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Endocrinology Vol. 147, No. 10 4843-4851
Copyright © 2006 by The Endocrine Society

Morphological Plasticity in the Neural Circuitry Responsible for Seasonal Breeding in the Ewe

Van L. Adams, Robert L. Goodman, A. K. Salm, Lique M. Coolen, Fred J. Karsch and Michael N. Lehman

Departments of Physiology and Pharmacology (V.L.A., R.L.G.) and Neurobiology and Anatomy (A.K.S.), West Virginia University Health Sciences Center, Morgantown, West Virginia 26506; Reproductive Sciences Program (F.J.K.), Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor Michigan 48109; and Department of Anatomy and Cell Biology (L.M.C., M.N.L.), University of Western Ontario, London, Ontario N6A 5C1, Canada

Address all correspondence and requests for reprints to: Dr. Robert L. Goodman, Department of Physiology and Pharmacology, PO Box 9229, West Virginia University, Morgantown, West Virginia. E-mail: bgoodman{at}hsc.wvu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An increase in the response of GnRH neurons to estrogen negative feedback is responsible for seasonal anestrus in the ewe, but the underlying neural mechanisms remain largely unknown. Neural plasticity may play an important role because the density of synaptic input to GnRH neurons changes with seasons. Moreover, the transition from breeding to anestrous season requires thyroid hormones, which are also required for neuronal development. In the first experiment, we examined whether the decrease in synapses on GnRH neurons is critical for the transition to anestrus by comparing synaptic input in thyroidectomized and thyroid-intact controls, using electron microscopic analysis. Thyroidectomized ewes remained in the breeding season, but the number of synaptic contacts on their GnRH cells was not different from those in thyroid-intact ewes that were anestrus. The next experiment tested whether there was a seasonal change in morphology of the A15 dopaminergic neurons that mediate estrogen negative feedback during anestrus by analyzing synapsin-positive close contacts onto A15 neurons with confocal microscopy. There was a 2-fold increase in these close contacts onto dendrites of A15 neurons in anestrus and a corresponding increase in the length of A15 dendrites at this time of year. The increase in dendritic length was blocked by thyroidectomy, but this procedure did not significantly affect synaptic input to A15 neurons. These results provide initial evidence that the seasonal change in synapses on GnRH neurons is not sufficient for the transition into anestrus but that plasticity of the A15 dopaminergic neurons mediating estrogen negative feedback may contribute to this seasonal alteration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY BIRDS AND mammals show annual fluctuations in fertility that result from seasonal changes in GnRH secretion (1). There is increasing evidence that in several species (2, 3, 4), the seasonal transition from breeding to nonbreeding seasons is dependent on thyroid hormones. Thus, thyroidectomy (THX) late in the breeding season prevents the onset of infertility, an effect that can be reversed with thyroid hormone replacement (5, 6, 7). In light of the well-known role of thyroid hormones in morphological development of the brain (8), these data raise the possibility that seasonal changes in GnRH secretion reflect morphological alterations in the neural circuitry controlling GnRH release. Seasonal plasticity is well established for the neural areas controlling singing in canaries and other songbirds (9). In a shorter time frame, there is also strong evidence in mammals that morphological alterations accompany physiological changes in secretion of oxytocin (10), vasopressin (11), and GnRH during the estrous cycle (12) and after ovariectomy (13).

Recent work in the ewe has identified some of the key components of the neural system controlling seasonal breeding in this species and opened the door to determining whether they show seasonal plasticity. Changes in sensitivity to estradiol (E2) negative feedback on tonic LH secretion drive seasonal breeding in the ewe (14, 15), and it is now clear that the A15 dopaminergic (DA) cell group in the retrochiasmatic area (RCh) plays a central role in this response (15, 16). These neurons are responsive to E2 during anestrus (17, 18, 19), when this steroid strongly inhibits GnRH secretion, but not during the breeding season (18) when E2 is a weak inhibitory steroid. Lesion studies have demonstrated that A15 neurons mediate E2 negative feedback during anestrus (20, 21). Although E2 stimulates their activity in anestrus, these DA neurons do not contain estrogen receptor-{alpha} (22, 23) or -ß (24), so that estrogen-responsive neural afferents to the A15 are likely to be involved in this system. Recently, two such groups have been identified in the ventromedial preoptic area (vmPOA) and the RCh. Estrogen receptor-{alpha}-containing neurons in each project to the A15 (25), and local administration of E2 to the vmPOA (26) or RCh (27, 28) during anestrus inhibits LH secretion via a DA system. Thus, we have proposed that a neural circuit consisting of estrogen-responsive neurons in the vmPOA and RCh, which project to inhibitory A15 DA neurons afferent to GnRH cells, mediates E2 negative feedback in anestrus and that this circuitry is not functional during the breeding season.

