Regulation of γ-aminobutyric acidB (GABAB) receptors in cerebral cortex during the estrous cycle

BRAIN RESEARCH ELSEVIER

Brain Research 640 (1994) 33-39

Research Report

Regulation of y-aminobutyric acidB (GABA B) receptors in cerebral cortex during the estrous cycle Muna I. AI-Dahan a, Mohammad H. Jalilian Tehrani b, Robert H. Thalmann c,. a Department of Cell Biology, b Department of Biochemistry, and c Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA

(Accepted 2 November 1993)

Abstract

We examined binding of the GABA B receptor agonist baclofen to brain synaptic membranes as a function of the natural variations in gonadal steroids that occur during the estrous cycle of the adult rat. We found that the binding of baclofen to neocortical membranes varied systematically as a function of the estrous cycle, with the lowest binding occurring during the estrus stage. Binding to archicortical (hippocampal) and hypothalamic preparations also varied with the estrous cycle, except that the lowest level of binding in these latter cases occurred during the diestrus stage. The variation of [3H]baclofen binding during the estrous cycle was different with respect to the binding of [3H]muscimol, an agonist for GABA A receptors, and [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist for serotonin 5-HT1A receptors that shares similar G proteins and effectors with GABA B receptors. Saturation binding studies of cortical GABA B receptors showed that apparent receptor density (Bmax) rather than affinity (K d) best accounted for the change in binding during the estrous cycle in that Bmax,like total specific binding, was at a minimum during the estrus stage. The robust regulation of GABA B receptors in neocortex was unexpected and its functional significance is at present unknown. However, the correlation of the menstrual cycle with mood and other behavioral changes, and the correlations of the estrous and menstrual cycles with seizure susceptibility, may somehow depend upon hormonal regulation of transmitter systems such as the one we have observed here. Key words: GABA B receptor; GABA A receptor; Serotonin 5-HT~A receptor; Neocortex; Hippocampus; Estrous cycle; Steroid

hormone; Progesterone; Estrogen

1. Introduction

The binding of the G A B A B agonist baclofen was studied as a function of the natural variations in ovarian steroids that occur during the estrous cycle of the adult rat. The results of such experiments must ipso facto reflect physiological changes in hormone levels, and will thus serve as a useful baseline against which to evaluate the results of hormone replacement experiments or any other simplifying experiment that must depart from in vivo conditions in order to precisely examine hormonal effects during the estrous cycle. Possible steroid effects upon G protein-linked conductances in hypothalamus [17] and hippocampus [9] p r o m p t e d us to examine whether natural variations of ovarian steroids would have an effect upon what are

* Corresponding author. Fax: (1) (713) 790-0545. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E 1518-8

perhaps the most widely distributed Gi/o protein-linked receptors in brain namely, the G A B A B receptors [6]. Presynaptically, these receptors powerfully regulate transmitter release from GABAergic and glutamatergic synapses [5,12,15,42], while postsynaptically, they are responsible for a G protein-mediated late inhibitory postsynaptic potential (IPSP) [14,27,39,40]. These receptors also have access to cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase via inhibition of adenylyl cyclase [41]. For comparison, the binding of muscimol, a G A B A A agonist [10] and 8 - O H - D P A T , a 5-HTIA agonist that activates similar G proteins and effectors as G A B A a receptors [27] was determined. G A B A A receptors are responsible for powerful and ubiquitous chloride-dependent postsynaptic inhibitory potentials in brain and for this reason are involved in many brain functions [13]. 5-HT1A receptors are widely distributed in brain [1,30], control potassium channels [27] and have been

34

M.I. Al-Dahan et al. / Brain Research 640 (1994) 33-39

( - ) - B a c l o f e n was given by Ciba-Geigy. Radioligands were purchased from New England Nuclear. All other reagents were purchased from Sigma.

