Castration rapidly decreases hypothalamic gamma-aminobutyric acidergic neuronal activity in both male and female rats.

The postcastration LH response is greater and somewhat more rapid in male than female rats. We have previously demonstrated that hypothalamic gamma-aminobutyric acid (GABA)ergic neuronal activity decreases following gonadectomy in male rats. To investigate whether these same hypothalamic GABA neurons decrease their activity postcastration in female rats, and whether more rapid and or greater postcastration decreases occur in male rats, we determined the timing and magnitude of the postcastration decreases in GABA turnover which are associated with the sexually dimorphic postcastration LH response. Adult male and 4-day cycling female rats were castrated between 0800 and 1000 h (females ovariectomized on diestrus day 1). Serum LH levels increased significantly by 12 h postcastration in both males and females with the magnitude of the increases being 6.2-fold in males and 2.8-fold in females. GABA turnover was determined in 16 microdissected brain structures by the GABA transaminase inhibition method at 0 h (sham-operated controls), 6 h, 12 h and 1, 2, 4 and 6 days postcastration. In male rats, in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], the rate of GABA turnover decreased significantly already by 6 h postcastration compared with the 0 h controls, and remained suppressed through 6 days. This rapid down regulation of DBB(ovlt) GABAergic neurons also occurred in female rats, however, the duration of the decrease was not as prolonged as in male rats. Similar changes occurred in the tuberoinfundibular GABAergic (TIGA) neurons projecting to the median eminence in both males and females. Down regulation of these GABAergic neurons precedes or is coincident with increased postcastration LH secretion in both sexes, and the duration of the decreases is consistent with the less robust postcastration LH response in female rats. In addition, the rate of GABA turnover decreased after castration in the interstitial (bed) nucleus of the stria terminalis, ventral aspect (INSTv), the medial preoptic nucleus, dorsomedial aspect (MPNdm) and the ventromedial nucleus, ventrolateral aspect (VMNvl) in male rats, and in the INSTv and VMNvl of female rats, while there was no effect of castration in other hypothalamic regions or control structures. The result in the female VMNvl is consistent with reports that GABA facilitates lordosis behavior in this hypothalamic structure. These findings are consistent with the hypothesis that discrete hypothalamic populations of sex steroid-sensitive GABAergic neurons mediate the postcastration LH responses in both male and female rats, and may underlie other sexually dimorphic adult phenotypes such as sex behavior.

Sex differences in GABA turnover and glutamic acid decarboxylase (GAD(65) and GAD(67)) mRNA in the rat hypothalamus.

GABAergic neurons are estimated to make up more than half of the neuronal population of the hypothalamus and they likely account for some of the structural and functional sexual dimorphisms observed in the mammalian brain. We previously reported sex differences in the rate of GABA turnover in discrete hypothalamic structures of adult rats. In the present study, we extended our search for sex differences in GABA turnover to additional structures, and further determined whether these differences were associated with differences in GAD(65) and or GAD(67) mRNA levels. Utilizing the GABA transaminase inhibition method, we determined GABA turnover in 14 microdissected brain regions. The rate of GABA turnover was about 2-fold greater in male than in diestrous day one (D(1)) female rats in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], anteroventral periventricular nucleus (AVPv), median eminence (ME), and dorsomedial portion of the ventromedial nucleus (VMNdm). A sex difference also was noted in the DBB(ovlt) for GAD(65) mRNA determined by microlysate RNase protection assay. Here, GAD(65) levels were almost 2-fold greater in male rats, which suggests that differences in the activity of this GAD enzyme isoform contributes to the difference in turnover in this area. Additionally, in the dorsomedial nucleus (DMN), the GAD(65) mRNA level was significantly higher in female rats, and in the medial amygdaloid nucleus (Am), GAD(67) mRNA was higher in male rats. These data reveal striking sexual dimorphisms in the rate of GABA turnover and in GAD mRNA levels in specific populations of hypothalamic GABAergic neurons. The functional relationships between these GABAergic neurons and sexually dimorphic phenotypes associated with these structures, such as gonadotropin secretion, reproductive behaviors, seizure threshold and others, warrant further investigation.

