percentage
of plasma membrane bearing synaptic modifications (Fig. 7A), the mean synaptic length (Fig. 7B), or the mean
number of synapses (Fig. 7C) or terminals (Fig. 7D) per 10 µm plasma
membrane.
In general, the dendrites of GnRH neurons received heavier innervation
than their somas (Table 2). Although seasonal
differences in the mean number of synapses per 10 µm plasma
membrane were seen for both dendritic and somatic plasma membranes,
these differences were more pronounced for axodendritic (F =
32.29; P < 0.001) than for axosomatic synapses (F
= 7.08; P < 0.05). The seasonal difference in
axosomatic inputs onto GnRH neurons was not associated with a
significant change in GnRH soma size; this did not differ among the
groups (Table 2). Finally, there were no significant differences in the
mean number of axodendritic or axosomatic inputs based on ovarian
status, although GnRH somas tended to be less densely innervated in
OVX+E ewes than in intact animals (F = 3.872; P =
0.07).
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Consistent with our previous observations, GnRH perikarya and
dendrites in the preoptic area were frequently ensheathed by glial
processes. Glial elements surrounding GnRH neurons most often appeared
as multiple, thin lamellae wrapped around immunoreactive perikarya and
dendrites (Figs. 3 and 4). Although we did not quantify the degree of
glial ensheathment, GnRH cells of both intact and OVX+E anestrous ewes
appeared to be more completely surrounded by glial processes (Figs. 3 and 4) than GnRH cells of either intact or OVX+E breeding season ewes
(Figs. 1 and 2). In contrast to the extensive degree of glial
ensheathment seen in GnRH cells, nearby nonimmunoreactive neurons were
rarely surrounded by glial processes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seasonal changes in GnRH neurons in this study were seen not only in intact animals, replicating our preliminary findings (27), but also in ovariectomized ewes bearing implants that maintained constant levels of estradiol. Consequently, these seasonal alterations in GnRH cells are unlikely to be due to changing levels of endogenous sex steroids, but may instead reflect changes in environmental photoperiod and/or the expression of an endogenous circannual rhythm (8, 9). Our observations, therefore, differ from one of the best studied examples of seasonal plasticity in the central nervous system, namely that of seasonal alterations in the ultrastructure and size of forebrain nuclei controlling song in canaries (28, 44). Unlike the changes described here, seasonal alterations in the number of synaptic inputs within nucleus robustus archistriatalis appear to be regulated by endogenous changes in sex steroids, although some seasonal differences in ultrastructural features persist in canaries treated chronically with testosterone implants (29). Interestingly, recent findings indicate that seasonal changes in the rate of cell proliferation in the adult canary telencephalon are independent of gonadal hormones (45).
Based on the observation of an increased number of synaptic inputs onto GnRH cells per unit membrane during the breeding season, we envision two possibilities: 1) new synapses are being formed; or 2) there is a decrease in the size of these cells resulting in less membrane but the same number of synapses. Our earlier light microscopic findings would seem to favor the latter interpretation because preoptic GnRH neurons appeared to possess fewer and shorter immunostained processes during the breeding season than in anestrous (32). However, changes in the appearance of immunostained processes at a light microscopic level may simply reflect the decreased content of immunoreactive peptide, rather than true morphological rearrangements. Furthermore, in the present study we found no significant seasonal changes in GnRH soma size, even though the mean number of axosomatic synapses increased in breeding season ewes. Thus, although changes in dendritic length could conceivably contribute to alterations in axodendritic inputs, our evidence suggests that new synapses are formed onto GnRH neurons during the breeding season at least at the level of their somas.
If the alterations we observed in synaptic inputs onto GnRH neurons are due to the expression of an endogenous circannual rhythm, then manipulations that prevent reproductive transitions by acting on the endogenous process itself should block these morphological changes as well. One example of such a manipulation in birds and sheep is thyroidectomy (46, 47). Evidence suggests that the endogenously driven reproductive transition from the breeding season to anestrus is dependent on the presence of thyroid hormone and that this dependence is expressed at the level of the GnRH neurosecretory system (47). Thyroidectomized ewes, unlike intact animals, fail to exhibit a seasonal decline in episodic secretion of GnRH into hypophyseal portal blood and thus fail to enter the nonbreeding season (48). T4 replacement reverses this effect and permits thyroidectomized ewes to exhibit an endogenously driven transition from the breeding season to anestrus (49). The potential involvement of the thyroid in seasonal structural plasticity is of particular interest because the presence of thyroid hormone is essential for the normal morphological maturation of the central nervous system (50, 51).
