Chronic hypersecretion of luteinizing hormone in transgenic mice disrupts both ovarian and pituitary function, with some effects modified by the genetic background.

When the pituitary or hypothalamus becomes resistant to steroid negative feedback, a vicious cycle ensues, resulting in chronic hypersecretion of luteinizing hormone (LH) from the pituitary and steroids from the ovaries. In women, LH hypersecretion is implicated in infertility, miscarriages, and development of granulosa cell tumors. Progress in defining the underlying mechanisms of LH toxicity, however, has been limited by the lack of well-defined animal models. To that end, we have developed a new transgenic mouse model (alpha-LHbetaCTP) wherein LH hypersecretion occurs chronically and results in several dire pathological outcomes. Chronic hypersecretion of LH was achieved by introducing a transgene containing a bovine alpha subunit promoter fused to the coding region of a chimeric LHbeta subunit. The alpha subunit promoter directs transgene expression only to gonadotropes. The LHbeta chimera contains the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit linked to the carboxyl terminus of bovine LHbeta. This carboxyl extension extends the half-life of LH heterodimers that contain the chimeric beta subunit. In intact alpha-LHbetaCTP females, serum LH is elevated five- to ten-fold in comparison to nontransgenic littermates. Levels of testosterone (T) and estradiol (E2) also are elevated, with an overall increase in the T-to-E2 ratio. These transgenic females enter puberty precociously but are anovulatory and display a prolonged luteal phase. Anovulation reflects the absence of gonadotropin-releasing hormone (GnRH) and the inability to produce a pre-ovulatory surge of LH. The ovaries are enlarged, with reduced numbers of primordial follicles and numerous, giant, hemorrhagic follicles. Despite the pathological appearance of the ovary, females can be superovulated and mated. Although pregnancy occurs, implantation is compromised due to defects in uterine receptivity. In addition, pregnancy fails at midgestation, reflecting a maternal defect presumably due to estrogen toxicity. When the transgene is in a CF-1 background, all females develop granulosa cell tumors and pituitary hyperplasia by five months of age. They die shortly thereafter due to bladder atony and subsequent kidney failure. When the transgene is placed in other strains of mice, their ovaries develop a luteoma rather than a granulosa cell tumor and the pituitary develops pituitary hyperplasia followed by adenoma. In summary, alpha-LHbetaCTP mice provide a direct association between abnormal secretion of LH and development of a number of ovarian and pituitary pathological responses.

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney.

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.

Chronic hypersecretion of luteinizing hormone in transgenic mice selectively alters responsiveness of the alpha-subunit gene to gonadotropin-releasing hormone and estrogens.

Steroid hormones can act either at the level of the hypothalamus or the pituitary to regulate gonadotropin subunit gene expression. However, their exact site of action remains controversial. Using the bovine gonadotropin alpha-subunit promoter linked to an expression cassette encoding the beta-subunit of LH, we have developed a transgenic mouse model where hypersecretion of LH occurs despite the presence of elevated ovarian steroids. We used this model to determine how hypersecretion of LH could occur when steroid levels are pathological. During transition from the neonatal period to adulthood, the endogenous LHbeta subunit gene becomes completely silent in these mice, whereas the alpha-directed transgene and endogenous alpha-subunit gene remain active. Interestingly, gonadectomy stimulates expression of the endogenous alpha and LHbeta subunit genes as well as the transgene; however, only the endogenous LHbeta gene retains responsiveness to 17beta-estradiol and GnRH. In contrast, LH levels remain responsive to negative regulation by androgen. Thus, alpha-subunit gene expression, as reflected by both the transgene and the endogenous gene, has become independent of GnRH regulation and, as a result, unresponsive to estradiol-negative feedback. This process is accompanied by a decrease in estrogen receptor alpha gene expression as well as an increase in the expression of transcription factors known to regulate the alpha-subunit promoter, such as cJun and P-LIM. These studies provide in vivo evidence that estrogen-negative feedback on alpha and LHbeta subunit gene expression requires GnRH input, reflecting an indirect mechanism of action of the steroid. In contrast, androgen suppresses alpha-subunit expression in both transgenic and nontransgenic mice. This suggests that androgens must regulate alpha-subunit promoter activity independently of GnRH. In addition to allowing the assessment of site of action of sex steroids on alpha-subunit gene expression, these studies also indicate that chronic exposure of the pituitary to LH-dependent ovarian hyperstimulation leads to a heretofore-undescribed pathological condition, whereby normal regulation of alpha, but not LHbeta, subunit gene expression becomes compromised.

