EB-primed Rats Research Articles

Participation of the nitric oxide pathway in lordosis induced by apelin-13 in female rats

The present study investigated the participation of the nitric oxide pathway in facilitating lordosis behavior induced by intrahypothalamic administration of apelin-13 in ovariectomized rats primed with estradiol benzoate (EB). The experiments involved the administration of a nitric oxide synthase inhibitor (L-NAME) or a nitric oxide-dependent, soluble guanylyl cyclase inhibitor (ODQ), and an inhibitor of protein kinase G (KT5823) to the ventromedial hypothalamus (VMH) of EB-primed rats 30 min before infusion of apelin-13 (0.75 μg/μl). This dose of apelin-13 consistently induces lordosis behavior at 30 min, 120 min, and 240 min following infusion. Results showed that injections of either L-NAME or KT5823 significantly reduced the lordosis induced by apelin at 120 and 240 min. However, VMH infusion of ODQ 30 min before apelin-13 infusion reduced but did not significantly inhibit, the lordosis elicited by this peptide at the same time points. We conclude that the nitric oxide pathway in the VMH plays an important role in lordosis induced by apelin-13 in EB-primed rats.

Apelin-13 facilitates lordosis behavior following infusions to the ventromedial hypothalamus or preoptic area in ovariectomized, estrogen-primed rats

In normal hormonal conditions, increased neuronal activity in the ventromedial hypothalamus (VMH) induces lordosis whereas activation of the preoptic area (POA) exerts an opposite effect. In the present work, we explored the effect of bilateral infusion of different doses of the apelin-13 (0.37, 0.75, 1.5, and 15 μg) in both brain areas on the expression of lordosis behavior. Lordosis quotient and lordosis reflex score were performed at 30, 120, and 240 min. Weak lordosis was observed following the 0.37 μg dose of apelin-13 at 30 min in the VMH of EB-primed rats; however, the rest of the doses induced significant lordosis relative to the control group. At 120 min, all doses induced lordosis behavior, while at 240 min, the highest dose of 15 μg did not induce significant differences. Interestingly, only the 0.75 μg infusion of apelin in the POA induced significant lordosis at 120 and 240 min. These results indicate that apelin-13 acts preferably in HVM and slightly in POA to initiate lordosis behavior in estrogen-primed rats.

Protein kinase inhibitors infused intraventricularly or into the ventromedial hypothalamus block short latency facilitation of lordosis by oestradiol.

An injection of unesterified oestradiol (E2 ) facilitates receptive behaviour in E2 benzoate (EB)-primed, ovariectomised female rats when it is administered i.c.v. or systemically. The present study tested the hypothesis that inhibitors of protein kinase A (PKA), protein kinase G (PKG) or the Src/mitogen-activated protein kinase (MAPK) complex interfere with E2 facilitation of receptive behaviour. In Experiment 1, lordosis induced by i.c.v. infusion of E2 was significantly reduced by i.c.v. administration of Rp-cAMPS, a PKA inhibitor, KT5823, a PKG inhibitor, and PP2 and PD98059, Src and MAPK inhibitors, respectively, between 30 and 240minutes after infusion. In Experiment 2, we determined whether the ventromedial hypothalamus (VMH) is one of the neural sites at which those intracellular pathways participate in lordosis behaviour induced by E2 . Administration of each of the four protein kinase inhibitors into the VMH blocked facilitation of lordosis induced by infusion of E2 also into the VMH. These data support the hypothesis that activation of several protein kinase pathways is involved in the facilitation of lordosis by E2 in EB-primed rats.

Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17β-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Tamoxifen and ICI 182,780 activate hypothalamic G protein-coupled estrogen receptor 1 to rapidly facilitate lordosis in female rats