Tris-citrate. The final pellets were resuspended in Tris buffer and frozen at - 70°C. We refer to this as the one-day preparation. Some of the 1-day preparations were used in binding assays (see below), while others were thawed the next day and subjected to further washing, as described below, before use in binding assays. We refer to the latter preparation as the 2-day preparation. Specifically, for the 2-day preparation, the pellets were resuspended in Tris buffer and incubated for 30 min at 37°C, and then centrifuged as above. The pellets were then washed four times with resuspension and centrifugation, twice with 50 m M Tris-citrate (pH 7.4) and twice with 50 m M Tris-HCl (pH 7.4). The final pellets were resuspended in Tris buffer and frozen at -700°C. Since there was no significant difference in one day vs. the two day preparation in our study of [3H]baclofen binding to neo cortex (t-test between results of the two protocols for each stage of the estrous cycle), both m e m b r a n e preparations were used and the results were combined for analysis. On the day of assay, m e m b r a n e suspensions were thawed and adjusted to 5 m g / m l protein and then treated with saponin at a final concentration of 0.5% for 3 min at 37°C. The saponin-treated membranes were centrifuged at 48,000X g for 10 rain. The pellets were washed three times with 50 m M Tris-HC1, each time by suspension and centrifugation as above. The final pellets were resuspended in 50 m M Tris buffer (pH 7.4) or 20 m M Hepes-Tris buffer (pH 7.1), and used immediately for the binding of [3H]baclofen or [3H]muscimol, respectively. Our preliminary results suggested that saponin treatment was necessary in order to remove endogenous GABA. Specific [3H]baclofen (20 nM) and [3H]muscimol(4 nM) binding to saponintreated m e m b r a n e s accounted for more than 60% of the total binding (not shown). For (3H]8-OH-DPAT binding, m e m b r a n e s were thawed and centrifuged at 45,000x g for 10 min. T h e pellets were then resuspended.in Tris-HCI buffer (pH 7.7) and incubated for 10 min at 37°C. The final pellets were resuspended in Tris-HCl buffer containing 0.1% ascorbic acid and 4 m M CaCI 2 and used immediately for binding.

2.2. Identification of the stages of the estrous cycle

2.4. [ 3H]Ligand binding

implicated in behavioral phenomena such as anxiety [31]. Although 5-HT1A and G A B A A receptors are thus important in their own right and have been little studied with respect to the estrous cycle, they were chosen for these experiments in order to alert us to effects that might be common either to the transmitter GABA or to the G protein-effector systems that are coupled to both G A B A B and 5-HT1A receptors. The neocortex and hippocampus are not as obviously related to sexual functions as are well recognized targets of ovarian steroids such as hypothalamus and amygdala [11,29,33,36,43]. Nevertheless, there is reason to expect that ovarian hormones may significantly affect synaptic components in the two former areas. For example, estrogen alters hippocampal synaptic anatomy [45] as well as synaptic responses [44]. Ovarian steroid effect upon areas including the cerebral cortex are suggested by the association of the estrous [46] and menstrual [26] cycles with seizure susceptibility, and by the correlation of the menstrual cycle with behavioral phenomena such as mood [19,23,32].

2. Materials and methods 2.1. Materials

Virgin female Sprague-Dawley rats, 150-200 g, were housed in a light (14 h light: 10 h dark; lights on at 07:00 h) and temperature (22°C) controlled environment and fed on a diet of Purina rat chow and tap water ad libitum. The stages of the estrous cycle of individual animals were determined by daily vaginal smears, taken between 10:00 and 11:00 h. Only rats showing at least three consecutive and regular 4-day cycles were selected.

2.3. Membrane preparations O n e hour after establishing the stage of the estrous cycle, the animal was sacrificed, its brain was dissected on an ice-cold ceramic tile and frozen at - 7 0 ° C . The division of the brain into regions followed the procedure of Jalilian-Tehrani et al. [16] to furnish whole cerebrums (brain without the portion of the brain stem caudal to thalamus) or portions of the cerebrum consisting primarily of neocortex or hypothalamus. Additionally, a portion consisting primarily of the hippocampal formation was dissected from the amygdala-hippocampal portion of the foregoing dissection. Normal male animals of the same age were used as a reference in some experiments. Crude synaptic m e m b r a n e s were prepared according to Al-Dahan and T h a l m a n n [2] with certain modifications. Briefly, tissues were homogenized in 50 m M Tris-citrate (pH 7.4) and 0.32 M sucrose and centrifuged at 1,000x g for 10 min. T h e supernatants were collected and recentrifuged at 48,000× g for 10 min. The pellets were then washed twice more by resuspension and centrifugation, once in 5 m M Tris-citrate and once in 50 m M

