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Endocrinology Vol. 143, No. 2 544-550
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Selective Regulation of Glutamic Decarboxylase Isoform 65, But Not Isoform 67, in the Bed Nucleus of the Stria Terminalis and the Preoptic Area of the Ewe Brain Across the Estrous Cycle

Sueli Pompolo, Christopher J. Scott and Iain J. Clarke

Prince Henry’s Institute of Medical Research (S.P., I.J.C.), Clayton 3168, Victoria, Australia; and Department of Physiology (C.J.S.), Monash University, 3800 Victoria, Australia

Address all correspondence and requests for reprints to: Dr. Sueli Pompolo, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, 3168, Victoria, Australia. E-mail: sueli.pompolo{at}med.monash.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{gamma}-Aminobutyric acid neurons in the preoptic area (POA) of the brain may regulate GnRH neurons. The level of expression of two isoforms (65 and 67) of glutamic acid decarboxylase (GAD) in the ewe brain was determined across the estrous cycle by in situ hybridization. GAD mRNA expression (cell number and silver grains/cell) was examined in the subdivisions of the bed nucleus of stria terminalis (BnST), in the diagonal band of Broca, and the POA. The number of cells expressing GAD65 and GAD67 mRNA did not change across the cycle. Within the rostro-dorsal BnST, the number of silver grains/cell for GAD65 mRNA was lower in the follicular phase than the luteal phase or at estrus. In the rostro-lateral division, expression was lower in the follicular phase. In the POA, the number of silver grains/cell for GAD65 mRNA was lower at estrus than during the luteal phase. The number of silver grains/cell for GAD67 mRNA did not change across the estrous cycle. GAD65 is thought to be the active enzyme during periods of high demand of GABA and our results are consistent with the GABA neurons of BnST being most active during the luteal phase of the estrous cycle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS THE brain hormone that is the primary regulator of reproduction. The cells that produce GnRH are controlled by various neuronal systems (neuropeptides and neurotransmitters) that modulate the pulsatile release of GnRH into the hypophysial portal blood vessels (1, 2, 3). In turn, GnRH regulates the synthesis and secretion of LH and FSH at the level of the anterior pituitary (2, 4). Gonadal steroids regulate the secretion of GnRH and gonadotropins across the estrous cycle (2, 3). Thus, during the luteal phase, high plasma levels of progesterone and low plasma estrogen levels exert negative feedback effects on GnRH secretion (2). In the absence of progesterone, rising plasma levels of estrogen during the follicular phase stimulate central and pituitary mechanisms leading to the generation of a preovulatory GnRH/LH surge through a process termed positive feedback (2). The neural mechanisms that transmit these steroid feedback signals to GnRH are largely unknown but are thought to involve stimulatory input to GnRH neurons (1, 3) as well as disinhibition or reduction of inhibitory input (2, 5, 6, 7).

In most species including the sheep, GnRH cells are found in a region extending from the diagonal band of Broca (dbB) through the preoptic area (POA) and into the hypothalamus (8). Although estrogen exerts feedback effects on GnRH secretion, original studies (9, 10) showed that GnRH cells do not to possess significant levels of ER. Some recent publications (11, 12, 13) suggest that there may be a low level of ER expression in some GnRH cells, but it is well accepted that the major effects of estrogen on GnRH involve action on afferent neurons to GnRH cells. Throughout the POA and dbB of the ewe brain there is a large number of non-GnRH neurons that express of ER{alpha} (14), and these are thought to mediate feedback effects on GnRH neurons. Among these are cells that produce {gamma}-aminobutyric acid (GABA), around 40% of which are immunoreactive for ER{alpha} in rats (15) and in sheep ( 3). Several morphological and functional studies support the notion that GABA-producing cells are important targets for steroids and may relay feedback information to GnRH neurons. Thus, synaptic contacts between GABA terminals and GnRH neurons have been described in rats (16), GABAA receptor subunits are expressed in GnRH neurons of female rats (17, 18, 19), and administration of a GABAA receptor agonist reduces GnRH mRNA expression (20). Because GABA is an inhibitory transmitter, the GABA-containing cells of the POA may mediate the negative feedback effects of estrogen and/or progesterone on GnRH neurons. Using push-pull perfusion or microdialysis, the extracellular concentrations of GABA were found to be elevated in the POA of estrogen-treated ovariectomized (OVX) rats compared with OVX controls and, on the other hand, low levels of GABA release were observed on the afternoon of the proestrus (5, 21, 22). In ewes, GABA levels in the POA also fall before and during the estrogen-induced LH surge but are increased by progesterone treatment of OVX ewes (23, 24). In rats and sheep, administration of GABA or GABA agonists into the medial preoptic area inhibit LH release (6, 25, 26, 27, 28) and block the estrogen-induced LH surge (7, 26, 29). In monkeys, Goldsmith and Thind (30) reported that there are no synaptic contacts were between GABA and GnRH neurons. Nevertheless, there is evidence that GABA is an inhibitory neurotransmitter responsible for restricting GnRH secretion before the onset of puberty in female rhesus monkey (31).