In the female rat, sexual receptivity (lordosis) can be facilitated by sequential activation of estrogen receptor (ER) α and G protein-coupled estrogen receptor 1 (GPER) by estradiol. In the estradiol benzoate (EB) primed ovariectomized (OVX) rat, EB initially binds to ERα in the plasma membrane that complexes with and transactivates metabotropic glutamate receptor 1a to activate β-endorphin neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). This activates MPN μ-opioid receptors (MOP), inhibiting lordosis. Infusion of non-esterified 17β-estradiol into the ARH rapidly reduces MPN MOP activation and facilitates lordosis via GPER. Tamoxifen (TAM) and ICI 182,780 (ICI) are selective estrogen receptor modulators that activate GPER. Therefore, we tested the hypothesis that TAM and ICI rapidly facilitate lordosis via activation of GPER in the ARH. Our first experiment demonstrated that injection of TAM intraperitoneal, or ICI into the lateral ventricle, deactivated MPN MOP and facilitated lordosis in EB-primed rats. We then tested whether TAM and ICI were acting rapidly through a GPER dependent pathway in the ARH. In EB-primed rats, ARH infusion of either TAM or ICI facilitated lordosis and reduced MPN MOP activation within 30min compared to controls. These effects were blocked by pretreatment with the GPER antagonist, G15. Our findings demonstrate that TAM and ICI deactivate MPN MOP and facilitate lordosis in a GPER dependent manner. Thus, TAM and ICI may activate GPER in the CNS to produce estrogenic actions in neural circuits that modulate physiology and behavior.

Sexual receptivity facilitated by unesterified estradiol: Dependence on estrogen and progestin receptors and priming dose of estradiol benzoate.

In some conditions, female sexual behavior in ovariectomized rats can be induced by continuous exposure of estradiol (E2) alone or by a single injection of a high dose of the long-lasting, esterified estradiol benzoate (EB). However, there are inconsistencies in the literature on the role of estrogens during priming or in the facilitation on female sexual behavior in EB-primed rats, as well as the cellular mechanisms involved. Either subcutaneous (sc) or intracerebral (icv) administration of some doses of free unesterified E2, induced lordosis in EB-primed rats. Either sc or icv injection of E2, immediately prior to testing, induced high levels of sexual receptivity when the female rats were primed with an EB sc injection of 2 μg EB. The roles of progesterone receptor (PR) and estrogen receptor on lordosis induced by sc or icv administration of E2 were explored. Tamoxifen or RU486 administrated sc or icv; each reduced lordosis induced by E2. Similarly, antisense oligonucleotides directed at PR-B or total PR (PR-A + PR-B) administrated icv immediately before EB injection inhibited lordosis induced by daily injections of EB. These results suggest that lordosis facilitated by free E2 is dependent on priming dose of EB. Furthermore both ERs and PRs are involved in this action of E2.

Lordosis of Rats Is Modified by Neurosteroidogenic Effects of Membrane Benzodiazepine Receptors in the Ventral Tegmental Area

Progestins modulate lordosis through actions in the ventral tegmental area (VTA). Whether neurosteroidogenesis of 5α-pregnan-3α-ol-20-one (3α,5α-THP), involving mitochondrial benzodiazepine receptors (MBR), is important for lordosis was investigated. Ovariectomized (Ovx), hormone-primed rats (experiments 1, 3, 5, 6) and rats in behavioral estrus (experiments 2 and 4) were unilaterally infused via chronic guide cannula to the VTA with a MBR agonist, N,N-dihexyl-2-(4-fluorophenyl) indole-30-acetamide (FGIN 1–27) or antagonist 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboximide (PK-11195). Experiment 1: Estradiol benzoate (EB)-primed (25 µg) rats administered 0 or 25 µg progesterone (P₄) SC showed increased lordosis when infused with 5.0 µg FGIN 1–27 to the VTA; those administered 100 or 200 µg P₄ SC exhibited greater lordosis when infused with 2.5 or 5.0 µg FGIN, relative to saline-infused rats. Experiment 2: Rats, near the termination of behavioral estrus, infused with 2.5 or 5.0 µg of FGIN 1–27 to the VTA, showed increased lordosis compared to that seen following vehicle administration. Experiment 3: EB-primed rats administered 200 or 500 µg P₄ SC showed decreased lordosis when infused with 100, 200, or 400 ng PK-11195, relative to saline-infused rats. Experiment 4: Rats infused at the peak of behavioral estrus with 100, 200, or 400 ng PK-11195 to the VTA exhibited reduced lordosis compared to that seen following vehicle administration. Experiment 5: 3α,5α-THP (100 ng) infusions to the VTA reinstated lordosis of hormone-primed rats infused with PK-11195 (100 ng) to the VTA. Experiment 6: FGIN 1–27 (5.0 µg) and PK-11195 (100 ng) infusions aimed at the VTA respectively increased and decreased midbrain levels of 3α,5α-THP compared to vehicle. Notably, the specific effects observed with infusions to the VTA were not seen with infusions to the control site, the substantia nigra. These data suggest that neurosteroidogenesis involving MBRs in the VTA mediates lordosis of hormone-primed or behavioral estrous rats.