One mechanism for the loss of response to E2 during the breeding season would be disruption of the synaptic connections comprising this circuit, thus producing a decrease in input to either GnRH neurons or DA cells in the A15 in breeding-season ewes. There is evidence for seasonal plasticity in input to GnRH neurons, but it is the opposite of that predicted by this simple analysis. Specifically, electron microscopic studies have demonstrated more synaptic contacts onto ovine GnRH perikarya (29) in the breeding season than in anestrus. Confocal microscopic analysis has confirmed this observation (30), and examination of specific neuronal phenotypes suggested there may be a net increase in stimulatory synapses during the breeding season (30, 31). Although these studies demonstrate seasonal plasticity in the input to GnRH neurons in the ewe, the physiological significance of these alterations remains to be determined, and no work has examined seasonal plasticity in the input to A15 DA neurons.

In the first experiment of this study, we addressed the physiological significance of the seasonal changes in afferents to GnRH neurons. In the second experiment, we determined whether there is a decrease in synaptic input to A15 DA neurons in the breeding season, and in the third experiment, we addressed the functional significance of the changes observed. To examine the relevance of seasonal morphological changes in A15 DA and GnRH neurons, we took advantage of the observations that thyroid hormones are necessary for the seasonal transition to anestrus. We reasoned that if this plasticity plays a critical role in seasonal breeding, then THX should block the morphological changes that occur in A15 DA and GnRH neurons between the breeding and anestrous seasons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal handling and surgical procedures
Mature, black-faced ewes of mixed breeding showing regular estrous cycles were maintained in an open barn that allowed exposure to ambient photoperiod and temperature at latitude 42° 18'N (University of Michigan; experiments 1 and 3) or at latitude 39° 38'N (West Virginia University; experiment 2). All animals had free access to water and were fed hay (experiments 1and 3) or a daily silage allowance (experiment 2). Ewes were moved indoors 1–3 d before all procedures and housed under artificial lighting adjusted so that photoperiod was similar to that outdoors.

THX were performed with aseptic techniques using gas anesthesia as previously described (5) and ovariectomies (OVX) were performed via midventral laporatomy using pentobarbital anesthesia. Physiological E2 concentrations were produced in OVX ewes by the sc insertion of a SILASTIC brand (Dow Corning, Midland, MI) implant containing crystalline E2, using two well-established estrogen treatments. In experiment 1, we employed a chronic treatment (e.g. several months) with 3-cm-long implants (14) because this model had been used in previous work demonstrating seasonal changes in synaptic contacts on GnRH neurons (29, 30). In experiments 2 and 3, a more acute treatment with shorter E2 implants that had produced seasonal changes in activity of A15 neurons (18) was used. Blood samples were collected by jugular venipuncture and allowed to clot overnight, and serum was harvested and stored at –20 C until assayed. All procedures involving animals were approved by the University of Michigan (experiments 1 and 3) or the West Virginia University (experiment 2) Animal Care and Use Committee.

Experimental protocols
Experiment 1: does THX prevent the decrease in synaptic input to GnRH neurons that occurs between the breeding season and anestrus?
Sixteen chronically OVX ewes were treated with 3-cm-long SILASTIC brand implants containing E2 in mid-breeding season (November). Eight of these animals were thyroid-intact controls, and the other eight had been THX for previous studies [surgical completeness confirmed by undetectable T4 concentrations (<1.6 ng/ml) in jugular blood samples]. All animals were killed in the middle of anestrus (May), and tissue was collected for electron microscopic (EM) analysis (see below). Serum LH was measured in daily samples collected the week before euthanasia to assess reproductive status.