GABA synthesis is catalyzed by glutamic acid decarboxylase (GAD), a rate-limiting enzyme that exists in two forms (GAD65 and GAD67) that are encoded by different genes (32). The two forms differ in molecular weight, amino acid sequence, cellular and subcellular location and their interaction with the GAD cofactor pyridoxal-5-phosphate. Accordingly, it has been hypothesized that GAD65 is mostly responsible for maintaining the pool of GABA in nerve terminals and is activated in response to increasing demand for the transmitter. On the other hand, GAD67 is tonically active and synthesizes GABA for nonvesicular release and/or metabolic purposes (32, 33).

Results from knockout mice suggest the GAD67 is important in the regulation of GABA levels and is especially relevant to the development of cleft palate (34); GAD65 knockouts are more susceptible to seizures than the controls. Nevertheless, GABA levels in the null mutants are similar to those in wild-type animals. Data from the rat suggests that estrogen controls the expression of both genes (35), and a reduction of both forms of GAD mRNA levels is seen during the afternoon of proestrus (36, 37).

The bed nuclei of stria terminalis (BnST) contains a large number of GABA cells and is a major source of input to the POA (38, 39). Electrical stimulation or lesioning of the BnST affects LH secretion and inhibits short photoperiod-induced testicular regression in hamsters (40, 41) and lactation in rats (42). The BnST is also an area that relays information from chemosensory areas to those that control sexual behavior (43) and has bidirectional connections with the amygdala (44) and the periventricular nucleus of hypothalamus (45).

The aim of the present work was to determine whether expression of the genes for GAD65 and GAD67 mRNA are regulated across the ovine estrous cycle in the BnST, POA, and dbB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Corriedale ewes of similar age and weight were maintained under natural pasture conditions at the Victorian Institute of Animal Science, Werribee, Victoria, Australia. The experiment was carried out according to the guidelines for the Care and Maintenance of Experimental Animals and was approved by the Monash Medical Center Animal Ethics Committee and the Animal Ethics Committee of the Victorian Institute of Animal Science before commencement of the study. The experiment was performed during the breeding season and estrous cycles were monitored using rams that had been fitted with brisket crayons that marked the ewes at the time of mating; the ewes were checked daily for estrous expression (46). In this species, the preovulatory LH surge and the onset of estrus are coincident (47). Estrous cycles were synchronized with an im injection of 125 µg of the synthetic luteolysin, Cloprostenol (Estrumate, Pitman-Moore, Sydney, New South Wales, Australia) (47). Three groups of animals (n = 4/group) were used, representing the luteal phase (d 10 of the cycle), the follicular phase or estrus. Follicular phase animals were killed 24 h after the injection of Cloprosterol. Estrous animals were killed 1–2 h after the onset of the behavioral estrus (approximately 48 h after Cloprostenol injection), which was determined by constant observation. Blood samples from the group of estrous ewes were taken before, at the onset of estrus and just before killing the animals (1–2 h after the onset of behavioral estrus). For the follicular and luteal phase groups, the blood samples were taken just before the animals were killed. Both ovaries of each animal were also inspected for the presence/absence of corpora lutea.