Characterization of the equine glycoprotein hormone alpha-subunit gene reveals divergence in the mechanism of pituitary and placental expression.

The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the human promoter, the cloned equine alpha-subunit promoter failed to direct trophoblast-specific expression in either tissue culture or transgenic mouse models, suggesting an entirely different mechanism for expression. In contrast, the equine alpha-subunit promoter was able to direct gonadotroph expression in both tissue culture and transgenic mouse models. In alphaT3-1 cells, 550 base pair (bp) was sufficient for expression. This expression involves promoter elements identified in other species as playing a role in gonadotroph expression, but mutation of these elements reveals differences in their relative contributions to promoter activity. In mice, 2800 bp of 5'-flanking sequence allowed specific expression in gonadotrophs but not in thyrotrophs or placenta. The pattern of estrogen regulation observed in transgenic mice matched neither the repression that has been observed with human and bovine alpha-subunit promoters in transgenic mice nor the stimulation in mRNA levels reported in mares, suggesting a unique mechanism that is not recapitulated in the transgenic model. Thus the equine alpha-subunit promoter uses a combination of conserved and unique features of gene regulation to direct its pattern of tissue-specific expression.

Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary.

A few years before reproductive senescence, primordial follicles are depleted from the ovary at a dramatically accelerated rate. It has been proposed that this depletion is due to transient increases in gonadotropin levels. To test this hypothesis, we used mice that produce chronically elevated levels of serum LH via expression of an LHbeta subunit transgene. Ovaries were collected from transgenic and control mice, and complete serial sections were prepared for histological examination. Each section was scanned for morphological abnormalities, and every fifth section was sampled to estimate the total number of primordial, primary, and large preantral follicles per ovary. Until 3 wk postpartum, ovaries from transgenic and control mice were morphologically similar. By 5 wk, control ovaries contained many healthy primordial, primary, and large preantral follicles as well as atretic follicles. Transgenic ovaries contained blood-filled cysts, misshapen granulosa cells, luteinized cells, and approximately 45% fewer primordial follicles than controls. By 3 mo, transgenic ovaries had about 68% fewer primordial follicles and 53% fewer primary follicles than controls. These results suggest that, in addition to having profound effects on growing follicles, chronically elevated LH levels deplete the primordial follicle pool and thus may hasten the onset of reproductive senescence.

Enhanced production of human immunodeficiency virus type 1 by in vitro-infected alveolar macrophages from otherwise healthy cigarette smokers.

Since cellular activation is required for replication of human immunodeficiency virus type 1 (HIV-1), the capacity of alveolar macrophages (AM) from smokers, which are relatively activated, and nonsmokers to support the production of HIV-1JR-FL was examined. Peak HIV-1 p24 antigen level in culture supernatants of infected AM from 13 smokers was significantly higher than that of 13 nonsmokers: 31,394 +/- 8295 versus 7037 +/- 2550 pg/mL (mean +/- SE; P < .002). This difference could not be explained on the basis of viral entry, extent of reverse transcription, or release of monokines, including tumor necrosis factor-alpha, interleukin-1 beta or -6, and granulocyte-macrophage colony-stimulating factor. HIV-1 production by blood monocytes from smokers and nonsmokers infected in vitro was negligible. Thus, cigarette smoking selectively increases the susceptibility of AM to productive infection with HIV-1. This finding provides a biologic plausibility to observations that smoking may enhance the progression of AIDS.