Experiment 2: are there seasonal changes in the synaptic input to and/or morphology of A15 DA neurons?
In this experiment, we used confocal microscopy to analyze tissue sections processed using dual immunocytochemistry for tyrosine hydroxylase (TH), as a marker for DA neurons, and synapsin I, as a marker for synaptic vesicles (32). Confocal microscopy allows a larger number of neurons to be examined for putative synaptic input than definitive EM identification of synapses, thus increasing the likelihood of sampling a statistically valid population. However, this approach cannot demonstrate the membrane specializations that identify actual synapses. Therefore, we first validated this approach using EM analysis after a dual-label preembedding technique (33) in which nickel-enhanced diaminobenzidine and unenhanced diaminobenzidine were used as labels for TH and synapsin I, respectively (Fig. 1Go). Close contacts were initially identified at the light level, making use of the color differences of these two chromogens. EM examination of thin sections of these close contacts from 12 TH-positive A15 neurons, as well as five adjacent non-TH neurons, demonstrated that every synapse contacting these cells identified at the EM level also contained synapsin I. For the main experiment, we examined synapsin-positive close contacts on DA neurons in the anestrous and breeding season using tissues collected from a previously described experiment (34). Briefly, anestrous (n = 6) and breeding-season (n = 8) ewes were OVX, and 3 wk later, half the animals in each group received a 0.5-cm-long E2 implant inserted sc. After 1 wk of E2 treatment, blood samples were collected every 12 min for 4 h to assess LH pulse patterns, and then tissue was collected for light microscopic analysis (see below).


Figure 1
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  		FIG. 1. Synapsin-positive terminals at the EM level. A, Synapsin-positive axon terminals (asterisks) contacting an unidentified dendrite (d) in the A15 region of an anestrous ewe; B, synapsin-positive terminals (at) contacting a portion of a TH-containing dendrite (d) in A15 region of the same animal. Magnification, approximately x20,000 (A) and x40,000 (B).

 
Experiment 3: is the seasonal plasticity in A15 DA neurons dependent on thyroid hormones?
Previous work using intracerebroventricular (icv) administration of T4 demonstrated that thyroid hormones act in the brain to allow the transition to anestrus (35). In this experiment, we used tissue collected from some of these animals to determine whether icv administration of T4 is sufficient for the seasonal changes in synaptic input and morphological changes in A15 DA neurons. Briefly, for the earlier study, ewes were OVX and THX in late November and early December and fitted with a chronic cannula into one lateral ventricle; 3-cm-long E2 implants were inserted sc at the time of OVX. T4 (9.6 µg/d; 100 µg/ml solution) or vehicle was then constantly infused icv from December 23 to April 4. This period spans the transition from breeding season to anestrus in thyroid-intact controls. Ewes receiving T4 icv showed the fall in LH concentrations indicating the onset of anestrus, whereas the ewes receiving vehicle did not (35). At the end of this earlier experiment, T4 treatment was stopped and E2 implants removed in six ewes from each group. At this time, estrogen implants were also removed from thyroid-intact controls (n = 6) from another experiment that had been OVX and given an E2 implant in August. Because T4 removal does not affect reproductive status once ewes have entered anestrus (36), this protocol resulted in three groups with different reproductive and thyroid axes at the time of tissue collection in June: 1) six thyroid-intact ewes were anestrus with normal T4 levels, 2) six THX ewes given T4 icv were anestrus with low T4 levels, and 3) six THX ewes given vehicle icv were breeding season with low T4 levels. A 0.5-cm-long E2 implant was then placed sc into three ewes in each group on May 28, and the animals were then killed 7 d later and tissue collected for light microscopic analysis. LH concentrations were monitored in jugular samples collected on d –2 and –1 before euthanasia.

Tissue collections
For EM analyses, ewes were anesthetized (approximately 1 g sodium pentobarbital iv) and intracranially perfused via both carotid arteries with 4% paraformaldehyde containing 0.2% glutaraldehyde and heparin as previously described (29). The preoptic and hypothalamic tissue block was dissected out, incubated in fixative at 4 C overnight, and then stored in 0.01 M phosphate buffer (PB) at 4 C. Coronal sections (50 µm) through both the POA and the medial basal hypothalamus were cut on a vibratome and stored at –20 C in a cryopreservative (37) solution for 3–6 months until processed for EM immunocytochemistry. Perfusion-fixed sections can be stored in cryopreservative for up to 1 yr without adversely affecting immunostaining for GnRH.

For light microscopic analyses, ewes were injected before perfusion twice (10 min apart) with 25,000 U heparin iv, followed by sodium pentobarbital (about 3 g iv). When the animal had stopped breathing, we quickly removed its head and perfused the brain via both internal carotid arteries with 6 liters of 4% paraformaldehyde in 0.1 M PB containing 10 U heparin/ml and 0.1% sodium nitrite. A block of tissue containing preoptic and hypothalamic tissue was dissected out, incubated in the same fixative overnight (4 C), and then infiltrated with 30% sucrose in PB. Thick coronal sections (50 µm) were cut using a freezing microtome and stored at –20 C in cryopreservative until processed for dual immunocytochemistry.

