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Estrogen Regulation of GABA Transmission in Rat Preoptic Area
Brain Research Bulletin, Vol. 44, No. 4, pp. 321–326, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 1 .00
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Estrogen Regulation of GABA Transmission in Rat Preoptic Area ALLAN E. HERBISON1 Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, UK ABSTRACT: The medial preoptic area represents a brain region where gonadal steroids act upon classical nuclear receptors to alter brain function. Of all the neuronal phenotypes shown to express estrogen receptors in the preoptic area, GABA neurones are the most abundant and known to be located in several nuclei of the medial preoptic area. Investigators utilising techniques capable of assessing endogenous GABA levels have shown that estrogen increases both basal and stimulated extracellular GABA concentrations within the preoptic area. Experiments have also shown that estrogen is able to modulate the actions of noradrenaline upon preoptic GABA neurones. The precise nature of estrogen’s stimulatory influence on preoptic GABA concentrations is not understood fully but appears to involve changes in both the release and reuptake of GABA. As estrogen does not influence glutamic acid decarboxylase activity or gene expression in the preoptic area, the subcellular mechanism(s) through which estrogen enhances GABA release remain unknown. Recent investigations indicate that estrogen upregulates transcription of the GAT-1 GABA transporter gene in the preoptic area, and that this may contribute the stimulatory effect of estrogen on extracellular GABA concentrations. Further studies have identified effects of estrogen on GABAA receptor expression and ligand binding and, together with the above observations, demonstrate a coordinated and multifaceted upregulation of the preoptic GABA network by estrogen. It is suggested that estrogen acts directly upon GABA neurones expressing estrogen receptors to alter the dynamics of inhibitory transmission within specific neuronal networks of the preoptic area. This is likely to be of functional significance to the ‘‘feedback’’ influence of estrogen on the neural regulation of reproduction. © 1997 Elsevier Science Inc.
way for understanding the nature of these actions has been to determine the neurochemical phenotype of preoptic neurones expressing estrogen, androgen, and progesterone receptors, and thereby examine the actions of gonadal steroids on specific neuronal cell types within the various subnuclei of the preoptic area. To date, most progress has been achieved in the preoptic area of the rat where at least six different neuropeptides including galanin [6], neurotensin [2,22], calcitonin gene-related peptide [23,57], enkephalin [17,57], substance P [41], and natriuretic peptide [54] have been identified within small (,10%) subpopulations of estrogen receptor (ER)-expressing neurones of the AVPv and MPN. The recent discovery of NADPH-diaphorase staining in ER-immunoreactive neurones of the preoptic area [42] also suggests the presence of neuronal nitric oxide synthase in some of these cells. However, the most abundant ER-expressing phenotype identified so far in the preoptic area is the population of neurones synthesizing the inhibitory amino acid gamma aminobutyric acid (GABA). I wish to focus here upon recent progress in understanding the effects and mode of action of estrogen on this neuronal cell population. NEUROANATOMICAL LOCATION OF PREOPTIC GABA NEURONES EXPRESSING ESTROGEN RECEPTORS The first evidence that GABA neurones in the central nervous system were targeted by estrogen was provided by Sar and colleagues in 1983 [48] when they reported that tritiated estradiol was concentrated in GABA neurones of the MPN and bed nucleus of the stria terminalis (BNST). In a more detailed study using the same experimental approach, Flugge and co-workers [13] were to demonstrate later that approximately 20% of all MPN neurones immunoreactive for glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, concentrated estradiol, and that this accounted for around 35% of all estrogen-accumulating cells in the MPN. They also reported ‘‘single’’ examples of estradiol accumulating GAD neurones in the septum, BNST, and medial forebrain bundle. More recent studies using ER immunocytochemistry have confirmed this pattern of ER expression in GAD-immunoreactive neurones in the rat (Fig. 1) [26] and sheep [25]. In particular, it is noteworthy that a quantitatively similar pattern of ER expression in GAD neurones is found in the central part of the presumptive MPN of the sheep [25]. Studies in the rat have highlighted, in addition, the synthesis of GAD in around 50% of ER-expressing cells in the perinuclear zone of the supraoptic nucleus [26] as well as within the more lateral anteroventral preoptic nucleus (Fig. 1).
KEY WORDS: Glutamic acid decarboxylase, GABA transporter, GABAA receptor, Noradrenaline, Messenger RNA.
INTRODUCTION As part of the limbic system, the medial preoptic area is involved in the regulation of a variety of autonomic, behavioural, and neuroendocrine functions. One of the most distinctive features of the medial preoptic area is its expression of receptors for the major gonadal steroid hormones testosterone, estrogen, and progesterone [22,46,48,50]. Within the medial preoptic area, neurones expressing these receptors are located principally in the anteroventral periventricular nucleus (AVPv), periventricular preoptic nucleus, and medial preoptic nucleus (MPN). Because of its expression of gonadal steroid receptors, the medial preoptic area is believed to be one of the most important locations within the brain where circulating gonadal steroids can influence neuronal activity. One path1
To whom requests for reprints should be addressed.
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FIG. 1. (A) High power photomicrograph of double-labelled estrogen receptor (black nuclei) and GAD (gray cytoplasmic staining) immunoreactive neurones in the medial preoptic nucleus of an ovariectomised rat. Open arrows indicate cells stained for GAD alone while filled arrows indicate two estrogen receptor-expressing GABA neurones. Scale bar represents 10 mm (B) Camera lucida diagram showing the location of GAD-immunoreactive (small dots), estrogen receptor-immunoreactive (large dots), and double-labelled (filled triangles) cells in the medial preoptic area (mpoa) at the level of the caudal anteroventral periventricular nucleus (avpv) of an ovariectomised rat. Note that double-labelled cells are found within the avpv, perinuclear zone (pnz) of the supraoptic nucleus (son), and in the anteroventral nucleus (avp). ac, anterior commisure; oc, optic chiasm.
Compared with the MPN, smaller numbers of double-labelled neurones are found in the AVPv, although, again, these cells represent approximately 30% of all GAD immunoreactive neurones identified within this structure (Fig. 1). Most of the GABA neurones within the hypothalamus are believed to be short-projecting interneurones [51] and, although not proven and in need of further investigation, it is assumed that these ER-expressing GABA neurones are active in modulating electrical activity locally within the preoptic area. EFFECTS OF ESTROGEN ON PREOPTIC GABA NEURONES Wuttke and co-workers were the first to show that estrogen was likely to enhance extracellular GABA concentrations in the preoptic area of the female rat [9,34,43]. In a series of studies these workers reported that estrogen exerted a stimulatory effect upon GABA turnover within the medial preoptic area [34] and that, when measured in vivo with the push–pull technique, endogenous GABA concentrations were approximately 50% higher in the preoptic area of diestrous rats compared with ovariectomized animals [43]. Subsequent studies in our laboratory using the in vitro brain slice [18] and in vivo microdialysis techniques [19,21], confirmed the stimulatory action of estrogen on basal GABA concentrations within the preoptic area, and showed further that
depolarisation-evoked GABA release from the preoptic area was also increased by estrogen treatment. Essentially, all these studies have focused on GABA release within the MPN and immediately surrounding preoptic tissue and it is not yet clear whether GABA terminals in other preoptic nuclei identified to contain estrogen-receptive GABA neurones are influenced by estrogen in a similar manner. Our own unpublished microdialysis observations (Yuri et al) indicate that depolarisationevoked GABA release is similarly elevated by estrogen within the AVPv and surrounding rostral preoptic area. Although results from both of the above laboratories are in clear agreement regarding the stimulatory effects of estrogen on extracellular GABA concentrations in the preoptic area, Fleischmann and co-workers [12] did not find any effect of estrogen treatment on basal or evoked GABA release in vitro. Reasons for the discrepancy may lie in their use of a synaptosome preparation, prepared from whole preoptic area, in which only GABA newly synthesized from radiolabelled glutamate was measured. In the male rat, Grattan and co-workers have shown that GABA turnover in the rostral preoptic area is likely to be stimulated by testosterone in a manner similar to that of estrogen in the female rat [14]. Interestingly, in their most recent report [15], they show that an androgen receptor antagonist reduces preoptic GABA turnover suggesting, therefore, that androgens and perhaps not
ESTROGEN AND PREOPTIC GABA NETWORK
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FIG. 2. Schematic diagram depicting the pathways of GABA synthesis and degradation within medial preoptic area nerve terminals in ovariectomised (left) and estrogen-treated (right) rats. The size of the arrows and lettering and numbers of receptors indicate points of the cycle and network that are up-regulated by estrogen. GAD, glutamic acid decarboxylase; GABA-Tp, GABA transporter (GAT-1); GABA-T, GABA transaminase; GABAAR, GABAA receptor (alpha 2, beta 3, gamma 1 subunits); Glu, glutamate.