Do GnRH neurons express the gene for the NMDA receptor?

Previous studies have revealed that in several animal models, N-methyl-D,L-Aspartate (NMA) stimulates LH secretion by acting at a suprapituitary site. In addition, NMDA receptor antagonists appear to block GnRH neuronal activation on the afternoon of proestrous as evidenced by the lack of c-Fos expression in the neurons and by the absence of an ovulatory LH surge. However, administration of NMA does not induce c-Fos or c-Jun expression in GnRH neurons. To better understand the effects of NMDA receptor activation on GnRH neuronal function, we examined whether GnRH neurons express the NMDA receptor in male rats, and in female rats during diestrus and proestrus, by performing double label in situ hybridization. An 35S-labeled cRNA probe for the NMDA receptor subunit (NMDAR1) was used to quantify NMDAR1 mRNA and a digoxigenin-labeled cRNA probe for GnRH was used to identify GnRH neurons. The data were quantified and expressed as grains/average cell area. In male and female rats, less than 5% of GnRH neurons expressed grain levels twice the minimum detectable level and were considered double-labeled. However, many non-GnRH neurons in the same areas as GnRH neurons expressed high levels of NMDAR1 mRNA. These results suggest that the effects of NMA on GnRH secretion are unlikely to be mediated solely by the activation of NMDA receptors on GnRH neurons. Given the widespread expression of NMDAR1 mRNA in the hypothalamus, it is possible that the stimulatory effects of NMA on GnRH neurons are indirect through activation of other neurons.

The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus.

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation, androgens and Müllerian-inhibiting substance, and is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene had normal numbers and location of GnRH neurons but exhibited grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restored pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction.

The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-F1-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.

Lactation-induced deficits in NMDA receptor-mediated cortical and hippocampal activation: changes in NMDA receptor gene expression and brainstem activation.

During lactation, there is an inhibition of cortical and hippocampal activation in response to N-methyl-D,L-aspartate (NMA), but not kainate, as assessed by induction of c-Fos expression. To study whether changes in NMDA receptor function may account for this inhibition, NMDA receptor subunit (NMDAR1) mRNA levels were measured by both Northern analysis and in situ hybridization. Analysis of NMDAR1 gene expression by Northern blot analysis did not reveal significant differences between cycling and lactating rats. Using in situ hybridization, NMDAR1 mRNA levels in several cortical and hippocampal areas appeared to be smaller in lactating rats, compared to cycling rats, although these differences reached significance only in the fronto-parietal cortex and piriform cortex. These subtle changes in NMDAR1 receptor subunit gene expression during lactation are not likely to account for the global lack of neuronal activation in response to NMA. However, it is possible that there may be changes in other NMDA receptor subunits that could account for the deficits in NMDA receptor activation. We also examined the activation state of afferent pathways in the brainstem that provide excitatory input to the cortex and hippocampus. During lactation, NMA induced c-Fos expression in similar areas of the brainstem as during the cycle, except in the locus coeruleus and dorsal raphe, where c-Fos expression was significantly less than that observed during the cycle. In contrast, no differences in the pattern of c-Fos expression in the brainstem in response to kainate were observed between cycling and lactating rats. The lack of NMA-induced activation of the locus coeruleus and dorsal raphe may contribute to the lack of cortical activation during lactation.

Use of Fos-related antigens (FRAs) as markers of neuronal activity: FRA changes in dopamine neurons during proestrus, pregnancy and lactation.