Hormonal and immunocytochemical procedures
LH concentrations were measured in duplicate 200-µl aliquots of serum by RIA as previously described (38); assay sensitivity averaged 0.6 ng/ml (NIH S24), and inter- and intraassay coefficients of variation of a pool that produced 60% displacement of iodinated LH were 11 and 21%, respectively. T4 was measured in selected samples to confirm ewes were THX using a commercially available kit (39) with a sensitivity of 1.6 ng/ml.

For EM visualization of synapses onto GnRH neurons, sections through the medial POA, the site where we previously observed seasonal changes (29), were processed for GnRH immunocytochemistry using a rabbit polyclonal antibody (1:10,000; 48 h at 4 C; LR-1, gift from R. Benoit) and a modified avidin-biotin-immunoperoxidase procedure as previously described (29). From each animal, 15–20 small pieces of tissue containing GnRH neurons were microdissected out of the sections by hand, postfixed, dehydrated, and flat embedded in plastic for EM processing. GnRH neurons were again visualized at the light microscopic level in thick (1-µm) sections, and semi-thin serial sections (70 nm) were cut to analyze these cells at an EM level. In addition, unidentified POA neurons located within 30–40 µm of individual GnRH cells were analyzed. Thin sections were examined with a JEOL 100CX electron microscope, and a series of overlapping, low-power electron micrographs (x4000) were taken to record each GnRH and unidentified neuron and their processes.

For light microscopic analysis, three sections encompassing the A15 area (experiment 2) or from the caudal portion of it (experiment 3) were selected from each animal to stain using a dual-fluorescence procedure for TH and synapsin (rostral A15 tissue from experiment 3 ewes had been used for other studies). Tissues from all treatment groups in each experiment were processed simultaneously; all incubations were at room temperature unless otherwise specified. After a 3-h wash in PB, sections were incubated in PB containing 0.2% Triton X-100 and 4% normal donkey serum (PBTX-NDS) for 1 h. They were then coincubated for 48 h at 4 C with 1:200 mouse monoclonal antibody against TH (Boehringer Manheim, Indianapolis, IN) and 1:800 rabbit polyclonal antibody against bovine brain synapsin I (Molecular Probes, Eugene, OR) in PBTX-NDS. After three 15-min washes in PB, tissues were sequentially incubated in PBTX-NDS with biotinylated donkey antimouse (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:200, 1 h), donkey antirabbit-CY2 (Jackson; 1:100, 30 min), and then CY3-conjugated streptavidin (Jackson; 1:200, 30 min). The sections were washed three times for 15 min and then mounted on gelatin-coated slides with 50% PB/50% glycerol and coverslipped.

Confocal images of sections through the A15 were acquired using an LSM-510 laser scanning microscope (Carl Zeiss, Thornwood, NY) equipped with Ar and He/Ne lasers. Each DA neuron selected was scanned with the appropriate excitation emission laser and filter configuration for detection of CY2 and CY3; scans at each wavelength were done independently to avoid bleed-through between channels. Two sets of images were taken along the z-plane (z-stack) for each neuron, consisting of 22 images in each channel that sliced the neuron from top to bottom at intervals of 1–1.5 µm determined by the confocal software based on the size of the neuron. These z-stacks were then converted to a series of tif files that could be analyzed using other software.

In experiment 2, images were taken of 10–12 DA neurons per animal at x63 magnification to count synapsin-positive contacts, and images were taken at x40 magnification to examine neural morphology. In experiment 3, images were taken of six to 12 neurons per animal at x40 magnification for both counting synapsin-positive contacts and examining neural morphology. Neurons with complete somata within the 50 µm-thick tissue section were selected for analysis.

Analysis of data
LH pulse frequencies were defined using previously described criteria (40) and differences in pulse frequency between groups assessed using the Wilcoxian-Mann-Whitney test (experiment 2). Mean LH concentrations were statistically analyzed by t test (Exp 1) or two-way ANOVA with Tukey’s posthoc evaluation (experiment 3).