Filtration technique with G F / C filters was used for the binding of all three radioligands. For [3H]baclofen binding, 120-160 /zg of m e m b r a n e protein was added to the incubation buffer containing 50 m M Tris-HCl (pH 7.4), 2.5 m M CaCI2, 0.25 m M E G T A and 20 nM [3H]baclofen in a total volume of 200 p,l. Control tubes used to define nonspecific binding also contained 1 m M GABA. T h e mixtures were incubated for 15 min at 22°C. For [3H]muscimol binding synaptic m e m b r a n e s (100-200 /.Lg) were added to tubes containing 20 m M Hepes-Tris buffer (pH 7.1) and 4 nM or 40 nM radioligand in a total volume of 0.8 ml and then incubated for 30 min at 4°C. Incubation was terminated by addition of 3.5 ml of ice-cold Tris-citrate Buffer (pH 7.1), followed by filtration. Filters were washed twice more and counted with liquid scintillation.Assay mixtures used to define non-specific binding also contained 1 m M GABA. [3H]8-OH-DPAT binding was carried out in a mixture containing 50 m M Tris-HCl (pH 7.7),10/zM pargyline, 0.25 nM radioligand and 200 p.g m e m b r a n e suspension in a total volume of 0.5 ml. Non-specific binding was defined as binding in the presence of 100/~M 5-hydroxytryptamine. The mixtures were incubated for 30 min at 22°C. Protein concentration was determined by the method of Lowry [181.

2.5. Analysis of data The saturation binding data of [3H]baclofen was analyzed by the iterative computer program L I G A N D [24]. All data comparing dif-

M.L Al-Dahan et aL/ Brain Research640 (1994)33-39 ferent stages of the estrous cyclewere analyzed by a one-wayanalysis of variance (ANOVA). One-way ANOVAs indicating significant overall variation among stages of the estrous cycle were followed by Neuman-Keuls tests for post hoc comparisons between individual stages of the estrous cycle. The results of the ANOVAs are given in the figure and table legends. Differences between individual stages that were significant at the 0.05 level are signified in each figure or table by P values.

3. Results

3.1. [3H]Baclofen binding to cerebrum during the stages of the estrous cycle Specific binding of [3H]baclofen to female rat cerebral membranes appeared to vary with the stage of the estrous cycle, with a tendency for the lowest level of binding to occur during the estrus stage and then increase toward a maximum at proestrus (Fig. 1). Although the overall variation with respect to the estrous cycle was not significant, the variation among stages of the estrous cycle was significant when analysed separately from the overall differences in level of binding among different membrane preparations (Fig. 1). To verify that our procedures were comparable to those in our previous experiments with males, these initial binding studies were also done using cerebral membranes from male animals of the same age and strain as the females. [3H]Baclofen binding in males appeared similar to binding during estrus and

E -8 v

35

metestrus, a time when the ovarian hormones would be at their lowest levels [4,7,25] (Fig. 1).

3.2. [ 3H]Baclofen binding to three separate regions within the cerebrum To identify a region within the cerebral hemispheres that might display this variation more robustly, we then separately examined the following three regions: (1) The neocortex, as the largest nominal region within the cerebrum, was chosen as a possible major contributor to the binding pattern observed in the cerebrum as a whole. This preparation excluded the archicortex (hippocampus) and the paleocortical area surrounding the amygdala. (2) The hypothalamus was examined because it contains estrogen and. progesterone receptors similar to the classical peripheral ones i n that they interact with the genome [8] and in that activation of genomic estrogen receptors produces an increase in progesterone receptors [20]. (3) Hippocampus was examined in order to include a well studied and functionally important area where it would be practical to initiate correlations of estrous stage with synaptic functions. Among these three regions, binding to neocortical membranes most closely resembled that to cerebral membranes in that there was significant variation across the estrous cycle (Fig. 2) that appeared to be minimal during estrus, and increased toward a maximum during the proestrus stage (Fig. 2). Variation in binding to hippocampal and hypothalamic membranes was also significant across stages of the estrous cycle (Fig. 2) but the pattern of binding as a function of the estrous cycle appeared quite different from the neocortex (Fig. 2), declining more slowly from a maximum value in proestrus to reach a minimum at diestrus, a time when binding in neocortex had already recovered to near its highest level.