estrogens are responsible for regulating preoptic GABA levels in the male. Whether androgen receptors are involved in modulating preoptic GABA levels in the female is not known. The only other effect of estrogen identified on preoptic GABA neurones is that of regulating the stimulatory action of noradrenaline on these cells. Neuroanatomical studies show that noradrenergic neurones synapse directly on preoptic GABA cells [33] and microdialysis studies indicate that noradrenaline elevates extracellular GABA concentrations in the preoptic area through an alpha adrenergic receptor mechanism [19]. In ovariectomized and shortterm (24 h) estrogen-treated rats, electrical activation of the A1 noradrenergic input increases extracellular GABA concentrations in the preoptic area but fails to do so in rats treated with estrogen for 72 h [19]. This interesting ‘‘switch’’ appears to result from changes occurring within the preoptic area as A1-stimulated noradrenaline release is not different between ovariectomised and 72 h estrogen-treated rats. Noradrenaline seems unlikely, however, to mediate the stimulatory effects of estrogen on extracellular GABA concentrations in the preoptic area as elevated basal GABA concentrations persist in the perifusate of isolated preoptic area brain slices [18]. Together, these findings indicate that estrogen modulates extracellular GABA concentrations as well as the influence of adrenergic inputs on preoptic GABA neurones. MECHANISMS OF ESTROGEN ACTION ON THE PREOPTIC GABA NETWORK In an early attempt to define the nature of estrogen’s influence on the preoptic GABA network we demonstrated that a significant increase in basal GABA concentrations within the preoptic area occurred with a latency of approximately 2 h following an intravenous bolus administration of estrogen to ovariectomized rats
[21]. Although entirely unphysiological, this study used an experimental protocol in which estrogen had been shown to alter the electrical firing of preoptic neurones in less than 5 min [56]. Hence, the 2 h latency observed in GABA levels suggested that rapid, possibly nongenomic, actions of estrogen were unlikely to play a major role in the stimulatory effect of estrogen on preoptic GABA neurones. If estrogen was to exert a more classical genomic influence upon GABA neurones to alter extracellular GABA concentrations, the principal targets would appear to be the GABAspecific proteins GAD, the GABA transporter, and/or GABA transaminase (Fig. 2) Glutamic Acid Decarboxylase The synthesis of GABA from glutamate in the nerve terminal occurs through two forms of GAD; GAD65, which exists mostly as the inactive apoenzyme but can be activated at times of increased need, and GAD67, which exists as the active holoenzyme and is responsible for most GABA synthesis [35]. Although GAD appeared to be a prime target for estrogen to influence GABA release in the preoptic area, several studies now indicate that estrogen is unlikely to influence GAD activity or GAD mRNA content within the medial preoptic area. Leigh and colleagues [32] reported that estrogen did not alter whole preoptic area GAD67 mRNA content, and we showed that cellular GAD67 mRNA expression within MPN neurones did not alter in response to ovariectomy [24]. This is in good agreement with results from several laboratories showing that GAD enzyme activity in the preoptic area is unaffected by estrogen [12,32,39,40,53]. This is in contrast to other brain regions where, regardless of whether they express ERs or not, estrogen has been shown to regulate both forms of GAD mRNA content [38,55]. This suggests that estrogen can alter GAD mRNA content
324 in some brain nuclei in an indirect manner, possibly involving the transsynaptic regulation of GAD gene expression. Although GAD65 mRNA expression in the preoptic area has not been evaluated in terms of estrogen regulation, the absence of any change in GAD activity within this region suggests that, like GAD67, it is not the site at which estrogen acts to alter extracellular GABA concentrations within the preoptic area. An important observation to come out of the preoptic GAD studies has been that GAD mRNA expression falls in the preoptic area on the afternoon prior to the endogenous [16,24] or ovarian steroid-induced [32,52] luteinizing hormone surge. It is possible that this decline in GAD mRNA expression is the molecular event underlying the known fall in extracellular GABA concentrations within the preoptic area [30], which appears necessary for the luteinizing hormone surge to occur [20]. Initial evidence suggests that this decline results from the actions of progesterone on GAD mRNA expression [32,52]. Hence, in the sense that progesterone receptor expression is often induced by estrogen exposure [5], this repressive effect of progesterone on the preoptic GABA neurones may represent an example of an indirect inductive action of estrogen within this network. GABA Transporter Of the GABA transporters identified to date, it appears as though GAT-1 and GAT-3 are the only types to be expressed in the rat preoptic area [8,28]. We estimate that GAT-1 is responsible for approximately 75% of GABA uptake in the medial preoptic area [28]. As the GABA transporter is the other principal determinant of extracellular GABA concentrations (Fig. 2), we hypothesised that estrogen may lower GABA uptake rates and thus underlie the stimulatory action of estrogen on GABA. This possibility of estrogen actions on GABA uptake was strengthened by the observation of a near perfect (one mismatch) estrogen response element 3 prime to the open reading frame of the GAT-1 gene [28]. In a series of in situ hybridisation and functional GABA uptake studies we demonstrated that estrogen caused an approximate 25% increase in GAT-1 mRNA expression within the MPN and AVPv and a similar increase in radiolabelled GABA uptake within the medial preoptic area [28]. Thus, contrary to our hypothesis, we found that estrogen was likely to enhance GABA transport within the preoptic area. However, as the direction of GABA transport in the nerve terminal can be reversed under conditions of sustained membrane depolarisation [1,3], the estrogen-dependent increment in GABA transporter activity that we identified may be underlie the facilitatory effect of estrogen on potassium-evoked GABA release within the preoptic area [18,19]. Other Sites of Estrogen Action? The results of the GABA transporter study indicate that at least one other site of estrogen action must exist to raise basal extracellular GABA concentrations. In recent experiments we have gone back to evaluate whether there is, in fact, enhanced basal GABA release in the presence of estrogen. Results of experiments undertaken with microdialysis and using GABA transporter blockers to evaluate release in the absence of uptake, show clearly that elevated GABA release occurs in the medial preoptic area in presence of estrogen (Fig. 2; Herbison, unpublished). The precise site of estrogen action in achieving this increased release is unknown. One possibility is that it may involve estrogen regulation of proteins involved more generally in vesicle transport and release within the terminal. Further, as a significant amount of GABA release within the brain is spontaneous [45] and calcium independent in nature [4], effects of estrogen on nonclassical release systems must also be considered. Actions of estrogen on GABA