Details of the plasma hormone levels in these sheep have been reported previously (46); plasma LH and progesterone levels were representative of the respective stages of the ovarian cycle. In particular, 4/4 of the group killed at estrus was experiencing an LH surge.

Tissue collection
The animals were injected with 25,000 IU of heparin 5 min before they were killed with an overdose of sodium pentobarbitone (Lethabarb, Virbac Australia, Qld, Australia). The heads were perfused, via internal carotid arteries, with 2 liters of 0.9% of saline containing 25,000 IU/liter of heparin, then 2 liters of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), then 1 liter of fixative containing 20% sucrose. Brains were removed, postfixed in 4% paraformaldehyde plus 30% sucrose for a further 14 d at 4 C. The hypothalamic/preoptic blocks were frozen on powdered dry ice and stored at -20 C. Sections (20 µm) were taken on a cryostat and stored in cryoprotectant solution at -20 C until used.

In situ hybridization
Sections were thoroughly rinsed in potassium PBS (0.02 M) and mounted on poly-L-lysine coated slides and air-dried overnight. The sections were then processed for in situ hybridization following a protocol described by Simmons et al. (48) using an 35S-labeled cRNA probe generated from a 615-bp cDNA fragment corresponding to nucleotides 597–1211 of the mouse GAD65 and 618-bp cDNA for GAD67 (corresponding to nucleotides 775-1392) (49). Details of probe synthesis, preparation of hybridization solution, hybridization conditions, and pre- and posthybridization washes are described elsewhere (50). Control sections were either pretreated with RNAase or hybridized with a GAD sense probe. The sections were then dipped in Ilford K5 photographic emulsion, stored in the dark for 6 d at 4 C, and then developed and counterstained with cresyl violet.

Data analysis
Cells expressing GAD 65 and GAD67 mRNA were examined in the dbB and POA, and rostral and caudal divisions of BnST. The stria terminalis clearly divides the BnST into posterior and anterior divisions. The anterior division or rostral sections are further divided into anterodorsal, anterolateral, and anteroventral areas. The posterior or caudal division of the BnST is also divided into several regions: the principal nucleus corresponding to dorsal portion, the rhomboid nucleus corresponding to the lateral portion, and the complex magnocellular plus dorsolateral area corresponding to ventral portion of the BnST (51). Within these rostral and caudal sections, we analyzed GAD expression in the dorsal, lateral, and ventral subdivisions of the BnST. Image analysis was carried out on autoradiograms using coded slides, and the operator was blind to the treatments. A single observer carried out the quantitative image analysis. In each area examined, the total number of cells that expressed either GAD65 and GAD67 mRNA was counted. In ten cells randomly chosen (or every cell if less than ten cells were found) from each area, the number of silver grains/cell was estimated by an image analysis system. Computer-assisted grain counting was performed under brightfield at 400x using a Fuji Photo Film Co., Ltd. (Tokyo, Japan) HC-2000 high-resolution digital camera and Analytical Imaging System 3.0 software (Imaging Research, Inc., St. Catherine’s, Ontario, Canada). We only counted cells that had a clearly discernible nucleus and for which the density of grains was more than five times the background.

Statistical analysis
Differences between luteal, follicular, and estrous phases of the cycle were determined by one-way ANOVA with paired comparisons by least significant differences. Homogeneity of variance was checked and transformation of data were not necessary.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GAD65 and GAD67 mRNA expressing cells were identified as clusters of silver grains over nuclei counterstained with cresyl violet (Fig. 1Go). When sections were hybridized with the sense strand, no hybridization (above background) was observed (not shown). Pretreatment of the sections with RNAase A also eliminated detection of GAD65 and GAD67 mRNA expression in tissue hybridized with the antisense probe (not shown).



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  		Figure 1. Photomicrograph of a cell expressing GAD67 mRNA in the rostro-dorsal BnST in the ovine brain. The arrow indicates the nucleus of the cell, over which silver grains are seen. An adjacent nucleus of a cell that did not express the gene (arrowhead). Scale bar, 10 µm.