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3.3. Binding of [3H]8-OH-DPAT and [3H]muscimol to neocortieal membranes

5 Pro

Est

Met

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Fig. 1. Specific binding of [3H]baclofen (20 nM) to cerebral membranes as a function of stages of the estrous cycle. This preparation consisted of the diencephalon and telencephalon. In this and subsequent figures, unless otherwise stated, the results represent the average + S.E.M. of three independent experiments, each carried out in triplicate. Pro, proestrus; Est, estrus; Met, metestrus; Di, diestrus. In the cerebrum, there was no significant overall varation among stages of the estrous cycle (one-way ANOVA) (P > 0.1; F = 0.85; df = 3,8). However, a two-way ANOVA suggested that variation among stages of the estrous cycle was significant when analysed separately from the overall differences in level of binding among different membrane preparations (Stages: P < 0.01; F = 12.70;df = 3; membrane preparation: P < 0.025; F = 7.05; df = 2).

We then measured binding of the 5-HT1A agonist [3H]8-OH-DPAT and the G A B A A agonist [3H]muscimol to neocortical membranes. These two additional receptor classes are important in their own right and have been little studied as a function of the estrous cycle. However, in this case they were chosen in order to alert us to effects that might be common either to the G protein-effector systems that are shared by both G A B A a and 5-HT1A receptors or to the transmitter GABA. Nevertheless, the binding pattern of each ligand was unique with respect to the others (Figs. 2 and

3). As in the case of [3H]baclofen, the variation of [3H]8-OH-DPAT) binding with respect to the estrous

M.I. Al-Dahan et al./'Brain Research 640 (1994) 33-39

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dence between two receptor families that share G proteins and effectors, and between two receptor families that share the same transmitter.

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3.4. Saturation binding study of [3H]baclofen binding during the estrous cycle

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To gain more information about the robust variation of [3H]baclofen binding to neocortical membranes with respect to the estrous cycle, we performed a saturation binding study using radioligand concentrations from 1-160 nM and subjected the results to a Scatchard analysis (Fig. 4; Table 1). The illustrated results were compiled from four independent experiments, each carried out in triplicate. At each stage, only a single binding site was recognized (Fig. 4) and there was no evidence of cooperativity of binding: the Hill numbers

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Fig. 2. Specific binding of [3H]baclofen (20 n M ) to scveral regions of the cerebrum as a function of the estrous cycle. Abbreviations are same as in Fig. 1. Neocortex (top panel): the overall variation a m o n g stagcs of the estrns cycle was significant ( A N O V A ) (P < 0.05; F = 3.17; df = 3,28). Probability values above the level of binding in each stage give the significance of the difference of that stage with respect to the estrus stage. Hippocampus (middle panel): thc overall variation a m o n g stages of the estrus cycle was significant (P <0.01; F = 7.56; df = 3,8;). Valucs above each stage give the significance of difference with respect to the diestrns stage. Hypothalamus (bottom panel): the overall variation among stages of the estrus cyclc was significant (P < 0.005; F = 7.71; df = 3,12). Values above cach stage are with respect to diestrus.

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cycle was significant (Fig. 3) but the specific pattern of binding over the cycle appeared to be quite different from that of [3H]baclofen. For example, although binding of [3H]baclofen approached its lowest level during the estrus stage (Fig. 2) the binding of [3H]8-OHDPAT) did not approach its lowest level until diestrus (Fig. 3). The variation of binding of 4 nM [3H]muscimol. with respect to the estrous cycle was not significant (Fig. 3). However, the binding of 40 nM was significant (Fig. 3) but again the association with the estrous cycle differed with that of [3H]baclofen. For example, binding of 40 nM [3H]muscimol was minimal at proestrus, while binding of [3H]baclofen was maximal during this same stage. Thus, with respect to agonist binding as a function of the estrous cycle, there was a lack of correspon-