Developmental sex differences in amino acid neurotransmitter levels in hypothalamic and limbic areas of rat brain.

GABA, glutamate and aspartate are the predominant amino acid neurotransmitters in the mammalian brain. We have previously reported a developmental sex difference in messenger RNA levels of glutamate decarboxylase, the rate-limiting enzyme in GABA synthesis [Davis A. M. et al. (1996) Horm. Behav. 30, 538-552]. Males were found to have significantly higher levels of messenger RNA in many steroid-concentrating regions of the hypothalamus and limbic system on day 1 of life. Therefore, in this study, we have examined levels of amino acid neurotransmitters during early postnatal development in many of the same or related brain areas. We found that levels of all three transmitters change as animals age. While both GABA and aspartate concentrations increase, glutamate levels decrease. In addition, there are sex differences in neurotransmitter levels in several areas examined, including the ventromedial and arcuate nuclei of the hypothalamus, and the CA1 region of the hippocampus. Sex differences for GABA occur only on postnatal days 1 and 5. However, sex differences in aspartate occur later in development (postnatal day 20). The CA1 region of males has a significantly greater concentration of GABA, glutamate and aspartate than females on postnatal day 1. In addition, treatment of females with testosterone propionate on the day of birth results in increased GABA levels, suggesting that these sex differences may be the result of hormone exposure during development. We hypothesize that these hormonally mediated sex differences in amino acid transmitters early in development contribute to the establishment of sexually dimorphic neuronal architecture in the adult.

Effects of prolactin on expression of the mRNAs encoding the immediate early genes zif/268 (NGF1-A), nur/77 (NGF1-B), c-fos and c-jun in the hypothalamus.

Prolactin (PRL) exerts a short-loop negative feedback effect on hypothalamic neurons which control its secretion from the anterior pituitary gland. The purpose of this study was to identify the location of hypothalamic neurons which respond to acute PRL exposure. Increasing evidence indicates that excitation of neurons often results in the rapid transcription of immediate early genes (IEGs). In the present study, quantitative in situ hybridization histochemistry (ISHH) was used to visualize the induction of mRNAs for four different IEGs: zif/268 (NGF1-A), nur/77 (NGF1-B), c-fos and c-jun. Three groups of male rats were compared: unmanipulated controls, rats injected s.c. with 2.4 mg ovine PRL (oPRL) suspended in polyvinylpyrrolidone (PVP), and PVP-injected controls. Animals were decapitated 0, 0.5, 1, 2, 3 or 4 h following injection. In all rats, the four probes labeled cells within the cortex, particularly the cingulate and piriform cortices, the hippocampus and the striatum. In the arcuate nucleus, there was a modest increase in the average number of cells/animal which expressed zif/268 mRNA following the injection of PVP and oPRL at all times studied. The average area of grains/cell representing zif/268 message also increased following the injection stimulus. The number of neurons expressing nur/77 mRNA was greater in PRL-treated rats compared with PVP-treated controls 0.5 and 1 h following injection. Nur/77-labeled neurons were co-extensive with the tuberoinfundibular dopaminergic (TIDA) neurons. The data suggest that cells located within the arcuate nucleus are involved in mediating PRL autofeedback on the brain.

Castration decreases single cell levels of mRNA encoding glutamic acid decarboxylase in the diagonal band of broca and the sexually dimorphic nucleus of the preoptic area.

Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.

Gonadal hormones alter hypothalamic GABA and glutamate levels.

GABA and glutamate levels were measured in brain sites important for lordotic responding and in other hypothalamic sites after gonadal hormone treatments sufficient to activate lordosis. Estradiol increased GABA and glutamate in the ventromedial nucleus and the vertical diagonal bands. Progesterone administration to estradiol primed females led to a rapid decline of the transmitters in these areas. Results are discussed in relation to neuroendocrine regulation.

Sex differences in the activity of gamma-aminobutyric acidergic neurons in the rat hypothalamus.