Evidence that Stimulation of Two Modalities of Pituitary Luteinizing Hormone Release in Ovarian Steroid-Primed Ovariectomized Rats May Involve Neuropeptide Y Y1 and Y4 Receptors

A large body of evidence indicates that neuropeptide Y (NPY) is involved in stimulation of basal and cyclic release of hypothalamic LHRH and pituitary LH. To identify the NPY receptor subtypes that mediate the excitatory effects of NPY in these two modalities of LH release, we studied the effects of 1229U91, a selective Y1 receptor antagonist and Y4 receptor agonist, in two experimental paradigms that reproduce the two modalities of LH secretion in steroid-primed ovariectomized (OVX) rats. Rats were ovariectomized and implanted with a permanent cannula into the lateral cerebroventricle. In the first experiment, rats received estradiol benzoate (EB, 30 μg/rat) on day 5, followed 2 days later with progesterone (2 mg/rat) at 1000 h to induce an afternoon LH surge. 1229U91 (30 μg/3 μl) or vehicle (control) was injected intracerebroventricularly into these rats either once at 1300 h or twice (15 μg/injection) at 1100 and 1200 h. Blood samples were collected before progesterone injection at 1000 h and at hourly intervals from 1300–1800 h via an intrajugular cannula implanted on the previous day. In control rats, serum LH levels rose significantly at 1400 h, and these high levels were maintained until 1700 h. After two injections of 1229U91, LH levels displayed a tendency to rise at 1300–1400 h, as in controls, but thereafter, decreased rapidly below the control range. In the second experiment, the acute effect of 1229U91 on LH release was evaluated in OVX rats pretreated with EB alone. Saline alone or saline containing 1, 3, 10, or 30 μg 1229U91 was injected intracerebroventricularly at 1000 h, and the effects on LH release were analyzed at 10, 20, 30, and 60 min. 1229U91 elicited a dose-dependent stimulation of LH release, with maximal response (950% of basal levels) occurring at 10 min after the 30-μg dose; elevated levels were maintained for 1 h. Because 1229U91 is a potent Y4 agonist with some affinity for Y5 receptors, these results raised the possibility that activation of Y4/Y5 receptors by 1229U91 may augment LH release. Therefore, we examined the effects of icv administration of rat pancreatic polypeptide, a Y4-selective agonist, and[ D-Trp32]-NPY, a Y5 agonist on LH release in EB-primed rats. Rat pancreatic polypeptide (0.5–2 μg/rat) stimulated LH release in a dose-related manner, and peak levels (280% of basal levels) were seen at 10–20 min; the response evoked by a higher dose (10 μg) was smaller than that induced by 0.5 or 2 μg.[ D-Trp32]-NPY was relatively less effective, because only the highest (10-μg) dose elicited a modest stimulation (244% of basal levels). These results demonstrate that 1229U91, a Y1 antagonist and Y4 agonist, evokes two types of responses; it suppresses the protracted ovarian steroid-induced LH surge, and acutely, it also stimulates LH. These results imply that normally two different types of NPY receptors may mediate the stimulation of LH release. Because 1229U91 is a Y1 receptor antagonist, inhibition of the steroid-induced LH surge by 1229U91 suggests that Y1 receptors may mediate the cyclic release of LH. On the other hand, acute stimulation of LH by 1229U91 implies that the Y4 agonist-like activity of 1229U91 may mediate the basal release of LH and that either NPY or a yet-to-be-identified endogenous Y4 receptor agonist may activate Y4 receptors in the hypothalamus to stimulate LH release.

Evidence that stimulation of two modalities of pituitary luteinizing hormone release in ovarian steroid-primed ovariectomized rats may involve neuropeptide Y Y1 and Y4 receptors.