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Fig. 3. Specific binding of [3H]muscimol and [3H]OH-DPAT to neocortical m e m b r a n e s as a function of the estrous cycle. Top panel: 4 nM [3H]muscimol did not reveal any change in binding as a function of the estrous cycle ( P > 0.1; F = 0.002; df = 3,9). Middle panel: 40 nM [3H]muscimol, however, did reveal an estrous-cycle associated pattern of binding ( P < 0.05; F ~ 3.91; df = 3,12). The values above each stage are with respect to the proestrns stage. Bottom panel: 0.25 n M [3H]OH-DPAT binding showed a clear estrous cycle-assodated pattern ( P < 0.005; F = 10.5; df = 3,8). values above each stage are with respect to the proestrus stage. Note that the binding of each of the three ligands, [3H]muscimol, [3H]OH-DPAT, and [3H]baclofen, was unique with respect to the other.

M.I. AI-Dahan et al. / Brain Research 640 (1994) 33-39 0.030

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Fig. 4. Scatchard analysis of the binding of [3H]baclofen to neocortical membranes at each stage of the estrous cycle. This figure was produced from the averages of the results of four independent experiments. Bronx, but not K d varied significantly among stages (see Table 1).

were near unity at each stage of the cycle (Table 1). Although no significant variation of affinity (K d) with respect to the estrous cycle was observed (Table 1), there was significant variation in apparent density of binding sites (Bin,x) (Fig. 4 and Table 1). The apparent density of binding sites (Bmax) rather than their affinity more closely followed the pattern of specific binding that was previously measured by our single point assays: Bmax was at a minimum during the estrus stage, and then increased to a maximum by the proestrus stage.

Table 1 Parameters of [3H]baclofen binding to neocortical membranes during the estrous cycle Stage

K d (nM)

Bmax (fmol/mg)

Hill number

Proestrus Estrus Metestrus Diestrus

42.9 + 7.3 28.5+2.5 40.1+2.7 30.2+3.3

716 + 62.3 ( P < 0.01) 424+58.1 662+ 79.0 ( P < 0.05) 669+46.5 ( P < 0.05)

1.05 + 0.06 0.92+0.07 0.99+0.02 0.98+0.03

These results represent the mean + S.E.M. of values derived from 4 independent experiments. The binding parameters were determined using the LIGAND program [23]. A N O V A revealed systematic variation in the case of Brnax ( P < 0.05; F = 4.02; df = 3,12) but not in the case of K d ( P > 0.05; F = 2.86; df = 3,12). Significance of differences of Bmax of each stage with respect to estrus is noted in parentheses.

We report here for the first time that there are systematic effects on GABA B receptors in cerebral cortex during the natural variations in ovarian steroids that occur during the estrous cycle. Estrous cycle-linked effects upon GABAB receptors were not limited to neocortex, but were also observed in archicortex (hippocampus), as well as in the hypothalamus. The effects of estrous cycle upon total specific binding to each of the three transmitter receptor families identified by [3H]baclofen, [3H]8-OH-DPAT, and [3H] muscimol must be interpreted cautiously without separate measurement of affinity and density in the case of each receptor. Nevertheless, the unique pattern of specific binding of each ligand to neocortex with respect to the estrous cycle was surprising. For example, in hypothalamus, the ovarian steroid estradiol has similar effects upon a potassium conductance that is elicited by both baclofen and a mu opiate [17]. Since mu opiates and baclofen bind to receptor families that are coupled to these channels through similar G proteins, it was suggested that the steroid might act via these G proteins [17]. Applying a similar hypothesis to our experiments, we predicted that the estrous cycle would be accompanied by similar affects on both GABA B and 5-HTIA receptor binding, since they also share G proteins and effectors [27]. This hypothesis, however, would seem inadequate to explain our results. In the case of GABA A sites probed with muscimol, we saw no variation in neocortex with a ligand concentration (4 nM) that should have primarily detected a high affinity muscimol site [3,10,38]. In agreement with O'Connor al. [28], higher muscimol concentrations (40 nM) that should include a substantial proportion of a lower affinity muscimol binding site [3,10,38] exposed an estrous cycle-associated binding pattern that was unique with respect to the other two ligands. At least in hypothalamus, estrogen treatment can increase levels of the synthetic enzyme GAD [21] which could in turn increase GABA levels. It is thus at least logically possible that the estrous cycle might also be accompanied by changes in GABA levels in the neo cortex and that such changes could secondarily cause the up and down regulation of GABA B receptors that we have observed. However, the lack of correspondence between binding to GABA A and GABA B receptors during the estrous cycle seems to suggest that such a factor could not by itself account for the estrous cycle effects on both GABA A and GABA B receptors. Although our results do not identify the hormone(s) responsible for the effects reported here, our working hypothesis begins with the ovarian steroids estrogen a n d / o r progesterone. Further, since baclofen binding in neocortex is lowest in estrus, when these hormones are at low levels, and highest during proestrus, when