In fact, changes in synaptic input to GnRH cells accompanying seasonal reproductive transitions have recently been reported in starlings (52), a species in which thyroid hormones are permissive to seasonal changes (46). There was an increase in the number of axosomatic inputs to GnRH cells in birds examined several months after becoming photorefractory to long day lengths, but no increase at the time of gonadal regression, when GnRH activity would be expected to be reduced. However, this study did not quantitatively assess axodendritic inputs onto GnRH cells or inputs to non-GnRH cells and examined only gonadally intact animals. Therefore, it is not possible to tell whether the photoperiodic changes observed in starlings were due to differences in gonadal steroids or to an intrinsic seasonal plasticity of the GnRH system.
The direction of the seasonal difference in synaptic inputs we observed was opposite that reported in starlings (52) or to what we might have expected on the basis of the heightened responsiveness of GnRH cells to estradiol negative feedback during anestrus (19). As estradiol negative feedback is probably conveyed to GnRH cells via afferents, an increase in the number of synaptic inputs to GnRH cells of anestrous ewes might be expected rather than a decrease. It is likely, however, that GnRH neurons that are contacted by a variety of neurochemical systems (53) receive a mix of both inhibitory and excitatory inputs. One possibility, consistent with the direction of the seasonal change we observed, would be that during anestrus there is a decrease in the number of excitatory inputs onto GnRH neurons and that unopposed inhibitory inputs predominate. In fact, although we did find a small subset of presynaptic terminals that contained pleomorphic vesicles, this type of input to GnRH neurons was only observed in the brains of breeding season ewes and not in anestrous ewes. As pleomorphic vesicles are generally thought to characterize inhibitory inputs (54, 55), this observation is consistent with the possibility that there is, in fact, less inhibitory input to GnRH neurons in anestrus. However, inhibitory GABAergic terminals in the hypothalamus almost always contain round vesicles (56), suggesting that the absence of flattened vesicles cannot be used as a simple criterion to define the excitatory nature of a presynaptic axon terminal. Furthermore, it is possible that the structural changes reported here may be unrelated to seasonal shifts in the negative feedback responsiveness to estradiol, but instead subserve other functions of GnRH neurons. For example, there are seasonal changes in the ability of estradiol to induce sexual receptivity in ewes (57, 58). If GnRH modulates estrous behavior in the ewe as it does in the rat (59), then the increased amount of synaptic input to GnRH neurons during the breeding season may serve to potentiate this role.
Seasonal changes in the synaptic inputs onto GnRH cells are likely to involve interactions between presynaptic terminals and not only postsynaptic GnRH somas and dendrites, but also glial cells. Glial elements play a pivotal role in many examples of plasticity and reinnervation in the central nervous system (60) and are intimately involved in structural plasticity involving the magnocellular neuroendocrine system at the level of the supraoptic nucleus and posterior pituitary (61, 62, 63). In the rhesus monkey, ovariectomy results in a significant increase in the degree of glial ensheathment of GnRH cells and a concomitant decrease in their innervation (64). GnRH neurons in anestrous ewes are frequently ensheathed by glial processes that separate them from potential inputs (41). Although we did not quantitatively compare their degree of glial ensheathment, the seasonal changes we observed are consistent with our impression that GnRH neurons in anestrous ewes tended to be more completely surrounded by glial processes than GnRH cells in breeding season animals.
In summary, these observations represent the first evidence that seasonality may induce morphological changes in the central nervous system that are not primarily due to changing levels of sex steroids, but instead may be either driven by changing environmental photoperiod and/or reflect the operation of an endogenous circannual pacemaker. Although the present observations were made at the level of GnRH cell bodies in the preoptic area, we also observed significant morphological rearrangements in GnRH neurons during the estrous cycle at the level of their terminals in the median eminence (65). Rearrangements in GnRH terminals in the median eminence have also been shown to occur after gonadectomy in the adult rat (66). Together with evidence of age-related changes in synaptic inputs to GnRH neurons in male rats (67) and steroid-associated alterations in monkeys (64), these results provide mounting evidence that the GnRH neuroendocrine system is capable of significant plasticity in the adult brain. Although the importance of such structural changes to the function of the GnRH neurosecretory system is not yet known, it is important to note that these findings of seasonal plasticity fit well with recent observations that there are seasonal differences in episodic GnRH secretion in both ovary-intact ewes (68, 69) and ovariectomized ewes bearing steroid implants (19).
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Acknowledgments |
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Footnotes |
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Received August 16, 1996.
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