On EM images, synapses were defined by the presence of a synaptic density and synaptic cleft. Because axon terminals that lack synaptic specializations but are in direct contact with GnRH neurons also vary with season (29), both types of contacts were analyzed. For experiment 1, we analyzed a total of 82 GnRH and 58 non-GnRH cells (5.1 ± 0.4 GnRH and 3.6 ± 0.4 non-GnRH neurons per ewe for each of the four groups). For each cell, we measured the total length of somatic and dendritic plasma membranes and calculated the mean number of synapses and axon terminals per 10 µm plasma membrane. In addition, for each cell, we calculated the percentage of plasma membrane in direct contact with glial processes. Astroglial processes were identified by the presence of intermediate filaments, arrayed in closely packed parallel bundles. Differences between the groups were tested for significance at P < 0.05 using two-way ANOVA. Post hoc analyses were performed using the Sheffe F test.

Confocal images (saved as tif files) were analyzed using MicroBrightfield’s (Colchester, VT) Neurolucida software. Red and green images were overlaid, and the perimeter of each neuron was identified by first tracing around the cell body of each layer of the stack from top to bottom and then tracing over the dendrites by matching the cursor size to the width of the dendrite and following the dendrite through the stack. Synapsin-positive boutons were defined as being in contact with the neuron if no pixels were visible between the red and green fluorescence. Close contacts were identified in individual z-slices and labeled using the asterisk marker. The software then used these tracings to produce a three-dimensional reconstruction of the neuron, with its associated synapsin-positive profiles. Once this reconstruction was complete, the software was used to calculate mean dendrite length, quantity of primary dendrites, surface area of cell body, surface area of dendrites, volume of cell body, and number of dendritic nodes (bifurcations). The total number of close contacts on soma and dendrites of each neuron was determined, using Neurolucida to rotate the three-dimensional reconstruction 360 degrees so that boutons not touching the neuron in all dimensions could be eliminated. The investigator was unaware of the particular treatment when analyzing all neurons. In experiments 2 and 3, treatment with E2 had no effect on any morphological variable, and therefore data from OVX and OVX plus E2 ewes were combined. Values for all neurons from a single animal were averaged and differences between groups determined by t test (experiment 2) or one-way ANOVA, with Tukey’s as a post hoc test (experiment 3).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: does THX prevent the decrease in synaptic input to GnRH neurons that occurs between the breeding season and anestrus?
As expected, THX ewes sustained high LH (7.58 ± 1.24 ng/ml) into May, thus being physiologically in the breeding season, whereas in thyroid-intact ewes, LH fell to very low levels (0.69 ± 0.03 ng/ml) typically observed in estrogen-treated OVX ewes during anestrus.

Both synaptic contacts with membrane specializations and axon terminals were evident on GnRH (Fig. 2Go) and non-GnRH (not shown) neurons. GnRH neurons in both THX and control ewes received significantly fewer synaptic inputs than unidentified POA neurons (Fig. 3AGo). Similarly, the mean number of axon terminals in direct contact with GnRH neurons was significantly less than that of contacts onto unidentified neurons (Fig. 3BGo). However, there was no significant effect of THX treatment on the mean number of synaptic or axon terminal inputs onto either GnRH cells or their unidentified neighbors (Fig. 3Go).


Figure 2
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  		FIG. 2. Examples of synaptic contacts/axon terminals onto GnRH cells in thyroid-intact (A) and thyroidectomized (B) ewes. A, Two axon terminals (A1 and A2) contact a portion of an immunoreactive GnRH dendrite (d). The lower terminal (A2) forms a synaptic specialization (arrows) with the postsynaptic dendrite. B, Two axon terminals synapsing upon a GnRH-positive dendritic spine. Both terminals possess synaptic modifications (cleft, densities) indicated by the arrows. Magnification, approximately x20,000 (A) and x40,000 (B).

 

Figure 3
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  		FIG. 3. Density of synapses (left) and axon terminals (right) on GnRH and non-GnRH neurons in the POA in thyroid-intact (white bars) and THX (shaded bars) ewes. *, P < 0.05 compared with non-GnRH neurons. There were no significant effects of thyroid status.

 
There was no significant difference between GnRH and unidentified cells in the percentage of somata membranes contacted by glial processes (Fig. 4AGo). In contrast, GnRH dendrites were contacted by surrounding glial cells to a significantly greater extent than neighboring unidentified POA cells (Fig. 4BGo). Again, there was no treatment effect of THX on the extent of glial ensheathment of either GnRH cells or their unidentified neighbors.


Figure 4
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  		FIG. 4. Glial ensheathment (percent plasma membrane contacted) of soma (left) and dendrites (right) of GnRH and non-GnRH neurons in the POA in thyroid-intact (white bars) and THX (shaded bars) ewes. *, P < 0.05 compared with non-GnRH neurons. There were no significant effects of thyroid status.