The rate of GABA turnover was determined in nine microdissected brain regions in adult male and female rats. In the medial preoptic nucleus (central aspect) and ventromedial nucleus (ventrolateral aspect) of the hypothalamus, areas involved in the regulation of gonadotropin secretion and sex behavior, GABAergic neuronal activity was about 2-fold greater in males than females. These results demonstrate a striking sexual dimorphism in the activity of specific populations of hypothalamic GABAergic neurons.

Prolactin- and testosterone-induced inhibition of LH secretion after orchidectomy: role of catecholaminergic neurones terminating in the diagonal band of Broca, medial preoptic nucleus and median eminence.

Central catecholaminergic neurones projecting to specific hypothalamic structures are involved in stimulating and inhibiting the activity of the GnRH-containing neurosecretory neurones. Both testosterone and elevated circulating prolactin (PRL) levels inhibit postcastration LH release. Three groups of adult male rats were orchidectomized and adrenalectomized, received corticosterone replacement and were: (i) administered purified ovine PRL (oPRL; 2400 microgram/s.c. injection) or (ii) its diluent, polyvinylpyrrolidone (PVP), every 12 h, or (iii) received physiological testosterone replacement for 2 days. At 0, 2 and 6 days postcastration, norepinephrine (NE), epinephrine (E) and dopamine (DA) turnover were estimated by the alpha-methyl-p-tyrosine method in three micro-dissected hypothalamic structures: the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis (DBB(ovlt)), the medial preoptic nucleus (MPN) and the median eminence (ME). In control (PVP-treated) rats, serum LH concentrations increased eightfold at 2 and 6 days postcastration and this rise was prevented by testosterone. oPRL treatment transiently suppressed LH secretion at 2 but not 6 days postcastration. Castration significantly decreased basal rat PRL (rPRL) levels at 2 and 6 days and testosterone administration partially prevented this effect. NE turnover in the ME and E turnover in the MPN increased markedly at 2 and 6 days postcastration, and testosterone replacement for 2 days prevented these increases. Thus, noradrenergic neurones innervating the ME and adrenergic neurones innvervating the MPN may drive postcastration LH secretion by providing stimulatory afferent input to the GnRH neurones. It was striking to observe that oPRL blocked the increases in both ME NE and MPN E turnover at 2 but not 6 days postcastration. Hence, oPRL may transiently suppress LH release by an inhibitory action on these NE and E neurones. DA turnover in the DBB(ovlt) was significantly decreased by 6 days postcastration. Testosterone-treated (2 days postcastration) and oPRL-treated (2 and 6 days postcastration) rats exhibited turnover values indistinguishable from day 0 controls. Hence, the A14 dopaminergic neurones, which synapse on GnRH neurones in the rostral preoptic area and may exert an inhibitory effect on them, are positively regulated by PRL and perhaps by testosterone as well. Autoregulatory feedback suppression of endogenous rPRL secretion by oPRL was observed both 2 and 6 days postcastration. In contrast to the A14 dopaminergic neurones, turnover in the A12 tuberoinfundibular dopaminergic (TIDA) neurones innervating the ME increased significantly by 6 days postcastration in control rats while oPRL administration further increased ME DA turnover at both 2 and 6 days. Hence, autofeedback regulation of rPRL secretion persists through at least 6 days of oPRL exposure temporally associated with markedly increased turnover in the TIDA neurones. In summary, our results support the hypothesis that the inhibitory effect of PRL on postcastration LH release is mediated by suppression of the activity of NE neurones innervating the ME and E neurones terminating in the MPN which, with time, become refractory to continued PRL exposure.

Prolactin- and testosterone-induced inhibition of LH secretion after orchidectomy: role of preoptic and tuberoinfundibular gamma-aminobutyric acidergic neurones.