HERBISON transaminase have not been assessed, although it is not clear how they would influence the release process (Fig. 2). GABA Receptors in the Preoptic Area A comprehensive evaluation of estrogen actions within the preoptic GABA network requires consideration of its potential for influencing GABA receptor functioning as well as GABA release. Previous studies have provided conflicting reports on whether estrogen alters GABAA receptor ligand binding in the preoptic area of the female [7,40]. In a recent study we have shown that the alpha 2, beta 3, and gamma 1 subunits of the GABAA receptor are all highly expressed by the great majority of MPN neurones and that up to two-thirds of these cells also synthesized ERs [27]. In situ hybridization analysis of cellular GABAA receptor subunit mRNA expression in the MPN demonstrated that estrogen replacement at diestrous levels for 7 days resulted in increased cellular alpha 2 and gamma 1 mRNA content within the MPN [27]. Together, these findings suggest that estrogen also has the ability to influence GABAA receptor mRNA expression in the MPN, and that if these changes are translated to a functional upregulation of GABAA receptor expression by these cells, then estrogen would indeed enhance both GABA release and GABAA receptor-mediated inhibition within the preoptic area (Fig. 2). Other studies demonstrated that elevated endogenous GABA levels in vivo do not themselves result in any autoregulation of GABAA receptor subunit mRNA expression in the preoptic area [10]. Precisely which MPN cells express these particular estrogen-regulated GABAA receptor subunits remains to be determined, but it seems likely that some of these cells would be the estrogen-receptive GABA neurones themselves. CONCLUSIONS AND PHYSIOLOGICAL SIGNIFICANCE Estrogen acts to increase GABA release and reuptake as well as GABAA receptor expression within the medial preoptic area (Fig. 2) and, at high concentrations, reduces the stimulatory effect of noradrenaline on preoptic GABA neurones. This latter action suggests that certain preoptic GABA neurones may form a relatively powerful estrogen-sensitive ‘‘gate’’ in terms of noradrenergic influences in this region. Although providing an excellent example of the multifaceted manner in which a gonadal steroid can influence GABA neurotransmission in a specific brain region, the functional implications of estrogen’s influence are not entirely clear. The elevated GABA release and reuptake coupled with the possibility of increased GABAA receptor expression on target neurones indicates that estrogen may ‘‘reshape’’ GABAergic transmission within the preoptic area. Future studies will be required to test this suggestion and, indeed, determine its functional significance within the preoptic area. At a network level, the overall effect of estrogen is to increase extracellular GABA concentrations in the preoptic area and, simplistically, this would suggest greater levels of inhibition within the preoptic networks utilizing estrogen-receptive GABA neurones. Previous studies that have manipulated GABA receptor functioning specifically within the preoptic area using microinfusion techniques have shown that preoptic GABA networks are involved in regulating reproductive hormone secretion [20,31,49], both male [11] and female [37] reproductive behavior and more general parameters such as locomotion and grooming [44]. At present, it is difficult to establish which preoptic networks involve estrogen-sensitive GABA neurones. In the case of luteinizing hormone secretion, it is suggested that estrogen-receptive GABA neurones of the preoptic area are involved in mediating part of the negative feedback actions of estrogen on gonadotrophin-releasing
ESTROGEN AND PREOPTIC GABA NETWORK hormone neurones [9,21,34]. Furthermore, the uncoupling of noradrenaline’s stimulatory influence upon preoptic GABA neurones by estrogen prior to the luteinizing hormone surge [19] would seem essential if the release profiles of these two neurotransmitters are to diverge as they do to enable the luteinizing hormone surge to occur [29]. Although some limited functional interpretations are possible at present, the challenge for the future is to establish precisely which subpopulations of GABA neurones within the preoptic nuclei are involved in which regulatory neuronal networks. As essentially all preoptic neurones express functional GABA receptors [36], this will be essential before the full functional significance of estrogen-dependent GABA signaling is realized within the preoptic area of the rat.
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The writer is indebted to colleagues within the Laboratory of Neuroendocrinology who have been involved in our contribution to research in this field but holds sole responsibility for the views expressed. AEH is a Lister Institute-Jenner Fellow.
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REFERENCES 1. Attwell, D.; Barbour, B.; Szatkowski, M. Nonvesicular release of neurotransmitter. Neuron 11:401– 407; 1993. 2. Axelson, J. F.; Shannon, W.; van Leeuwen, F. W. Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus. Neurosci. Lett. 136:5–9; 1992. 3. Belhage, B.; Hansen, G. H.; Schousboe, A. Depolarization by K1 and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: Vesicular versus non-vesicular release of GABA. Neuroscience 54:1019 –1034; 1993. 4. Bernath, S. Calcium-independent release of amino acid neurotransmitters: Fact or artifact? Prog. Neurobiol. 38:57–91; 1992. 5. Blaustein, J. D.; Turcotte, J. C. Estradiol-induced progestin receptor immunoreactivity is found only in estrogen receptor-immunoreactive cells in guinea pig brain. Neuroendocrinology 49:454 – 461; 1989. 6. Bloch, G. J.; Kurth, S. M.; Akesson, T. R.; Micevych, P. E. Estrogenconcentrating cells within cell groups of the medial preoptic area: Sex differences and co-localization with galanin-immunoreactive cells. Brain Res. 595:301–308; 1992. 7. Canonaco, M.; Tavolaro, R.; Maggi, A. Steroid hormones and receptors of the GABAA supramolecular complex. Neuroendocrinology 57:974 –984; 1993. 8. Clark, J. A.; Deutch, A. Y.; Gallipoli, P. Z.; Amara, S. G. Functional expression and CNS distribution of a b-alanine-sensitive neuronal GABA transporter. Neuron 9:337–348; 1992. 9. Demling, J.; Fuchs, E.; Baumert, M.; Wuttke, W. Preoptic catecholamine, GABA, and glutamate release in ovariectomised and ovariectomised estrogen-primed rats utilizing a push-pull cannula technique. Neuroendocrinology 41:212–218; 1985. 10. Fenelon, V. S.; Herbison, A. E. In vivo regulation of specific GABAA receptor subunit messenger RNAs by increased GABA concentrations in rat brain. Neuroscience 71:661– 670; 1996. 11. Fernandez–Guasti, A.; Larrson, K.; Beyer, C. Comparison of the effects of different isomers of bicuculline infused in the preoptic area on male rat sexual behavior. Experientia 41:1414 –1416; 1985. 12. Fleischmann, A.; Makman, M. H.; Etgen, A. M. Ovarian steroids increase veratridine-induced release of amino acid neurotransmitters in preoptic area synaptosomes. Brain Res. 507:161–163; 1990. 13. Flugge, G.; Oertel, W. H.; Wuttke, W. Evidence for estrogen-receptive GABAergic neurons in the preoptic/anterior hypothalamic area of the rat brain. Neuroendocrinology 43:1–5; 1986. 14. Grattan, D. R.; Selmanoff, M. Castration-induced decrease in the activity of medial preoptic and tuberoinfundibular GABAergic neurons is prevented by testosterone. Neuroendocrinology 60:141–149; 1994. 15. Grattan, D. R.; Rocca, M. S.; Sagrillo, C. A.; McCarthy, M. M.; Selmanoff, M. Antiandrogen microimplants into the rostral medial
22.
23.
24.
25.
26.
27.
28.
29. 30.
31.
32.
33.
34.