 
Experiment 2: are there seasonal changes in the synaptic input and/or morphology of A15 DA neurons?
LH data from these ewes have already been reported (34). In the animals used in this experiment, LH pulse frequency was significantly inhibited by E2 in OVX anestrous ewes (from 4.6 ± 0.3 to 2.0 ± 0.0 pulses/4 h) but not in OVX breeding-season animals (4.6 ± 0.3 before vs. 4.8 ± 0.6 pulses/4 h during E2 treatment).

TH-positive neurons identified by immunofluorescence had properties similar to those described previously using conventional light immunocytochemistry (18, 41); cell somas were 25–35 µm in diameter, and two to three primary dendrites were usually evident (Fig. 5Go). Not surprisingly, a large number of synapsin-positive boutons were evident (Fig. 5Go), but the ones contacting TH-positive neurons could be identified in single z-slices through the neurons and by rotating the reconstructed neuron with the Neurolucida software (Figs. 5Go and 6Go) The total number of synapsin-positive close contacts on A15 neurons was significantly higher in anestrus (56.6 ± 4.7) than in the breeding season (45.2 ± 2.3). This increase in close contacts was due to changes in input to dendrites, not to somata (Fig. 7Go).


Figure 5
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  		FIG. 5. Single optical slices through the A15 region of a breeding season (top) and an anestrous (bottom) ewe stained for TH (red) and synapsin (green). Synapsin-positive close contacts are illustrated in the enlarged insets at bottom right of each panel, The arrowhead indicates a doublet of boutons not in contact with the nearby dendrite. Bar, 20 µm.

 

Figure 6
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  		FIG. 6. Rotation of a single reconstructed neuron (gray) and boutons (black) that appear to be in contact allows identification of boutons not in actual contact. A–C, The same neuron shown at three different angles. Arrowheads indicate a bouton that appears to be in close contact (B) but is not touching the dendrite (A). This bouton is not visible in C because it is behind the dendrite.

 

Figure 7
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  		FIG. 7. Differences in synapsin-positive close contacts on the dendrites (left) and soma (right) of A15 DA neurons from breeding-season (white bars) and anestrous (shaded bars) ewes. *, P < 0.05 vs. breeding season.

 
No significant change in cell body volume occurred between the breeding and the anestrous season (Fig. 8Go). In contrast, mean dendrite length was almost three times longer in anestrus (Fig. 8Go), and there was a corresponding increase in surface area of the dendritic tree in anestrus (data not shown). Because of this increase in membrane area, there was no significant seasonal difference in the density of synaptic contacts on dendrites between anestrous (0.015 ± 0.001) and breeding-season (0.022 ± 0.005) ewes. There was no seasonal difference in number of primary dendrites, but there was more extensive branching in anestrus, as indicated by a significant increase in number of bifurcations in the dendritic tree (Fig. 8Go).


Figure 8
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  		FIG. 8. Differences in morphological characteristics of soma (left) and dendrites (right) of A15 DA neurons from breeding-season (white bars) and anestrous (shaded bars) ewes. *, P < 0.05 vs. breeding season.

 
Experiment 3: is the seasonal plasticity in A15 DA neurons dependent on thyroid hormones?
Estradiol produced a marked suppression of LH in the thyroid-intact and THX ewes given T4 icv, indicating that these animals were in anestrus, whereas it produced only a modest inhibition of LH secretion in the THX ewes given vehicle icv, indicating that these ewes had remained in the breeding season (Table 1Go).


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  		TABLE 1. Effects of E2 treatment on mean (±SEM) LH concentrations in THX and thyroid-intact OVX ewes during the anestrous season

 
The number of close contacts on dendrites in the vehicle-treated THX ewes was slightly lower than in the other two groups, but this was not statistically significant (Fig. 9Go). In contrast, the mean length of dendrites was significantly longer in intact and THX ewes given T4 icv than in THX ewes given vehicle icv. No significant differences were observed in other morphological variables.


Figure 9
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  		FIG. 9. Effect of thyroid status during the transition to anestrus on dendritic length (left) and number of synapsin-positive close contacts (right). Treatment during the transition resulted in two groups that were anestrus (shaded bars) and one that was in the breeding season (white bars). See text for details. *, P < 0.05 vs. groups in anestrus.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies examined the possible role of seasonal plasticity in two neural systems clearly implicated in seasonal breeding in the ewe: 1) GnRH neurons and 2) A15 DA neurons that mediate the seasonal changes in E2 negative feedback. The data suggest that plasticity in input to GnRH neurons is not sufficient for seasonal breeding in ewes. They also identify seasonal changes in morphology and synapses onto A15 DA neurons that may play some role in the annual changes in fertility in this species.