This manuscript describes the use of staining of Fos-related antigens (FRAs) as markers for changes in neuronal activity. The model system consisted of the tuberoinfundibular dopamine (TIDA) neurons located in the arcuate nucleus of the hypothalamus. Under normal conditions, these neurons are devoid of c-Fos staining even though the neurons are tonically active and can express FRAs. During specific neuroendocrine states the neurons undergo changes in activity, as described by other studies. At times when the activity is relatively high as in pregnancy and during proestrus, approximately 50%-60% of the TIDA neurons expressed FRA immunoreactivity. Moreover changes over the course of proestrus paralleled known shifts in TIDA activity (declining as the day progressed). At times when TIDA activity is suppressed, such as during lactation, FRA staining in TIDA neurons was markedly reduced or absent. Upon removal of the suckling stimulus, FRA staining rose to reach peak expression 12-24 h after pup removal (without coordinate induction of c-Fos). These data suggest that FRA staining can serve as a useful marker of activity in the TIDA neurons which permits not only assessment of stimulated activity but also suppressed function in the neurons. A cautionary note in using this approach along with acquisition of serial blood samples for hormone measurement is that surgical procedures for monitoring plasma hormone levels are associated with strong long-lived induction of FRAs (and c-Fos) in many neurons (including the TIDA neurons) that can confound interpretation of FRA staining.

Effects of N-methyl-D-aspartate receptor activation on cFos expression in luteinizing hormone-releasing hormone neurons in female rats.

N-Methyl-D,L-aspartic acid (NMA), an agonist of N-methyl-D-aspartate (NMDA) excitatory amino acid receptors, stimulates the secretion of LH by increasing the release of LHRH. During proestrus, LHRH neurons express cFos in association with the LH surge. To determine the involvement of NMDA receptors in the activation of LHRH neurons on proestrus, we treated animals with an NMDA receptor blocker, MK-801. Treatment with MK-801 (0.3 mg/kg, sc) at 1130 h blocked both the LH and PRL surges and cFos expression in LHRH neurons. These data suggest that NMDA receptors are involved in the regulation of LHRH neuronal activation during the LH surge. We then determined whether NMA treatment could restore LH secretion and cFos expression in LHRH neurons in animals whose endogenous proestrous LH surges were blocked with pentobarbital. In the pentobarbital-blocked rats, NMA failed to induce cFos expression in LHRH neurons and increase LH secretion, but it did result in an increase in PRL secretion. To determine if NMA treatment alone could induce cFos expression in LHRH neurons, diestrous rats were treated with NMA by either systemic (40 mg/kg BW; four injections, 10 min apart) or third ventricular (2 micrograms in 2 microliters; four injections, 10 min apart) injections. NMA administration (regardless of the route of administration) caused an increase in LH secretion and significant cFos expression in many regions of the brain, including sites where the LHRH perikarya are concentrated. However, neither systemic nor intraventricular administration of NMA induced cFos expression in LHRH neurons. Thus, even though NMA results in increased activity of LHRH neurons, as evidenced by increased LH secretion, NMDA receptor activation alone appears to be insufficient to induce cFos expression in the LHRH neurons.

Altered luteinizing hormone and prolactin responses to excitatory amino acids during lactation.