preoptic area decrease g-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male. Endocrinology 137:4167– 4173; 1996. Grattan, D. R.; Rocca, M. S.; Strauss, K. I.; Sagrillo, C. A.; Selmanoff, M.; McCarthy, M. M. GABAergic neuronal activity and mRNA levels for both forms of glutamic acid decarboxylase (GAD65 and GAD67) are reduced in the diagonal band of Broca during the afternoon of proestrus. Brain Res. 733:46 –55; 1996. Gu, G.; Simerly, B. Hormonal regulation of opioid peptide neurons in the anteroventral periventricular nucleus. Horm. Behav. 28:503–511; 1994. Herbison, A. E.; Heavens, R. P.; Dyer, R. G. Oestrogen and noradrenaline modulate endogenous GABA release from slices of the rat medial preoptic area. Brain Res. 486:195–200; 1989. Herbison, A. E.; Heavens, R. P.; Dyer, R. G. Oestrogen modulation of excitatory A1 noradrenergic input to rat medial preoptic gamma aminobutyric acid neurones demonstrated by microdialysis. Neuroendocrinology 52:161–168; 1990. Herbison, A. E.; Dyer, R. G. Effect on luteinizing hormone secretion of GABA receptor modulation in the medial preoptic area at the time of proestrous luteinising hormone surge. Neuroendocrinology 53:317– 320; 1991. Herbison, A. E.; Heavens, R. P.; Dye, S.: Dyer, R. G. Acute action of oestrogen on medial preoptic gamma-aminobutyric acid neurons: Correlation with oestrogen negative feedback on luteinizing hormone secretion. J. Neuroendocrinol. 3:101–106; 1991. Herbison, A. E.; Theodosis, D. T. Localisation of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone in the male and female rat. Neuroscience 50:283–298; 1992. Herbison, A. E.; Theodosis, D. T. Immunocytochemical identification of oestrogen receptors in preoptic neurones containing calcitonin generelated peptide in the male and female rat. Neuroendocrinology 56: 761–764; 1992. Herbison, A. E.; Augood, S. J.; McGowan, E. M. Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy. Mol. Brain Res. 14:310 –316; 1992. Herbison, A. E.; Robinson, J. E.; Skinner, D. C. Distribution of estrogen receptor-immunoreactive cells in the preoptic area of the ewe: Co-localization with glutamic acid decarboxylase but not luteinizing hormone-releasing hormone. Neuroendocrinology 57:751–759; 1993. Herbison, A. E. Immunocytochemical evidence for oestrogen receptors within GABA neurones located in the perinuclear zone of the supraoptic nucleus and GABAA receptor b2/3 subunits on supraoptic oxytocin neurones. J. Neuroendocrinol. 6:5–11; 1994. Herbison, A. E.; Fenelon, V. S. Estrogen regulation of GABAA receptor subunit mRNA expression in preoptic area and bed nucleus of the stria terminalis of female rat brain. J. Neurosci. 15:2328 –2337; 1995. Herbison, A. E.; Augood, S. J.; Simonian, S. X.; Chapman, C. Regulation of GABA transporter activity and mRNA expression by estrogen in rat preoptic area. J. Neurosci. 15:8302– 8309; 1996. Herbison, A. E. Noradrenergic regulation of cyclic GnRH secretion. Rev. Reprod. 2:1– 6; 1997. Jarry, H.; Perschl, A.; Wuttke, W. Further evidence that preoptic anterior hypothalamic GABAergic neurons are part of the GnRH pulse and surge generator. Acta Endocrinol. 118:573–579; 1988. Lamberts, R.; Vijayan, E.; Graf, M.; Mansky, T.; Wuttke, W. Involvement of preoptic-anterior hypothalamic GABA neurons in the regulation of pituitary LH and prolactin release. Exp. Brain Res. 52:356 – 362; 1983. Leigh, A. J.; Carter, N. D.; Horton, R.; Silverlight, J. J.; Wilson, C. A. Ovarian steroid regulation of glutamic acid decarboxylase gene expression in individual hypothalamic nuclei. J. Neuroendocrinol. 2: 433– 438; 1990. Leranth, C.; MacLusky, N. J.; Shanabrough, M.; Naftolin, F. Catecholaminergic innervation of luteinizing hormone-releasing hormone and glutamic acid decarboxylase immunopositive neurons in the rat medial preoptic area. Neuroendocrinology 48:591– 602; 1988. Mansky, T.; Mestres–Ventura, P.; Wuttke, W. Involvement of GABA in the feedback action of estradiol on gonadotropin and prolactin
326
35. 36. 37.
38.
39.
40. 41.
42.
43. 44. 45. 46.
HERBISON release: Hypothalamic GABA and catecholamine turnover rates. Brain Res. 231:353–364; 1982. Martin, D. L.; Rimvall, K. Regulation of gamma-aminobutyric acid synthesis in the brain. J. Neurochem. 60:395– 407; 1993. Mayer, M. L. Electrophysiological analysis of inhibitory synaptic mechanisms in the preoptic area of the rat. J. Physiol. 316:327–346; 1981. McCarthy, M. M.; Malik, K. F.; Feder, H. H. Increased GABAergic neurotransmission in medial hypothalamus facilitates lordosis but has opposite effects in the preoptic area/anterior hypothalamus. Brain Res. 507:40 – 43; 1990. McCarthy, M. M.; Kaufman, L. C.; Brooks, P. J.; Pfaff, D. W.; Schwartz-Giblin, S. Estrogen modulation of mRNA levels for two forms of glutamic acid decarboxylase (GAD) in the female rat brain. J. Comp. Neurol. 360:685– 697; 1995. Munaro, N. I.; Dotti, C.; Taleisnik, S. Glutamic acid decarboxylase activity in the hypothalamus of the rat: Effects of estrogens. In: Donoso, A. O.; Racagni, G., eds. GABA and endocrine function. Raven: New York; 1986:201–207. O’Connor, L. H.; Nock, B.; McEwen, B. S. Regional specificity of gamma-aminobutyric acid receptor regulation by estradiol. Neuroendocrinology 47:473– 481; 1988. Okamura, H.; Yokosuka, M.; Hayashi, S. Induction of substance P-immunoreactivity by estrogen in neurons containing estrogen receptors in the anteroventral periventricular nucleus of female but not male rats. J. Neuroendocrinol. 6:609 – 615; 1994. Okamura, H.; Yokusuka, M.; Hayashi, S. Estrogenic induction of NADPH-diaphorase activity in the preoptic neurons containing estrogen receptor immunoreactivity in the female rat. J. Neuroendocrinol. 6:597– 601; 1994. Ondo, J.; Mansky, T.; Wuttke. W. In vivo GABA release from the medial preoptic area of diestrous and ovariectomized rats. Exp. Brain Res. 46:69 –72; 1982. Osborne, P. G.; Mataga, N.; Onoe, H.; Watanabe, Y. Behavioral activation by stimulation of a GABAergic mechanism in the preoptic area of rat. Neurosci. Lett. 158:201–204; 1993. Otis, T. S.; Staley, K. J.; Mody, I. Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res. 545:142–150; 1991. Pfaff, D.; Keiner, M. Atlas of estradiol-concentrating cells in the
47.
48. 49.
50.
51. 52.
53. 54.
55. 56. 57.
central nervous system of the female rat. J. Comp. Neurol. 151:121– 158; 1973. Sar, M. Distribution of progestin-concentrating cells in rat brain: Colocalization of [3H]ORG.2058, a synthetic progestin, and antibodies to tyrosine hydroxylase in hypothalamus by combined autoradiography and immunocytochemistry. Endocrinology 123:1110 –1118; 1988. Sar, M.; Stumpf, W. E.; Tappaz, M. L. Localization of 3H estradiol in preoptic GABA neurons. Fed. Proc. 42:495; 1983. Seltzer, A. M.; Donoso, A. O. Restraining action of GABA on estradiol-induced LH surge in the rat: GABA activity in brain nuclei and effects of GABA mimetics in the medial preoptic nucleus. Neuroendocrinology 55:28 –34; 1992. Simerly, R. B.; Chang, C.; Muramatsu, M.; Swanson, L. W. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: An in situ hybridization study. J. Comp. Neurol. 294:76 –95; 1990. Tappaz, M. L.; Brownstein, M. J. Origin of glutamate-decarboxylase (GAD)-containing cells in discrete hypothalamic nuclei. Brain Res. 132:95–106; 1977. Unda, R.; Brann, D. W.; Mahesh, V. B. Progesterone suppression of glutamic acid decarboxylase (GAD67) mRNA levels in the preoptic area: Correlation to the luteinizing hormone surge. Neuroendocrinology 62:562–570; 1995. Wallis, C. J.; Luttge, W. G. Influence of estrogen and progesterone on glutamic acid decarboxylase activity in discrete regions of rat brain. J. Neurochem. 34:609 – 613; 1980. Watson, R. E.; Hutchinson, R. K.; Langjub, M. C.; Landis, J. W.; Seksaria, S.; Rainey, D. M.; Keil, L. C. Colocalization of natriuretic peptide and estrogen receptor immunoreactivities in preoptic nuclei in the female rat. J. Neuroendocrinol. 6:79 – 87; 1994. Weiland, N. G. Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus. Endocrinology 131:2697–2702; 1993. Yagi, K. Changes in firing rates of single preoptic and hypothalamic units following an intravenous administration of estrogen in the castrated female rat. Brain Res. 53:343–352; 1973. Yuri, K.; Kawata, M. Estrogen receptor-immunoreactive neurons contain calcitonin gene-related peptide, methionine-enkephalin or tyrosine hydroxylase in the female rat preoptic area. Neurosci. Res. 21:135– 141; 1994.