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) may play an important role in the regulation of LH-releasing hormone secretion. The present study examined the effect of prolactin on GABAergic neuronal activity in microdissected brain regions of the orchidectomized rat, to determine whether inhibition of LH secretion after castration by acute hyperprolactinaemia was associated with prolactin-induced changes in GABAergic neuronal activity. The effects of prolactin were contrasted with the effects of testosterone on hypothalamic GABAergic neurones after orchidectomy. GABA concentrations were measured by high pressure liquid chromatography in eight microdissected brain regions in untreated rats and 60 min after inhibition of the GABA catabolic enzyme GABA transaminase by injection of amino-oxyacetic acid (AOAA). The rate of GABA accumulation in microdissected brain regions following injection of AOAA was taken as an index of GABAergic neuronal activity. Rats were divided into seven experimental groups: intact controls, 2 days after castration, 2 days after castration with prolactin treatment (2.5 mg ovine prolactin injected s.c. every 12 h, starting at the time of castration), 2 days after castration with testosterone replacement (30 mm silicone elastomer implant containing crystalline testosterone), 6 days after castration, 6 days after castration with prolactin treatment, and 6 days after castration with testosterone replacement. Both 2 and 6 days after castration, plasma LH was markedly elevated above levels in intact rats, and AOAA-induced GABA accumulation was significantly decreased in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, in the medial preoptic nucleus and in the median eminence. Hyperprolactinaemia significantly reduced LH levels 2 days but not 6 days after castration. GABAergic neuronal activity, however, was not significantly affected by prolactin at either time. Testosterone replacement blocked the postcastration elevation in plasma LH and prevented the castration-induced suppression of GABAergic neuronal activity both 2 and 6 days after castration. There were no castration- or hormone-induced changes in GABAergic neurones observed in the medial or lateral septum, caudate nucleus, cingulate cortex or arcuate nucleus. These results demonstrate that the activity of GABAergic neurones terminating in the rostral hypothalamus and the median eminence is positively regulated by testosterone, and that these steroid-sensitive GABAergic neurones may be important in the negative-feedback control of LH secretion. Inhibition of LH secretion by hyperprolactinaemia, however, may not be mediated by changes in GABAergic neuronal activity.

Castration-induced decrease in the activity of medial preoptic and tuberoinfundibular GABAergic neurons is prevented by testosterone.

We recently determined that castration specifically decreased GABA turnover in discrete rostral and mediobasal hypothalamic structures. This study aimed to investigate whether testosterone could stimulate GABAergic neuronal activity in these hypothalamic GABAergic neurons in the castrate rat, and to compare the effects of episodic testosterone replacement with the constant levels provided by subcutaneous testosterone implants. Animals were divided into 4 experimental groups: intact, 48 h castrate, 48 h castrate+testosterone capsules (2 x 30 mm Silastic implants, 1.57 mm ID, 3.18 mm OD) and 48 h castrate+testosterone injections (100 micrograms/injection s.c., every 8 h). GABA concentrations were measured in 4 microdissected brain regions either before or 60 min after inhibition of the GABA degrading enzyme, GABA transaminase, by injection of aminooxyacetic acid (AOAA, 100 mg/kg i.p.). The rate of GABA accumulation in the tissue following injection of AOAA was used as an index of GABAergic neuronal activity. Castration resulted in a 10-fold increase in serum LH concentrations compared with intact rats. Either mode of testosterone administration completely prevented this castration-induced LH rise. In the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, the medial preoptic nucleus and in the median eminence, GABA turnover was significantly reduced by castration to approximately 50% that of intact rats. Either testosterone implants or testosterone injections prevented this castration-induced decrease in GABA turnover, such that the turnover rates were not significantly different from intact rats. There was no effect of castration with or without testosterone replacement in the cingulate cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic male rats.

We have examined the changes that occur in neuronal expression of LHRH mRNA in response to castration and hyperprolactinemia in male rats. Single cell levels of LHRH mRNA were determined by quantitative in situ hybridization histochemistry using an 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Nine days postcastration, a 10.4-fold increase in mean plasma LH titers was observed which was associated with significantly increased LHRH mRNA in rostral hypothalamic neuronal cell bodies. Both increases were blocked in rats rendered hyperprolactinemic by the presence of the 7315a PRL-secreting pituitary tumor. The location and number of neurons expressing LHRH mRNA were unchanged, indicating that these differences were attributable to amounts of mRNA expressed per neuron. Experimental differences occurred in LHRH perikarya situated throughout the rostral hypothalamus from the organum vasculosum of the lamina terminalis to the caudal extent of the medial preoptic nucleus. These results suggest that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of LHRH in the rostral hypothalamus.