 
Cells expressing GAD65 and GAD67 mRNA were found in rostral and caudal regions of the preoptic region. More specifically, GAD-expressing cells were observed in the dbB, in the medial and lateral POA, and in the dorsal, lateral, and ventral portions of the BnST (Fig. 2Go). The distribution of cells expressing both forms of GAD mRNA was similar throughout the rostral and caudal sections of the preoptic region, but this was not analyzed. The effect of the stage of cycle on the number of cells expressing GAD65 and GAD67 and the number of silver grains per cell is shown in Figs. 3Go, C and D, and 4Go, C and D, respectively. There was no effect of the stage of the cycle on the number of GAD65 or GAD67 mRNA-containing cells.



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  		Figure 2. Drawings of rostral (A and B) and caudal (C and D) sections of septo/preoptic area of ovine brain. Sections A and C represent the distribution of cells expressing GAD65 mRNA, and sections B and D show the distribution of cells expressing GAD67 mRNA. Scale bar, 10 µm.

 


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  		Figure 3. Mean (±SEM) GAD65 mRNA expression (silver grains/cells) and the number of cells (mean ± SEM) expressing GAD65 mRNA rostral (A and C) and caudal sections (B and D) of the septo/preoptic area of ovine brain. *, P < 0.05; **, P < 0.003; ***, P < 0.001.

 


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  		Figure 4. Mean (±SEM) GAD67 mRNA expression (silver grains/cells) and the number of cells (mean ± SEM) expressing GAD67 mRNA rostral (A and C) and caudal sections (B and D) of the septo/preoptic area of ovine brain.

 
The number of silver grains/cell in cells hybridized with the GAD65 probe in the rostral forebrain was similar at the three stages of the estrous cycle in the dbB and the ventral BnST. In the rostro-dorsal BnST the number of silver grains/cell for GAD65 mRNA was lower in the follicular phase than either the luteal phase (P < 0.05) or at estrus [P < 0.003 (Fig. 3AGo)]. In the rostro-lateral BnST, the number of silver grains/cell for GAD65 mRNA was greater (P < 0.05) in the luteal phase than in the follicular phase of the estrous cycle. In the POA, the level of expression was lower (P < 0.001) during estrus than during the luteal phase (Fig. 3BGo). The level of expression (silver grains/cell) for GAD67 mRNA did not change across the estrous cycle (Fig. 4Go, A and B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report that, in the ewe, the expression of GAD65 mRNA within individual cells of the rostro-dorsal and rostro-lateral BnST is decreased during the follicular phase of the estrous cycle. Further, we show that there is a reduction in expression of GAD65 mRNA in the POA at estrus. In contrast, no changes in GAD65 mRNA expression were seen in the rostro-ventral or caudal subdivisions of BnST or the dbB. In addition, no cyclic changes were observed in the expression of GAD67 mRNA, and the number of cells expressing GAD65 and GAD67 mRNA did not change across the estrous cycle. Thus, we report that cyclic changes in GAD expression are isoform-specific and occur in specific regions of the BnST and POA; these changes may relate to regulation of the GnRH neurons. The present study cannot, however, provide definitive evidence of this, and the cyclic changes may well subserve some other function, e.g. behavior; further work is required to ascertain the significance of our results.

Based on the principle of disinhibition (reduction of an inhibitory input), one would expect a reduction of negative input to GnRH neurons during the period leading up to the preovulatory GnRH/LH surge. There is strong evidence that the GABAergic system exerts an inhibitory action on GnRH secretion (see Introduction), and in sheep (23) and rats ( 5) extracellular GABA levels fall in the preoptic/septal area before and during the estrogen-induced LH surge. Furthermore, infusion of GABA into the POA blocks the LH surge in proestrous rats (29). The present results suggest that the reduction in GABA release in the POA at the time of the LH surge may be due, at least in part, to a reduction in GABA synthesis in the POA and/or the rostral subdivisions of the BnST. Thus, reduced mRNA levels for GAD in the mid-follicular phase of the cycle (present study) could translate into reduced enzyme activity in the mid- to late-follicular phase and onwards into the surge phase, leading to reduced GABA activity at the time of the surge (21). In this regard, it is notable that the fall in levels of extracellular GABA that was observed in the POA during the presurge period in the ewe (23) (approximately 22% of the levels in the luteal phase) was similar to the reduction that we observed in the expression of GAD65 mRNA (16–27%).