PII S0361-9230(97)00210-4
Estrogen Regulation of GABA Transmission in Rat Preoptic Area ALLAN E. HERBISON1 Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, UK ABSTRACT: The medial preoptic area represents a brain region where gonadal steroids act upon classical nuclear receptors to alter brain function. Of all the neuronal phenotypes shown to express estrogen receptors in the preoptic area, GABA neurones are the most abundant and known to be located in several nuclei of the medial preoptic area. Investigators utilising techniques capable of assessing endogenous GABA levels have shown that estrogen increases both basal and stimulated extracellular GABA concentrations within the preoptic area. Experiments have also shown that estrogen is able to modulate the actions of noradrenaline upon preoptic GABA neurones. The precise nature of estrogen’s stimulatory influence on preoptic GABA concentrations is not understood fully but appears to involve changes in both the release and reuptake of GABA. As estrogen does not influence glutamic acid decarboxylase activity or gene expression in the preoptic area, the subcellular mechanism(s) through which estrogen enhances GABA release remain unknown. Recent investigations indicate that estrogen upregulates transcription of the GAT-1 GABA transporter gene in the preoptic area, and that this may contribute the stimulatory effect of estrogen on extracellular GABA concentrations. Further studies have identified effects of estrogen on GABAA receptor expression and ligand binding and, together with the above observations, demonstrate a coordinated and multifaceted upregulation of the preoptic GABA network by estrogen. It is suggested that estrogen acts directly upon GABA neurones expressing estrogen receptors to alter the dynamics of inhibitory transmission within specific neuronal networks of the preoptic area. This is likely to be of functional significance to the ‘‘feedback’’ influence of estrogen on the neural regulation of reproduction. © 1997 Elsevier Science Inc.
way for understanding the nature of these actions has been to determine the neurochemical phenotype of preoptic neurones expressing estrogen, androgen, and progesterone receptors, and thereby examine the actions of gonadal steroids on specific neuronal cell types within the various subnuclei of the preoptic area. To date, most progress has been achieved in the preoptic area of the rat where at least six different neuropeptides including galanin [6], neurotensin [2,22], calcitonin gene-related peptide [23,57], enkephalin [17,57], substance P [41], and natriuretic peptide [54] have been identified within small (,10%) subpopulations of estrogen receptor (ER)-expressing neurones of the AVPv and MPN. The recent discovery of NADPH-diaphorase staining in ER-immunoreactive neurones of the preoptic area [42] also suggests the presence of neuronal nitric oxide synthase in some of these cells. However, the most abundant ER-expressing phenotype identified so far in the preoptic area is the population of neurones synthesizing the inhibitory amino acid gamma aminobutyric acid (GABA). I wish to focus here upon recent progress in understanding the effects and mode of action of estrogen on this neuronal cell population. NEUROANATOMICAL LOCATION OF PREOPTIC GABA NEURONES EXPRESSING ESTROGEN RECEPTORS The first evidence that GABA neurones in the central nervous system were targeted by estrogen was provided by Sar and colleagues in 1983 [48] when they reported that tritiated estradiol was concentrated in GABA neurones of the MPN and bed nucleus of the stria terminalis (BNST). In a more detailed study using the same experimental approach, Flugge and co-workers [13] were to demonstrate later that approximately 20% of all MPN neurones immunoreactive for glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, concentrated estradiol, and that this accounted for around 35% of all estrogen-accumulating cells in the MPN. They also reported ‘‘single’’ examples of estradiol accumulating GAD neurones in the septum, BNST, and medial forebrain bundle. More recent studies using ER immunocytochemistry have confirmed this pattern of ER expression in GAD-immunoreactive neurones in the rat (Fig. 1) [26] and sheep [25]. In particular, it is noteworthy that a quantitatively similar pattern of ER expression in GAD neurones is found in the central part of the presumptive MPN of the sheep [25]. Studies in the rat have highlighted, in addition, the synthesis of GAD in around 50% of ER-expressing cells in the perinuclear zone of the supraoptic nucleus [26] as well as within the more lateral anteroventral preoptic nucleus (Fig. 1).
KEY WORDS: Glutamic acid decarboxylase, GABA transporter, GABAA receptor, Noradrenaline, Messenger RNA.
INTRODUCTION As part of the limbic system, the medial preoptic area is involved in the regulation of a variety of autonomic, behavioural, and neuroendocrine functions. One of the most distinctive features of the medial preoptic area is its expression of receptors for the major gonadal steroid hormones testosterone, estrogen, and progesterone [22,46,48,50]. Within the medial preoptic area, neurones expressing these receptors are located principally in the anteroventral periventricular nucleus (AVPv), periventricular preoptic nucleus, and medial preoptic nucleus (MPN). Because of its expression of gonadal steroid receptors, the medial preoptic area is believed to be one of the most important locations within the brain where circulating gonadal steroids can influence neuronal activity. One path1
To whom requests for reprints should be addressed.
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FIG. 1. (A) High power photomicrograph of double-labelled estrogen receptor (black nuclei) and GAD (gray cytoplasmic staining) immunoreactive neurones in the medial preoptic nucleus of an ovariectomised rat. Open arrows indicate cells stained for GAD alone while filled arrows indicate two estrogen receptor-expressing GABA neurones. Scale bar represents 10 mm (B) Camera lucida diagram showing the location of GAD-immunoreactive (small dots), estrogen receptor-immunoreactive (large dots), and double-labelled (filled triangles) cells in the medial preoptic area (mpoa) at the level of the caudal anteroventral periventricular nucleus (avpv) of an ovariectomised rat. Note that double-labelled cells are found within the avpv, perinuclear zone (pnz) of the supraoptic nucleus (son), and in the anteroventral nucleus (avp). ac, anterior commisure; oc, optic chiasm.
Compared with the MPN, smaller numbers of double-labelled neurones are found in the AVPv, although, again, these cells represent approximately 30% of all GAD immunoreactive neurones identified within this structure (Fig. 1). Most of the GABA neurones within the hypothalamus are believed to be short-projecting interneurones [51] and, although not proven and in need of further investigation, it is assumed that these ER-expressing GABA neurones are active in modulating electrical activity locally within the preoptic area. EFFECTS OF ESTROGEN ON PREOPTIC GABA NEURONES Wuttke and co-workers were the first to show that estrogen was likely to enhance extracellular GABA concentrations in the preoptic area of the female rat [9,34,43]. In a series of studies these workers reported that estrogen exerted a stimulatory effect upon GABA turnover within the medial preoptic area [34] and that, when measured in vivo with the push–pull technique, endogenous GABA concentrations were approximately 50% higher in the preoptic area of diestrous rats compared with ovariectomized animals [43]. Subsequent studies in our laboratory using the in vitro brain slice [18] and in vivo microdialysis techniques [19,21], confirmed the stimulatory action of estrogen on basal GABA concentrations within the preoptic area, and showed further that
depolarisation-evoked GABA release from the preoptic area was also increased by estrogen treatment. Essentially, all these studies have focused on GABA release within the MPN and immediately surrounding preoptic tissue and it is not yet clear whether GABA terminals in other preoptic nuclei identified to contain estrogen-receptive GABA neurones are influenced by estrogen in a similar manner. Our own unpublished microdialysis observations (Yuri et al) indicate that depolarisationevoked GABA release is similarly elevated by estrogen within the AVPv and surrounding rostral preoptic area. Although results from both of the above laboratories are in clear agreement regarding the stimulatory effects of estrogen on extracellular GABA concentrations in the preoptic area, Fleischmann and co-workers [12] did not find any effect of estrogen treatment on basal or evoked GABA release in vitro. Reasons for the discrepancy may lie in their use of a synaptosome preparation, prepared from whole preoptic area, in which only GABA newly synthesized from radiolabelled glutamate was measured. In the male rat, Grattan and co-workers have shown that GABA turnover in the rostral preoptic area is likely to be stimulated by testosterone in a manner similar to that of estrogen in the female rat [14]. Interestingly, in their most recent report [15], they show that an androgen receptor antagonist reduces preoptic GABA turnover suggesting, therefore, that androgens and perhaps not
ESTROGEN AND PREOPTIC GABA NETWORK
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FIG. 2. Schematic diagram depicting the pathways of GABA synthesis and degradation within medial preoptic area nerve terminals in ovariectomised (left) and estrogen-treated (right) rats. The size of the arrows and lettering and numbers of receptors indicate points of the cycle and network that are up-regulated by estrogen. GAD, glutamic acid decarboxylase; GABA-Tp, GABA transporter (GAT-1); GABA-T, GABA transaminase; GABAAR, GABAA receptor (alpha 2, beta 3, gamma 1 subunits); Glu, glutamate.