The results of experiment 1 clearly demonstrate that THX late in the breeding season does not affect the synaptic input or glial ensheathment of GnRH neurons in anestrus. Because these THX ewes remained in the breeding season in a neuroendocrine sense, the results support the possibility that the seasonal changes previously observed in synaptic contacts (29) and glial ensheathment (31) are not causally related to seasonal breeding. One limitation of this experiment is that we did not include a thyroid-intact breeding-season group as a positive control. However, the magnitude of seasonal changes in synaptic input is almost 2-fold and has now been observed in three separate experiments (29, 31). It is thus very likely that the levels of input observed in both the groups in this study, which are similar to those of anestrous ewes in previous work, would also differ significantly from intact breeding-season animals. On the other hand, seasonal changes in glial ensheathment are not of the same magnitude and have been reported in only one experiment (31), using a different methodology, so we are reluctant to draw any firm conclusions from the lack of differences in this variable between THX and thyroid-intact ewes. Thus we conclude that the seasonal changes in synaptic input are not sufficient for seasonal changes in GnRH secretion. They may be necessary, in combination with other seasonal changes in the neural circuitry mediating E2 negative feedback, or may be related to other possible functions of GnRH neurons, such as seasonal differences in sexual receptivity (42, 43, 44). It is unlikely that they are related to seasonal changes in control of the LH surge because this system does not vary with season in the ewe (42).

The second experiment identified seasonal plasticity in the A15 DA neurons that are a critical component of the neural circuitry mediating seasonal changes in E2 negative feedback. Because these inhibitory neurons transmit the E2 negative feedback signal from estrogen-responsive afferents in the vmPOA and RCh to GnRH neurons only in anestrus (see introductory section), it is tempting to speculate that the increase in their dendritic trees and the synaptic input to them in anestrus plays an important role in the seasonal changes in estrogen negative feedback. However, there are two important caveats that need to be considered in interpreting these observations. First, synaptic contacts were identified at the light microscopic, rather than the more traditional EM, level using synapsin I as a marker. Synapsin I is an actin-binding protein that is found exclusively in presynaptic vesicles (32), and EM analysis of its localization in the A15 (Fig. 1Go) and the suprachiasmatic nucleus (45) indicate that it is found in all synaptic vesicles. Moreover, this approach indicated seasonal changes in synaptic input to GnRH neurons (30) that were very similar to those observed with EM analysis (29). Therefore, confocal microscopic analysis of synapsin I appears to be a reliable method for determining synaptic input that allows considerably more neurons to be examined than is feasible at the EM level.

The second factor that complicates interpretation of these results is the use of TH immunoreactivity to identify components of the A15 neurons. This approach raises the possibility that the seasonal increases in dendritic length and synaptic contacts reflect an increase in expression of this enzyme in anestrus that allows better visualization of the dendrites and their synaptic contacts. However, TH mRNA levels are higher in the breeding season (46), and there is a corresponding increase in A15 TH-immunopositive neurons during the breeding season compared with anestrus (18). Thus, although this possibility cannot be completely excluded, the data on TH expression do not support it.

The results of experiment 3 suggest that the increase in dendritic length of A15 neurons during the transition from breeding to anestrous seasons is dependent on thyroid hormones, whereas the seasonal increase in synaptic input is not. In this experiment, in contrast to experiment 2, the neurons used for analysis were selected from the caudal portion of the Al5 nucleus because more rostral sections had been used for other studies. Thus, the lack of differences in synaptic input in experiment 3 may reflect differences within the A15 nucleus, similar to regional differences within the robustus archistriatalis, a nucleus involved in singing in the canary (47). The lack of a breeding-season group as a positive control also raises the same limitations to interpretation of these data as those in experiment 1 (see above). Finally, these results do not preclude an important role for seasonal changes in a subset of synaptic input to A15 neurons. For example, we have recently observed an increased number of glutamatergic boutons on A15 neurons in anestrus (Singh S. R., C. J. McManus, L. M. Coolen, M. N. Lehman, and R. L. Goodman, unpublished data). Thus, additional work appears to be warranted before concluding that seasonal changes in synaptic input to A15 neurons are not required for the transition to anestrus.