The fall in GAD65 mRNA in the BnST during the follicular phase and in the POA at the onset of estrus probably reflects a mechanism to reduce the production/secretion of GABA because this isoform is the one mostly responsible for maintaining a pool of releasable GABA in nerve terminals and is activated in response to increased demand (32). In terms of an acute regulation of the levels of GABA in nerve terminals, available for release, it would seem appropriate that GAD65 be regulated. On the other hand, GAD67 is constitutive and we did not see any changes in expression of mRNA levels for this isoform. Our data contrast with earlier data from the rat showing that both isoforms are regulated by estrogen (35, 37, 52). GAD67 is thought to contribute to the formation of GABA in dendrites and cell bodies and to be involved in cellular metabolism (32). Nevertheless, the fact that consistent results from laboratories mentioned above, have shown regulation of both isoforms in the rat suggests that there may be a species difference in the cyclic alterations in expression of the two genes.

The observation that GAD65 mRNA levels change across the estrous cycle suggests regulation by estrogen and/or progesterone. With regard to progesterone, this steroid appears to up-regulate the GABAergic system in the POA of the ewe (24). Accordingly, the lower levels of GAD65 mRNA expression in the rostral BnST during the follicular phase of the estrous cycle would be due to the lower levels of circulating progesterone at that time. In addition, the vast majority of GAD-containing cells in the POA of the pubertal female monkey contain progesterone receptors (53), indicating that progesterone may act directly on GABAergic cells in this region. Our data raise the possibility that this action of progesterone may be to alter GAD65 transcription and/or mRNA stability. Progesterone receptor-containing cells in guinea pig (54, 55) are also ER positive, although the reverse is not always the case. It is noteworthy, though, that cells in the BnST do not contain progesterone receptors (50, 56, 57), suggesting that progesterone does not have a direct action on GAD65-containing cells in this region.

There is good evidence that cells of the BnST are integrally involved in regulation of GnRH neurons (40) and a high percentage express ER (22, 58). Unpublished data from this lab (Stackpole, C., I. J. Clarke, and S. Pompolo, in preparation) also show a reduction in terminal staining for dopamine-ß-hydroxylase (indicating noradrenaline release) in the same region of the BnST during the follicular phase and estrus, further suggesting that this is important area for the control of reproduction. The degree of colocalization of GABA and ER in the BnST is unknown, although there is 40% colocalization in the POA (see Introduction). Our results suggest that GAD65-containing cells of the rostro-lateral and rostro-dorsal divisions of the BnST could modulate GnRH neurons. Although studies in sheep (39) and rat (38) have shown projections from the dorsal and lateral BnST to the POA, there are no direct inputs to GnRH neurons (39). This suggests that regulation is likely to be via interneurons located in the POA. Analysis of this pathway would be substantially assisted by identification of an antibody that would immunohistochemically stain GAD-containing cells in the sheep brain, but we have not been able to obtain such an antibody.

In conclusion, this work conducted in the female sheep provides further information on the regulation of GABAergic neurons across the estrous cycle. Firstly we show that the expression of GAD65, but not GAD67, mRNA is regulated. Secondly, we show that a decrease in GAD65 expression in the follicular phase is restricted to the dorsal and lateral divisions of the rostral BnST and to the POA at estrus. Thirdly, the changes across the cycle are in terms of expression level/cell rather than a change in the detectable number of cells. This is consistent with a role for the GABAergic system in the preoptic region to inhibit GnRH/LH secretion, mediating, in part, the negative feedback actions of estrogen and/or progesterone.


   Acknowledgments
 
The authors are grateful for the technical assistance of Mr. Bruce Doughton, Ms. Karen Briscoe, and Ms. Alix Rao, the artistic assistance of Ms. Sue Panckridge, and to Dr. A. Tilbrook for statistical advice.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia.

Abbreviations: BnST, Bed nucleus of stria terminalis; dbB, diagonal band of Broca; GABA, {gamma}-aminobutyric acid; GAD, glutamic acid decarboxylase; OVX, ovariectomized; POA, preoptic area.

Received July 19, 2001.

Accepted for publication October 4, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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