estrogens are responsible for regulating preoptic GABA levels in the male. Whether androgen receptors are involved in modulating preoptic GABA levels in the female is not known. The only other effect of estrogen identified on preoptic GABA neurones is that of regulating the stimulatory action of noradrenaline on these cells. Neuroanatomical studies show that noradrenergic neurones synapse directly on preoptic GABA cells [33] and microdialysis studies indicate that noradrenaline elevates extracellular GABA concentrations in the preoptic area through an alpha adrenergic receptor mechanism [19]. In ovariectomized and shortterm (24 h) estrogen-treated rats, electrical activation of the A1 noradrenergic input increases extracellular GABA concentrations in the preoptic area but fails to do so in rats treated with estrogen for 72 h [19]. This interesting ‘‘switch’’ appears to result from changes occurring within the preoptic area as A1-stimulated noradrenaline release is not different between ovariectomised and 72 h estrogen-treated rats. Noradrenaline seems unlikely, however, to mediate the stimulatory effects of estrogen on extracellular GABA concentrations in the preoptic area as elevated basal GABA concentrations persist in the perifusate of isolated preoptic area brain slices [18]. Together, these findings indicate that estrogen modulates extracellular GABA concentrations as well as the influence of adrenergic inputs on preoptic GABA neurones. MECHANISMS OF ESTROGEN ACTION ON THE PREOPTIC GABA NETWORK In an early attempt to define the nature of estrogen’s influence on the preoptic GABA network we demonstrated that a significant increase in basal GABA concentrations within the preoptic area occurred with a latency of approximately 2 h following an intravenous bolus administration of estrogen to ovariectomized rats
[21]. Although entirely unphysiological, this study used an experimental protocol in which estrogen had been shown to alter the electrical firing of preoptic neurones in less than 5 min [56]. Hence, the 2 h latency observed in GABA levels suggested that rapid, possibly nongenomic, actions of estrogen were unlikely to play a major role in the stimulatory effect of estrogen on preoptic GABA neurones. If estrogen was to exert a more classical genomic influence upon GABA neurones to alter extracellular GABA concentrations, the principal targets would appear to be the GABAspecific proteins GAD, the GABA transporter, and/or GABA transaminase (Fig. 2) Glutamic Acid Decarboxylase The synthesis of GABA from glutamate in the nerve terminal occurs through two forms of GAD; GAD65, which exists mostly as the inactive apoenzyme but can be activated at times of increased need, and GAD67, which exists as the active holoenzyme and is responsible for most GABA synthesis [35]. Although GAD appeared to be a prime target for estrogen to influence GABA release in the preoptic area, several studies now indicate that estrogen is unlikely to influence GAD activity or GAD mRNA content within the medial preoptic area. Leigh and colleagues [32] reported that estrogen did not alter whole preoptic area GAD67 mRNA content, and we showed that cellular GAD67 mRNA expression within MPN neurones did not alter in response to ovariectomy [24]. This is in good agreement with results from several laboratories showing that GAD enzyme activity in the preoptic area is unaffected by estrogen [12,32,39,40,53]. This is in contrast to other brain regions where, regardless of whether they express ERs or not, estrogen has been shown to regulate both forms of GAD mRNA content [38,55]. This suggests that estrogen can alter GAD mRNA content
324 in some brain nuclei in an indirect manner, possibly involving the transsynaptic regulation of GAD gene expression. Although GAD65 mRNA expression in the preoptic area has not been evaluated in terms of estrogen regulation, the absence of any change in GAD activity within this region suggests that, like GAD67, it is not the site at which estrogen acts to alter extracellular GABA concentrations within the preoptic area. An important observation to come out of the preoptic GAD studies has been that GAD mRNA expression falls in the preoptic area on the afternoon prior to the endogenous [16,24] or ovarian steroid-induced [32,52] luteinizing hormone surge. It is possible that this decline in GAD mRNA expression is the molecular event underlying the known fall in extracellular GABA concentrations within the preoptic area [30], which appears necessary for the luteinizing hormone surge to occur [20]. Initial evidence suggests that this decline results from the actions of progesterone on GAD mRNA expression [32,52]. Hence, in the sense that progesterone receptor expression is often induced by estrogen exposure [5], this repressive effect of progesterone on the preoptic GABA neurones may represent an example of an indirect inductive action of estrogen within this network. GABA Transporter Of the GABA transporters identified to date, it appears as though GAT-1 and GAT-3 are the only types to be expressed in the rat preoptic area [8,28]. We estimate that GAT-1 is responsible for approximately 75% of GABA uptake in the medial preoptic area [28]. As the GABA transporter is the other principal determinant of extracellular GABA concentrations (Fig. 2), we hypothesised that estrogen may lower GABA uptake rates and thus underlie the stimulatory action of estrogen on GABA. This possibility of estrogen actions on GABA uptake was strengthened by the observation of a near perfect (one mismatch) estrogen response element 3 prime to the open reading frame of the GAT-1 gene [28]. In a series of in situ hybridisation and functional GABA uptake studies we demonstrated that estrogen caused an approximate 25% increase in GAT-1 mRNA expression within the MPN and AVPv and a similar increase in radiolabelled GABA uptake within the medial preoptic area [28]. Thus, contrary to our hypothesis, we found that estrogen was likely to enhance GABA transport within the preoptic area. However, as the direction of GABA transport in the nerve terminal can be reversed under conditions of sustained membrane depolarisation [1,3], the estrogen-dependent increment in GABA transporter activity that we identified may be underlie the facilitatory effect of estrogen on potassium-evoked GABA release within the preoptic area [18,19]. Other Sites of Estrogen Action? The results of the GABA transporter study indicate that at least one other site of estrogen action must exist to raise basal extracellular GABA concentrations. In recent experiments we have gone back to evaluate whether there is, in fact, enhanced basal GABA release in the presence of estrogen. Results of experiments undertaken with microdialysis and using GABA transporter blockers to evaluate release in the absence of uptake, show clearly that elevated GABA release occurs in the medial preoptic area in presence of estrogen (Fig. 2; Herbison, unpublished). The precise site of estrogen action in achieving this increased release is unknown. One possibility is that it may involve estrogen regulation of proteins involved more generally in vesicle transport and release within the terminal. Further, as a significant amount of GABA release within the brain is spontaneous [45] and calcium independent in nature [4], effects of estrogen on nonclassical release systems must also be considered. Actions of estrogen on GABA