A large body of evidence indicates that neuropeptide Y (NPY) is involved in stimulation of basal and cyclic release of hypothalamic LHRH and pituitary LH. To identify the NPY receptor subtypes that mediate the excitatory effects of NPY in these two modalities of LH release, we studied the effects of 1229U91, a selective Y1 receptor antagonist and Y4 receptor agonist, in two experimental paradigms that reproduce the two modalities of LH secretion in steroid-primed ovariectomized (OVX) rats. Rats were ovariectomized and implanted with a permanent cannula into the lateral cerebroventricle. In the first experiment, rats received estradiol benzoate (EB, 30 microg/rat) on day 5, followed 2 days later with progesterone (2 mg/rat) at 1000 h to induce an afternoon LH surge. 1229U91 (30 microg/3 microl) or vehicle (control) was injected intracerebroventricularly into these rats either once at 1300 h or twice (15 microg/injection) at 1100 and 1200 h. Blood samples were collected before progesterone injection at 1000 h and at hourly intervals from 1300 -1800 h via an intrajugular cannula implanted on the previous day. In control rats, serum LH levels rose significantly at 1400 h, and these high levels were maintained until 1700 h. After two injections of 1229U91, LH levels displayed a tendency to rise at 1300-1400 h, as in controls, but thereafter, decreased rapidly below the control range. In the second experiment, the acute effect of 1229U91 on LH release was evaluated in OVX rats pretreated with EB alone. Saline alone or saline containing 1, 3, 10, or 30 microg 1229U91 was injected intracerebroventricularly at 1000 h, and the effects on LH release were analyzed at 10, 20, 30, and 60 min. 1229U91 elicited a dose-dependent stimulation of LH release, with maximal response (950% of basal levels) occurring at 10 min after the 30-microg dose; elevated levels were maintained for 1 h. Because 1229U91 is a potent Y4 agonist with some affinity for Y5 receptors, these results raised the possibility that activation of Y4/Y5 receptors by 1229U91 may augment LH release. Therefore, we examined the effects of icv administration of rat pancreatic polypeptide, a Y4-selective agonist, and [D-Trp32]-NPY, a Y5 agonist on LH release in EB-primed rats. Rat pancreatic polypeptide (0.5-2 microg/rat) stimulated LH release in a dose-related manner, and peak levels (280% of basal levels) were seen at 10-20 min; the response evoked by a higher dose (10 microg) was smaller than that induced by 0.5 or 2 microg. [D-Trp32]-NPY was relatively less effective, because only the highest (10-microg) dose elicited a modest stimulation (244% of basal levels). These results demonstrate that 1229U91, a Y1 antagonist and Y4 agonist, evokes two types of responses; it suppresses the protracted ovarian steroid-induced LH surge, and acutely, it also stimulates LH. These results imply that normally two different types of NPY receptors may mediate the stimulation of LH release. Because 1229U91 is a Y1 receptor antagonist, inhibition of the steroid-induced LH surge by 1229U91 suggests that Y1 receptors may mediate the cyclic release of LH. On the other hand, acute stimulation of LH by 1229U91 implies that the Y4 agonist-like activity of 1229U91 may mediate the basal release of LH and that either NPY or a yet-to-be-identified endogenous Y4 receptor agonist may activate Y4 receptors in the hypothalamus to stimulate LH release.

Attenuation of dopamine uptake in vivo following priming with estradiol benzoate

Dopamine (DA) uptake and clearance were examined using in vivo voltammetry following injection of DA (200 μM) into the nucleus accumbens of ovariectomized (OVX) or OVX-estrogen-primed rats (estradiol benzoate, EB, 10 μg 48 and 24 h prior to experiment). The rate of DA uptake was significantly attenuated in steroid-treated animals: this decrease was accompanied by a significant increase in DA clearance time. Quinpirole (0.5 mg/kg) modulated the kinetics of DA uptake in OVX but not EB-primed rats. These data suggest that DA clearance can be regulated by physiological doses of EB.