We have used excitatory amino acids as tools to elucidate changes in hypothalamic function associated with lactation, focusing on the regulation of luteinizing hormone (LH) and prolactin secretion. In these studies, we have compared the responsiveness to NMA (N-methyl-D,L-aspartate), an agonist for the N-methyl-D-aspartate (NMDA) receptor, with that of kainate, an agonist for another type of glutamate receptor, the kainate receptor. To address the issue of the permeability of the blood-brain barrier to either NMA or kainate, systemic and central administration of the drugs were compared. Four injections of either drug were administered at 10-min intervals to cycling or lactating rats suckling 8 pups. All of these treatment significantly stimulated LH secretion in cycling rats. However, neither systemic injections of NMA (40 mg/kg) or kainate (2.5-3.5 mg/kg), nor third-ventricular administration of NMA (2 micrograms/2 microliters) or kainate (0.2-0.3 micrograms/2 microliters) stimulated LH secretion during lactation. In contrast, LH responses to NMA were observed in lactating animals suckling 2 pups. These data demonstrate that the intensity of the suckling stimulus determines the degree of gonadotropin-releasing hormone (GnRH) neuronal inhibition during lactation. Recovery of the LH response to NMA in animals suckling 8 pups was not observed after treatment with RU 486 to block the effects of progesterone. Thus, the elevated levels of progesterone during lactation do not appear to play a role in inhibiting GnRH neuronal responsiveness. Removal of the 8-pup suckling stimulus for 24 h also did not restore the LH response to NMA. However, treatment with RU 486 and removal of the suckling stimulus for 24 h did restore LH responses to NMA, suggesting that progesterone may play a role in prolonging the recovery of GnRH neuronal responsiveness. The prolactin responses to NMA and kainate changed with the reproductive state of the animal and the site of administration. Central injections of either drug stimulated prolactin release in both cycling and lactating animals. In contrast, whereas systemic administration of NMA stimulated prolactin secretion in cycling animals, kainate had no effect. In the lactating animals, systemic administration of either drug inhibited prolactin secretion. Thus, the difference in the prolactin responses to systemic administration of the drugs may not only be due to a difference in the distribution of kainate and NMDA receptors but also to the steady state level of activity of the prolactin-releasing and -inhibiting factors which is determined by the reproductive state of the animal.(ABSTRACT TRUNCATED AT 400 WORDS)

Cortical refractoriness to N-methyl-D,L-aspartic acid (NMA) stimulation in the lactating rat: recovery after pup removal and blockade of progesterone receptors.

We have previously reported that lactating rats, unlike cycling rats, are refractory to N-methyl-D,L-aspartic acid (NMA), but not kainate, in terms of behavioral responses and activation of cFos expression in the neocortex and hippocampus. To study the factors involved in the suppression of cortical activation in lactating rats in response to NMA, we examined the effects of removing either the suckling stimulus and/or progesterone. The degree of cFos expression was used as a marker for cortical activation. Whereas control suckled animals exhibited little or no cFos activation in the piriform cortex in response to NMA, cycling rats showed a high degree of activation. Blockade of the effects of progesterone or removal of the pups for 24 h, resulted in a moderate level of cFos intensity in response to NMA. Total recovery was observed only in animals who had their pups removed for 24 h and the effects of progesterone were blocked. In general, similar results were obtained in the hippocampus except that the total recovery of hippocampal activation took longer than the cortex. Thus, the deficits in cortical activation depend on the presence of both the suckling stimulus and progesterone. However, progesterone alone cannot induce these cortical deficits since pregnant rats showed no deficits in cortical activation in response to NMA when compared to cycling rats. Therefore, the suckling stimulus is required for the inhibition of NMDA-receptor mediated activation of the cortex and hippocampus. The effects of progesterone appear to act synergistically with the effects of suckling.

LHRH neurons express cJun protein during the proestrous surge of luteinizing hormone.

LHRH neurons express cFos on the afternoon of proestrus in association with the ovulatory LH surge. This study determined whether LHRH neurons express another proto-oncogene product, Jun, during the estrous cycle. By using immunocytochemical double staining techniques, localization of Jun proteins and LHRH was compared with expression of cFos in LHRH neurons. LHRH neurons expressed Jun proteins on the afternoon of proestrus with detection of Jun through the morning of estrus. The time course of Jun expression in LHRH neurons during proestrus suggests a common stimulus for both Jun and cFos expression in LHRH neurons, with Jun proteins persisting somewhat longer than cFos. Pentobarbital, which blocks the preovulatory LH surge and cFos expression in LHRH neurons, blocked Jun expression in LHRH neurons; the LHRH neurons similarly expressed Jun and cFos on the following afternoon at the time of the expected delayed LH surge. There was a similarity in the patterns (in terms of numbers and distribution) of Jun-positive and cFos-positive LHRH neurons. Both were localized in the preoptic area and anterior hypothalamus; LHRH neurons above the anterior commissure or rostral to the OVLT did not express Jun or cFos. For the first time, these data provide direct evidence that both proto-oncogene products, Jun and cFos, are expressed in LHRH neurons in association with the proestrous LH surge. Taken together, these results suggest that changes in gene expression mediated by Jun-cFos heterodimers may accompany LHRH activation on the afternoon of proestrus.