HERBISON transaminase have not been assessed, although it is not clear how they would influence the release process (Fig. 2). GABA Receptors in the Preoptic Area A comprehensive evaluation of estrogen actions within the preoptic GABA network requires consideration of its potential for influencing GABA receptor functioning as well as GABA release. Previous studies have provided conflicting reports on whether estrogen alters GABAA receptor ligand binding in the preoptic area of the female [7,40]. In a recent study we have shown that the alpha 2, beta 3, and gamma 1 subunits of the GABAA receptor are all highly expressed by the great majority of MPN neurones and that up to two-thirds of these cells also synthesized ERs [27]. In situ hybridization analysis of cellular GABAA receptor subunit mRNA expression in the MPN demonstrated that estrogen replacement at diestrous levels for 7 days resulted in increased cellular alpha 2 and gamma 1 mRNA content within the MPN [27]. Together, these findings suggest that estrogen also has the ability to influence GABAA receptor mRNA expression in the MPN, and that if these changes are translated to a functional upregulation of GABAA receptor expression by these cells, then estrogen would indeed enhance both GABA release and GABAA receptor-mediated inhibition within the preoptic area (Fig. 2). Other studies demonstrated that elevated endogenous GABA levels in vivo do not themselves result in any autoregulation of GABAA receptor subunit mRNA expression in the preoptic area [10]. Precisely which MPN cells express these particular estrogen-regulated GABAA receptor subunits remains to be determined, but it seems likely that some of these cells would be the estrogen-receptive GABA neurones themselves. CONCLUSIONS AND PHYSIOLOGICAL SIGNIFICANCE Estrogen acts to increase GABA release and reuptake as well as GABAA receptor expression within the medial preoptic area (Fig. 2) and, at high concentrations, reduces the stimulatory effect of noradrenaline on preoptic GABA neurones. This latter action suggests that certain preoptic GABA neurones may form a relatively powerful estrogen-sensitive ‘‘gate’’ in terms of noradrenergic influences in this region. Although providing an excellent example of the multifaceted manner in which a gonadal steroid can influence GABA neurotransmission in a specific brain region, the functional implications of estrogen’s influence are not entirely clear. The elevated GABA release and reuptake coupled with the possibility of increased GABAA receptor expression on target neurones indicates that estrogen may ‘‘reshape’’ GABAergic transmission within the preoptic area. Future studies will be required to test this suggestion and, indeed, determine its functional significance within the preoptic area. At a network level, the overall effect of estrogen is to increase extracellular GABA concentrations in the preoptic area and, simplistically, this would suggest greater levels of inhibition within the preoptic networks utilizing estrogen-receptive GABA neurones. Previous studies that have manipulated GABA receptor functioning specifically within the preoptic area using microinfusion techniques have shown that preoptic GABA networks are involved in regulating reproductive hormone secretion [20,31,49], both male [11] and female [37] reproductive behavior and more general parameters such as locomotion and grooming [44]. At present, it is difficult to establish which preoptic networks involve estrogen-sensitive GABA neurones. In the case of luteinizing hormone secretion, it is suggested that estrogen-receptive GABA neurones of the preoptic area are involved in mediating part of the negative feedback actions of estrogen on gonadotrophin-releasing
ESTROGEN AND PREOPTIC GABA NETWORK hormone neurones [9,21,34]. Furthermore, the uncoupling of noradrenaline’s stimulatory influence upon preoptic GABA neurones by estrogen prior to the luteinizing hormone surge [19] would seem essential if the release profiles of these two neurotransmitters are to diverge as they do to enable the luteinizing hormone surge to occur [29]. Although some limited functional interpretations are possible at present, the challenge for the future is to establish precisely which subpopulations of GABA neurones within the preoptic nuclei are involved in which regulatory neuronal networks. As essentially all preoptic neurones express functional GABA receptors [36], this will be essential before the full functional significance of estrogen-dependent GABA signaling is realized within the preoptic area of the rat.
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The writer is indebted to colleagues within the Laboratory of Neuroendocrinology who have been involved in our contribution to research in this field but holds sole responsibility for the views expressed. AEH is a Lister Institute-Jenner Fellow.
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REFERENCES 1. Attwell, D.; Barbour, B.; Szatkowski, M. Nonvesicular release of neurotransmitter. Neuron 11:401– 407; 1993. 2. Axelson, J. F.; Shannon, W.; van Leeuwen, F. W. Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus. Neurosci. Lett. 136:5–9; 1992. 3. Belhage, B.; Hansen, G. H.; Schousboe, A. Depolarization by K1 and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: Vesicular versus non-vesicular release of GABA. Neuroscience 54:1019 –1034; 1993. 4. Bernath, S. Calcium-independent release of amino acid neurotransmitters: Fact or artifact? Prog. Neurobiol. 38:57–91; 1992. 5. Blaustein, J. D.; Turcotte, J. C. Estradiol-induced progestin receptor immunoreactivity is found only in estrogen receptor-immunoreactive cells in guinea pig brain. Neuroendocrinology 49:454 – 461; 1989. 6. Bloch, G. J.; Kurth, S. M.; Akesson, T. R.; Micevych, P. E. Estrogenconcentrating cells within cell groups of the medial preoptic area: Sex differences and co-localization with galanin-immunoreactive cells. Brain Res. 595:301–308; 1992. 7. Canonaco, M.; Tavolaro, R.; Maggi, A. Steroid hormones and receptors of the GABAA supramolecular complex. Neuroendocrinology 57:974 –984; 1993. 8. Clark, J. A.; Deutch, A. Y.; Gallipoli, P. Z.; Amara, S. G. Functional expression and CNS distribution of a b-alanine-sensitive neuronal GABA transporter. Neuron 9:337–348; 1992. 9. Demling, J.; Fuchs, E.; Baumert, M.; Wuttke, W. Preoptic catecholamine, GABA, and glutamate release in ovariectomised and ovariectomised estrogen-primed rats utilizing a push-pull cannula technique. Neuroendocrinology 41:212–218; 1985. 10. Fenelon, V. S.; Herbison, A. E. In vivo regulation of specific GABAA receptor subunit messenger RNAs by increased GABA concentrations in rat brain. Neuroscience 71:661– 670; 1996. 11. Fernandez–Guasti, A.; Larrson, K.; Beyer, C. Comparison of the effects of different isomers of bicuculline infused in the preoptic area on male rat sexual behavior. Experientia 41:1414 –1416; 1985. 12. Fleischmann, A.; Makman, M. H.; Etgen, A. M. Ovarian steroids increase veratridine-induced release of amino acid neurotransmitters in preoptic area synaptosomes. Brain Res. 507:161–163; 1990. 13. Flugge, G.; Oertel, W. H.; Wuttke, W. Evidence for estrogen-receptive GABAergic neurons in the preoptic/anterior hypothalamic area of the rat brain. Neuroendocrinology 43:1–5; 1986. 14. Grattan, D. R.; Selmanoff, M. Castration-induced decrease in the activity of medial preoptic and tuberoinfundibular GABAergic neurons is prevented by testosterone. Neuroendocrinology 60:141–149; 1994. 15. Grattan, D. R.; Rocca, M. S.; Sagrillo, C. A.; McCarthy, M. M.; Selmanoff, M. Antiandrogen microimplants into the rostral medial
22.
23.
24.
25.
26.
27.
28.
29. 30.
31.
32.
33.
34.