Catecholamine turnover rates in discrete hypothalamic areas and associated changes in median eminence luteinizing hormone-releasing hormone and serum gonadotropins on proestrus and diestrous day 1.

We have correlated catecholamine [CA; i.e. norepinephrine (NE), dopamine (DA), and epinephrine (E)] turnover rates in discrete hypothalamic nuclei and in the median eminence (ME), with concentration changes in ME LHRH and serum LH, FSH, PRL, estradiol, and progesterone levels at various times during proestrus and diestrous day 1 in 4-day cyclic rats. CA concentrations were measured with a radioenzymatic assay at 0, 60, and 120 min after ip injection of 400 mg/kg alpha-methyl-p-tyrosine, and rate constants and turnover rates were calculated. In a separate assay NE, DA, and E were separated by two-dimensional thin layer chromatography, and concentrations and turnover rates of CAs were calculated. The microdissected hypothalamic nuclei examined for NE turnover rates included the medial preoptic nucleus (MPN), suprachiasmatic nucleus (SCN), arcuate nucleus (AN), and ME. DA turnover rates also were measured in the MPN, ME, and AN. ME LHRH and serum hormone concentrations were measured by RIA. Between 0900--1200 h, proestrous serum estradiol was elevated, but other serum hormones were basal, and CA turnover rates in the brain were low. However, ME LHRH concentrations increased significantly between 0900--1200 h on proestrus. Between 1200--1500 h, serum LH, FSH, PRL, and progesterone levels increased and ME LHRH levels declined significantly; during this time interval (1200--1400 h), a significant rise in ME NE and DA turnover rates occurred. Between 1500--1700 h on proestrus, while serum gonadotropins were still rising toward peak concentrations, increased ME NE turnover rates were maintained, but increased NE turnover rates also were evident in MPN, SCN, and AN. During this same time interval (1500--1700 h), a marked decline in ME and AN DA turnover rates occurred, although such rates remained unchanged within the MPN. There were no corresponding changes in MPN E turnover rates at any of the time intervals studied. The increased turnover rates of ME NE coupled with the concomitant decline in ME LHRH levels and the rise in plasma LH and FSH levels suggest that increased NE release may be important in initiating preovulatory LH and FSH surges. These changes in brain neurotransmitters and serum hormones are not the result of a diurnal rhythm, since corresponding changes in CA turnover rates or serum gonadotropins did not occur between 0900--1100 h and 1500--1700 h diestrous day 1.

Aromatization and 5alpha-reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat.

The in vitro aromatization and 5alpha-reduction of androgens to estrogens and dihydrotestosterone (DHT) were determined in incubations of microdissected brain regions of male and female gonadectomized, adrenalectomized rats. Metabolites formed from [1alpha,2alpha-3H]androstenedione or [1alpha,2alpha-3H]testosterone were purified by celite liquid-liquid partition chromatography, silica gel chromatography and recrystallization to stable 3H/14C ratios. The medial preoptic nucleus-anterior hypothalamic nucleus exhibited the highest aromatase activity and the second highest conversion to DHT. The lateral preoptic and lateral hypothalamic nuclei showed little aromatase activity yet exhibited high rates of formation of DHT. The medial basal hypothalamus showed the second highest level of aromatase activity but consistently formed the lowest amount of DHT. The discrect anatomical localization of these enzymatic conversions is suggestive of their being involved in the physiological actions of androgens.

Concentrations of dopamine and norepinephrine in discrete hypothalamic nuclei during the rat estrous cycle.

The norepinephrine (NE) and dopamine (DA) concentrations of the medial preoptic nucleus (MPN), suprachiasmatic nucleus (SCN), arcuate nucleus (AN), ventromedial nucleus (VMN) and median eminence (ME) were measured during the estrous cycle. Nuclei were microdissected and assayed using the catechol-0-methyl transferase enzymatic method. A significant increase in NE was observed between the morning and afternoon of proestrus (P) in the SCN. In contrast, no significant changes in DA were detected in any area on P. No change in NE or DA was found between the morning and afternoon of diestrus 1 (D1). These findings are consistent with NE-containing neurons being involved in the surge of LH on the afternoon of proestrus.