Lactation inhibits hippocampal and cortical activation of cFos expression by nivida but not kainate receptor agonists.

Lactation in the rat activates afferent neuronal pathways that cause marked changes in hormone secretion and maternal behavior. Previously, we used excitatory amino acids (EAAs) to challenge the neuroendocrine axis of the lactating rat and showed altered hypothalamic responsiveness to EAAs. In conducting those studies, we noted that the typical hyperactive behavior associated with NMA (N-methyl-d,l-aspartate) treatment was completely absent in lactating animals. In these studies we have examined the effects of lactation on cortical responsiveness to EAAs by using cFos expression as a marker of neuronal activation. Lactation inhibited hippocampal and cortical cFos induction in response to NMA, which was consistent with the absence of behavioral responses. However, NMA responsiveness was observed in other areas of the brain. Recovery of cortical activation in response to NMA was not observed until 24 h after removal of the suckling stimulus. In contrast, treatment with kainate (an agonist for a different type of glutamate receptor) induced similar patterns of cFos expression and behavioral responses (wet-dog shakes) in cycling and lactating rats. These data demonstrate that lactation, a physiological condition, can inhibit cortical activation that is mediated by NMDA but not kainate receptors. In so doing, lactation represents a novel model system in which to study mechanisms of NMDA receptor inactivation and subsequent consequences on hippocampal and cortical function.

Differences in the luteinizing hormone and prolactin responses to multiple injections of kainate, as compared to N-methyl-D,L-aspartate, in cycling rats.

We have previously reported that repetitive iv injections of NMA [N-methyl-D,L-aspartate, the mixed analog acting on the N-methyl-D-aspartate (NMDA) receptor] can induce a consistent increase in LH and PRL secretion in cycling rats, but not in lactating rats. To further explore the use of excitatory amino acids (EAAs) as tools for understanding the regulation of the neuroendocrine reproductive axis, we have examined the effects of multiple injections of kainate, an agonist to another subclass of EAA receptor, on LH and PRL secretion in cycling rats. Recent studies suggest that kainate receptors may be more abundant than NMDA receptors in the hypothalamus. Five iv injections of kainate were administered at 50-min intervals to diestrous or estrous rats. Blood samples were collected every 10 min and assayed for LH and PRL. LH, but not PRL secretion, was stimulated by this regimen of kainate treatment. Surprisingly, the LH response to kainate, unlike NMA, decreased with repetitive injections of the drug. The response to the last pulse of kainate was approximately 30-40% of the first pulse. This decline in LH responsiveness to kainate was not due to desensitization at the level of the pituitary or to refractoriness of GnRH neurons, since further stimulation of LH release could be obtained by the administration of GnRH or NMA. The mechanisms responsible for the diminishing GnRH response to kainate remain unclear. However, we speculate that it might be due to the delayed activation of inhibitory inputs to GnRH neurons or to the desensitization of kainate receptors. On the other hand, the absence of a PRL response to kainate, in contrast to the stimulatory effect of NMA, most likely reflects differences in the distribution of kainate and NMDA receptors on dopamine neurons and neurons containing PRL-releasing factors, or on extrahypothalamic afferent neuronal populations projecting to the hypothalamus. In conclusion, the effects of systemic injections of kainate on LH and PRL secretion differed from NMA in that the LH response could not be sustained with multiple injections and PRL was unresponsive to kainate stimulation.