preoptic area decrease g-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male. Endocrinology 137:4167– 4173; 1996. Grattan, D. R.; Rocca, M. S.; Strauss, K. I.; Sagrillo, C. A.; Selmanoff, M.; McCarthy, M. M. GABAergic neuronal activity and mRNA levels for both forms of glutamic acid decarboxylase (GAD65 and GAD67) are reduced in the diagonal band of Broca during the afternoon of proestrus. Brain Res. 733:46 –55; 1996. Gu, G.; Simerly, B. Hormonal regulation of opioid peptide neurons in the anteroventral periventricular nucleus. Horm. Behav. 28:503–511; 1994. Herbison, A. E.; Heavens, R. P.; Dyer, R. G. Oestrogen and noradrenaline modulate endogenous GABA release from slices of the rat medial preoptic area. Brain Res. 486:195–200; 1989. Herbison, A. E.; Heavens, R. P.; Dyer, R. G. Oestrogen modulation of excitatory A1 noradrenergic input to rat medial preoptic gamma aminobutyric acid neurones demonstrated by microdialysis. Neuroendocrinology 52:161–168; 1990. Herbison, A. E.; Dyer, R. G. Effect on luteinizing hormone secretion of GABA receptor modulation in the medial preoptic area at the time of proestrous luteinising hormone surge. Neuroendocrinology 53:317– 320; 1991. Herbison, A. E.; Heavens, R. P.; Dye, S.: Dyer, R. G. Acute action of oestrogen on medial preoptic gamma-aminobutyric acid neurons: Correlation with oestrogen negative feedback on luteinizing hormone secretion. J. Neuroendocrinol. 3:101–106; 1991. Herbison, A. E.; Theodosis, D. T. Localisation of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone in the male and female rat. Neuroscience 50:283–298; 1992. Herbison, A. E.; Theodosis, D. T. Immunocytochemical identification of oestrogen receptors in preoptic neurones containing calcitonin generelated peptide in the male and female rat. Neuroendocrinology 56: 761–764; 1992. Herbison, A. E.; Augood, S. J.; McGowan, E. M. Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy. Mol. Brain Res. 14:310 –316; 1992. Herbison, A. E.; Robinson, J. E.; Skinner, D. C. Distribution of estrogen receptor-immunoreactive cells in the preoptic area of the ewe: Co-localization with glutamic acid decarboxylase but not luteinizing hormone-releasing hormone. Neuroendocrinology 57:751–759; 1993. Herbison, A. E. Immunocytochemical evidence for oestrogen receptors within GABA neurones located in the perinuclear zone of the supraoptic nucleus and GABAA receptor b2/3 subunits on supraoptic oxytocin neurones. J. Neuroendocrinol. 6:5–11; 1994. Herbison, A. E.; Fenelon, V. S. Estrogen regulation of GABAA receptor subunit mRNA expression in preoptic area and bed nucleus of the stria terminalis of female rat brain. J. Neurosci. 15:2328 –2337; 1995. Herbison, A. E.; Augood, S. J.; Simonian, S. X.; Chapman, C. Regulation of GABA transporter activity and mRNA expression by estrogen in rat preoptic area. J. Neurosci. 15:8302– 8309; 1996. Herbison, A. E. Noradrenergic regulation of cyclic GnRH secretion. Rev. Reprod. 2:1– 6; 1997. Jarry, H.; Perschl, A.; Wuttke, W. Further evidence that preoptic anterior hypothalamic GABAergic neurons are part of the GnRH pulse and surge generator. Acta Endocrinol. 118:573–579; 1988. Lamberts, R.; Vijayan, E.; Graf, M.; Mansky, T.; Wuttke, W. Involvement of preoptic-anterior hypothalamic GABA neurons in the regulation of pituitary LH and prolactin release. Exp. Brain Res. 52:356 – 362; 1983. Leigh, A. J.; Carter, N. D.; Horton, R.; Silverlight, J. J.; Wilson, C. A. Ovarian steroid regulation of glutamic acid decarboxylase gene expression in individual hypothalamic nuclei. J. Neuroendocrinol. 2: 433– 438; 1990. Leranth, C.; MacLusky, N. J.; Shanabrough, M.; Naftolin, F. Catecholaminergic innervation of luteinizing hormone-releasing hormone and glutamic acid decarboxylase immunopositive neurons in the rat medial preoptic area. Neuroendocrinology 48:591– 602; 1988. Mansky, T.; Mestres–Ventura, P.; Wuttke, W. Involvement of GABA in the feedback action of estradiol on gonadotropin and prolactin
326
35. 36. 37.
38.
39.
40. 41.
42.
43. 44. 45. 46.
HERBISON release: Hypothalamic GABA and catecholamine turnover rates. Brain Res. 231:353–364; 1982. Martin, D. L.; Rimvall, K. Regulation of gamma-aminobutyric acid synthesis in the brain. J. Neurochem. 60:395– 407; 1993. Mayer, M. L. Electrophysiological analysis of inhibitory synaptic mechanisms in the preoptic area of the rat. J. Physiol. 316:327–346; 1981. McCarthy, M. M.; Malik, K. F.; Feder, H. H. Increased GABAergic neurotransmission in medial hypothalamus facilitates lordosis but has opposite effects in the preoptic area/anterior hypothalamus. Brain Res. 507:40 – 43; 1990. McCarthy, M. M.; Kaufman, L. C.; Brooks, P. J.; Pfaff, D. W.; Schwartz-Giblin, S. Estrogen modulation of mRNA levels for two forms of glutamic acid decarboxylase (GAD) in the female rat brain. J. Comp. Neurol. 360:685– 697; 1995. Munaro, N. I.; Dotti, C.; Taleisnik, S. Glutamic acid decarboxylase activity in the hypothalamus of the rat: Effects of estrogens. In: Donoso, A. O.; Racagni, G., eds. GABA and endocrine function. Raven: New York; 1986:201–207. O’Connor, L. H.; Nock, B.; McEwen, B. S. Regional specificity of gamma-aminobutyric acid receptor regulation by estradiol. Neuroendocrinology 47:473– 481; 1988. Okamura, H.; Yokosuka, M.; Hayashi, S. Induction of substance P-immunoreactivity by estrogen in neurons containing estrogen receptors in the anteroventral periventricular nucleus of female but not male rats. J. Neuroendocrinol. 6:609 – 615; 1994. Okamura, H.; Yokusuka, M.; Hayashi, S. Estrogenic induction of NADPH-diaphorase activity in the preoptic neurons containing estrogen receptor immunoreactivity in the female rat. J. Neuroendocrinol. 6:597– 601; 1994. Ondo, J.; Mansky, T.; Wuttke. W. In vivo GABA release from the medial preoptic area of diestrous and ovariectomized rats. Exp. Brain Res. 46:69 –72; 1982. Osborne, P. G.; Mataga, N.; Onoe, H.; Watanabe, Y. Behavioral activation by stimulation of a GABAergic mechanism in the preoptic area of rat. Neurosci. Lett. 158:201–204; 1993. Otis, T. S.; Staley, K. J.; Mody, I. Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res. 545:142–150; 1991. Pfaff, D.; Keiner, M. Atlas of estradiol-concentrating cells in the
47.
48. 49.
50.
51. 52.
53. 54.
55. 56. 57.
central nervous system of the female rat. J. Comp. Neurol. 151:121– 158; 1973. Sar, M. Distribution of progestin-concentrating cells in rat brain: Colocalization of [3H]ORG.2058, a synthetic progestin, and antibodies to tyrosine hydroxylase in hypothalamus by combined autoradiography and immunocytochemistry. Endocrinology 123:1110 –1118; 1988. Sar, M.; Stumpf, W. E.; Tappaz, M. L. Localization of 3H estradiol in preoptic GABA neurons. Fed. Proc. 42:495; 1983. Seltzer, A. M.; Donoso, A. O. Restraining action of GABA on estradiol-induced LH surge in the rat: GABA activity in brain nuclei and effects of GABA mimetics in the medial preoptic nucleus. Neuroendocrinology 55:28 –34; 1992. Simerly, R. B.; Chang, C.; Muramatsu, M.; Swanson, L. W. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: An in situ hybridization study. J. Comp. Neurol. 294:76 –95; 1990. Tappaz, M. L.; Brownstein, M. J. Origin of glutamate-decarboxylase (GAD)-containing cells in discrete hypothalamic nuclei. Brain Res. 132:95–106; 1977. Unda, R.; Brann, D. W.; Mahesh, V. B. Progesterone suppression of glutamic acid decarboxylase (GAD67) mRNA levels in the preoptic area: Correlation to the luteinizing hormone surge. Neuroendocrinology 62:562–570; 1995. Wallis, C. J.; Luttge, W. G. Influence of estrogen and progesterone on glutamic acid decarboxylase activity in discrete regions of rat brain. J. Neurochem. 34:609 – 613; 1980. Watson, R. E.; Hutchinson, R. K.; Langjub, M. C.; Landis, J. W.; Seksaria, S.; Rainey, D. M.; Keil, L. C. Colocalization of natriuretic peptide and estrogen receptor immunoreactivities in preoptic nuclei in the female rat. J. Neuroendocrinol. 6:79 – 87; 1994. Weiland, N. G. Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus. Endocrinology 131:2697–2702; 1993. Yagi, K. Changes in firing rates of single preoptic and hypothalamic units following an intravenous administration of estrogen in the castrated female rat. Brain Res. 53:343–352; 1973. Yuri, K.; Kawata, M. Estrogen receptor-immunoreactive neurons contain calcitonin gene-related peptide, methionine-enkephalin or tyrosine hydroxylase in the female rat preoptic area. Neurosci. Res. 21:135– 141; 1994.