38

M.I. Al-Dahan et al. / Brain Research 640 (1994) 33-39

both h o r m o n e s reach their peak [4,7,25], we suggest that the responsible hormone(s) increases rather than decreases G A B A B receptors. However, a logical possibility remains that some other h o r m o n e or peptide that varies with the estrous cycle might be responsible for some or all of these effects. It is not clear why such robust regulation of G A B A B receptors should occur in areas such as the neo cortex that are not as often associated with sexual function as are areas such as the hypothalamus. Notwithstanding, cyclic effects such as this one may s o m e h o w account for the correlation of the menstrual cycle with m o o d and other behavioral changes [19,23,32], and for correlations of the estrous [46] and menstrual [26] cycles with seizure susceptibility. It is necessary to establish normal physiological baselines for h o r m o n e effects against which to compare the results of more simplified and rigorous studies. H o r m o n e replacement paradigms may not match the concentrations of h o r m o n e s that are achieved normally in brain, although experiments specifically directed to this end have done so (e.g. see [22]) with respect to estradiol and biogenic amines. However, even if h o r m o n e levels are matched, the temporal characteristics of the exposure may differ from specific physiological ones. For example, in the usual paradigms of ovariectomy with h o r m o n a l replacement several weeks later, steroids may reach lower levels for longer periods of time than occur during the 4 - 5 day estrous cycle. Ensuing regulatory responses of c o m p o n e n t s of the system such as receptors and enzymes could thus differ between a given experimental system and the intact animal. However, such simplified experimental paradigms b e c o m e more powerful if it is possible to evaluate their results in the framework of a known physiological effect. For example, any detailed explanation of the molecular basis of G A B A B receptor regulation during the estrous cycle should make it possible to reconstitute the effects that we report here. A final question is why such robust effects of the estrous cycle were discovered in a neocortical preparation where ovarian steroid receptors have been detected at low levels, if at all [11,29,33,43]. A m o n g the possibilities are: (1) T h e cortical steroid receptors responsible for these effects may not be recognized by ligands that are based on homology with receptors elsewhere. For example the neocortical progesterone sites reported by Parsons et al. [29] were not inducible by estrogen, and may differ in other ways from classic progesterone receptors. (2) A related possibility is that the steroid receptors responsible for these results are novel membrane-associated molecules that have little or no homology with the m o r e classic types [20,34,37]. (3) T h e cortical effects may be the result of synaptic input from receptor-bearing neurons elsewhere. (4)

T h e cortical effects may be caused by some molecule other than estrogen or progesterone that varies systematically with the estrous cycle. (5) Finally, low levels of detectable steroid receptor do not preclude a direct action of h o r m o n e u p o n cortical neurons, by analogy with the existence of low levels of detectable a n d r o g e n receptors on striated muscle (e.g. see ref. 35). These results do not specify the m o d e of action of the h o r m o n e or h o r m o n e s that are involved. However, it is important to note that whatever mechanisms produce this cyclical regulation of G A B A B receptor must function as a result of normal h o r m o n a l variations in the animal, and do not d e p e n d u p o n an adventitiously chosen temporal pattern of h o r m o n e administration, nor upon a concentration of steroid which may not normally occur. Supported by NIH Grant NS 21713 to R.H.T. Generous access was provided to laboratory facilities of Eugene Barnes. The authors thank James H. Clark for helpful suggestions during these experiments and for critically reading earlier versions of this manuscript.

Acknowledgments.

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