In contrast to the ambiguity in interpreting the synaptic input, clear differences in dendritic length were evident, with A15 neurons from animals that remained in the breeding season having shorter dendrites than those in the two groups that were anestrus when tissue was collected. Because T4 concentrations were low in both groups of THX ewes, the difference in dendritic length in these two groups must reflect actions of T4 during the transition to anestrus. Moreover, they are due to actions of this hormone within the brain because they occurred in ewes given T4 icv. However, it is unlikely that the changes in dendritic morphology are caused by direct actions of T4 on A15 neurons, because local administration of T4 to the RCh did not overcome the effects of THX (48). Instead, T4 acted in two other areas, the vmPOA and premammillary region, that contain cells projecting to A15 DA neurons (48, 49). These data raise the possibility that the increase in dendritic length in anestrus occurs in response to signals from afferent inputs from these two areas. Thyroid-hormone-dependent neurotrophins that have been identified in extrahypothalamic areas in rats (50, 51) are possible candidates for such a signal (52). Finally, it is important to keep in mind that although the correlation between the length of A15 dendrites and seasonal reproductive status supports the hypothesis that these changes play a role in the transition into anestrus, they do not demonstrate that the increased length of dendrites in anestrus is responsible for the onset of infertility. A more direct test of this hypothesis awaits development of a method for disrupting these morphological changes.

The seasonal changes in morphology observed in ovine GnRH and A15 neurons and songbird nuclei (9) are in the middle of a continuum of physiological plasticity observed in neuroendocrine and nonneuroendocrine systems that can be influenced by gonadal and thyroid hormones. At one extreme are organizational events that occur once during development, such as the well-known masculinizing actions of testicular steroids (53) and actions of thyroid hormone on development of the cerebellum (54) and hippocampus (55). At the acute end of this continuum are morphological and physiological changes seen in models of learning and memory, such as increases in spine density (56) and long-term potentiation (LTP) in the hippocampus (57) and synaptogenesis in the motor cortex induced by learning a motor skill (58). Although the latter are thought to follow Hebbian mechanisms (59), many of them can be influenced by hormones. For example, estrogen stimulates hippocampal spine density (56), and thyroid hormones have both organizational (55) and activational (57) actions on LTP. The seasonal changes in the songbird appear to be most similar to hippocampal spine density because they are driven by seasonal changes in gonadal steroids (9), whereas those in the ewe can occur even if estradiol levels are held constant with estrogen implants. Thus, the neural plasticity underlying seasonal breeding in ewes most closely resembles developmental changes in the substrates responsible for induction of LTP, because thyroid hormones must be present during a critical period for them to occur. Of course, they differ from LTP in that they occur annually rather than just once in the lifespan of an individual.

In summary, these studies have identified seasonal plasticity in the A15 DA neurons that mediate seasonal changes in E2 negative feedback, some of which are dependent on the presence of thyroid hormones during the transition to anestrus. In contrast, the seasonal plasticity of GnRH neurons is independent of thyroid hormones. Because the transition from breeding to anestrous seasons is thyroid-hormone dependent, these results suggest that seasonal plasticity in GnRH neurons is not sufficient for seasonal breeding, but support a role for plasticity of A15 DA neurons.


   Acknowledgments
 
We thank Dr. Geoffrey Dahl, Dr. Catherine Viguie, and Mr. Douglas Doop for assistance in preparation of experimental animals and collection of nervous tissue at the University of Michigan. We appreciate the assistance of Mr. Jeff Altemus with the confocal microscopic work and Ms. Reenie Fitzgerald for her dedicated work supporting the electron microscopic portion of this study.


   Footnotes
 
This work was supported by the National Institutes of Health (HD17864) to R.L.G., National Science Foundation (IBN-9810822) to M.N.L., and U.S. Department of Agriculture (94-37203-0760) to F.J.K.

Present address for V.L.A.: Marshall University School of Medicine, Huntington, West Virginia 25755.

Disclosure statement: The authors have nothing to declare.

First Published Online July 20, 2006

Abbreviations: DA, Dopaminergic; E2, estradiol; EM, electron microscopic; icv, intracerebroventricular; LTP, long-term potentiation; OVX, ovariectomy; PB, phosphate buffer; PBTX-NDS, PB containing 0.2% Triton X-100 and 4% normal donkey serum; RCh, retrochiasmatic area; TH, tyrosine hydroxylase; THX, thyroidectomy; vmPOA, ventromedial preoptic area.

Received March 30, 2006.

Accepted for publication July 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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