The neurosteroids, progesterone and 3α,5α-THP, enhance sexual motivation, receptivity, and proceptivity in female rats

The effects of progesterone (P) and the neurosteroid and P metabolite, 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) on ovariectomized (ovx), estradiol-3-benzoate (EB)-primed rats on sexual motivation, receptivity, and proceptivity were examined. Changes in central P and 3α,5α-THP were measured following administration of EB, EB+P, EB+3α,5α-THP, or EB+inhibitor of 5α-reductase or P metabolism (epostane and finasteride)+P (Expt. 1). Partner preference was measured as the duration of time females in these different hormonal treatments spent in proximity to a male vs. female conspecific (Expt. 2). Receptivity (lordosis quotients and ratings) and proceptivity (darting, hopping, ear wiggling, and pacing), for different hormone treatments were assessed (Expt. 3 and Expt. 4, respectively). Conditioned place preference following hormone treatments and paced mating enabled assessment of sexual motivation (Expt. 5). Central P and 3α,5α-THP were measured in various combinations of hormone/mating conditions (Expt. 6). Studies revealed that 3α,5α-THP has a significant role in these reproductive measures. Brain concentrations of 3α,5α-THP were significantly higher in animals receiving EB+P or EB+3α,5α-THP compared to animals receiving EB alone, or EB+P in conjunction with an inhibitor of P metabolism. EB+P and EB+3α,5α-THP significantly increased time spent in proximity to the male, receptivity and proceptivity. When administered to ovx, EB-primed rats, the progestin metabolite, 3α,5α-THP, had effects on these behaviors similar to P. Epostane, an inhibitor of P and 3α,5α-THP biosynthesis, and finasteride, an inhibitor of P metabolism to 3α,5α-THP, administered to EB+P animals reduced male partner preference, proceptive, and receptive behaviors to levels seen in EB+vehicle animals. Notably, whole brain 3α,5α-THP levels were significantly increased and whole brain P levels were significantly reduced in paced mated rats compared to standard mated, and receptive non-mated animals. These studies suggest that P and 3α,5α-THP may have some common effects on reproductive behavior, e.g., sexual motivation, receptivity, and proceptivity.

Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge.

Neuropeptide-Y (NPY), a hypothalamic peptide, is involved in stimulation of LHRH and LH surges during proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. The NPY neurons that reside in the arcuate nucleus of the medial basal hypothalamus (MBH) and accumulate 17 beta-estradiol participate in the initiation of LHRH and LH surges. To determine whether NPY synthesis is altered in conjunction with the LH surge, we studied the dynamic changes in prepro-NPY mRNA levels in the MBH in association with the LH surge elicited by estradiol benzoate (EB) alone or by progesterone (P) in EB-primed ovx rats. Five days after ovariectomy, rats received oil or EB (30 micrograms/rat) at 1000 h on day 0. On day 2, these rats were injected with either oil or P (2 mg/rat) at 1000 h. Rats were killed before (1000 h) and at 2-h intervals after oil or P injection. The MBHs were dissected out and processed for determination of prepro-NPY mRNA levels by solution hybridization/RNase protection assay using a cRNA probe. Although in control ovx rats, prepro-NPY mRNA levels remained unchanged between 1000-1600 h, prepro-NPY mRNA levels showed dynamic changes in steroid-primed rats. In the EB-primed rats, prepro-NPY mRNA levels rose significantly (100%) at 1200 and 1400 h before the LH rise at 1600 h, and the levels remained elevated up to 1800 h. After P injection to the EB-primed rats, this response was further augmented, with a slightly different temporal pattern. Prepro-NPY mRNA levels rose at 1400 h (600%) before the onset of the LH rise at 1600 h and declined steadily to significantly lower values at 1800 h, coincident with the highest rate of LH secretion. These studies demonstrate dynamic shifts in hypothalamic NPY gene expression in association with the LH (LHRH) surge, and that maximal increases occur before the onset of the LH rise, but thereafter, NPY gene expression diverged in the two ovarian-steroid treatment models. These findings along with previous evidence of similar antecedent increases in NPY content in the median eminence, followed by release, suggest that augmented NPY synthesis and release are two temporally dissociable neural events for the LHRH and LH surges.

Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge

Neuropeptide-Y (NPY), a hypothalamic peptide, is involved in stimulation of LHRH and LH surges during proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. The NPY neurons that reside in the arcuate nucleus of the medial basal hypothalamus (MBH) and accumulate 17 beta-estradiol participate in the initiation of LHRH and LH surges. To determine whether NPY synthesis is altered in conjunction with the LH surge, we studied the dynamic changes in prepro-NPY mRNA levels in the MBH in association with the LH surge elicited by estradiol benzoate (EB) alone or by progesterone (P) in EB-primed ovx rats. Five days after ovariectomy, rats received oil or EB (30 micrograms/rat) at 1000 h on day 0. On day 2, these rats were injected with either oil or P (2 mg/rat) at 1000 h. Rats were killed before (1000 h) and at 2-h intervals after oil or P injection. The MBHs were dissected out and processed for determination of prepro-NPY mRNA levels by solution hybridization/RNase protection assay using a cRNA probe. Although in control ovx rats, prepro-NPY mRNA levels remained unchanged between 1000-1600 h, prepro-NPY mRNA levels showed dynamic changes in steroid-primed rats. In the EB-primed rats, prepro-NPY mRNA levels rose significantly (100%) at 1200 and 1400 h before the LH rise at 1600 h, and the levels remained elevated up to 1800 h. After P injection to the EB-primed rats, this response was further augmented, with a slightly different temporal pattern. Prepro-NPY mRNA levels rose at 1400 h (600%) before the onset of the LH rise at 1600 h and declined steadily to significantly lower values at 1800 h, coincident with the highest rate of LH secretion. These studies demonstrate dynamic shifts in hypothalamic NPY gene expression in association with the LH (LHRH) surge, and that maximal increases occur before the onset of the LH rise, but thereafter, NPY gene expression diverged in the two ovarian-steroid treatment models. These findings along with previous evidence of similar antecedent increases in NPY content in the median eminence, followed by release, suggest that augmented NPY synthesis and release are two temporally dissociable neural events for the LHRH and LH surges.

Interaction of estradiol, alpha-melanocyte-stimulating hormone, and dopamine in the regulation of sexual receptivity in the female rat.

Ovariectomized and adrenalectomized rats kept in a reversed lighting system received either 5 micrograms (once) or 1 microgram estradiol benzoate (EB) daily for 2, 3, or 4 days. These treatments induced sexual receptivity in a proportion of the rats, and alpha-melanocyte-stimulating hormone (alpha-MSH; 20 micrograms/rat s.c.) enhanced this proportion. In rats made receptive by 5 micrograms EB alone, the dopamine (DA) activity in preoptic area and arcuate nucleus was significantly reduced, but the alpha-MSH concentrations were not affected 54-56 h after EB treatment. Addition of alpha-MSH significantly increased the DA activity in ventromedial nucleus (VMN) and zona incerta, but this action is unlikely to account for its stimulation of sexual receptivity, since this effect was not blocked by 0.1 mg/kg haloperidol. Treatment with 100 mg/kg Dopa, which induces a presynaptic DA action, stimulated the receptivity in the EB-primed rats and selectively increased the concentration of alpha-MSH in the VMN, while 50 mg/kg Dopa plus 50 mg/kg benserazide, which induces a postsynaptic DA activity, inhibited the receptivity and reduced the alpha-MSH levels in the VMN. It is suggested that estradiol stimulates the female sexual receptivity by reducing the activity of a dopaminergic system in arcuate nucleus and preoptic area. alpha-MSH does not appear to affect this action, although it enhances the effect of steroids on the sexual behaviour, probably at the level of the VMN. The secretion of alpha-MSH may be under a dopaminergic inhibitory control, and the peptide may autoregulate its own secretion via this dopaminergic system which is sited in zona incerta and VMN.

Phorbol esters and forskolin infused into midbrain central gray facilitate lordosis.

Several peptides, when infused into the MCG, facilitate lordosis in estrogen-primed female rats. Since these peptides can act through cAMP and/or protein kinase C, and these second messenger systems have been implicated in neuromodulation, this study examined if pharmacological agents which stimulate these systems would facilitate lordosis. Ovariectomized female Fisher rats were given bilateral cranial cannulae targeted to the MCG, or cortex dorsal to MCG, and allowed at least a week to recover. Forty-eight hours after injection of 1.25 micrograms estradiol benzoate (EB), 1 microliter of each of the following was infused into the MCG (n=8-12): 1) forskolin (5 micrograms/microliter 50% DMSO); 2) phorbol-20-oxo-20-deoxy-12,13-dibutyrate (PBu; 5 micrograms/microliter 50% DMSO); 3) both forskolin and PBu (2.5 micrograms of each/microliter); 4) vehicle (50% DMSO). In a separate study of identical design, 1 microliter of another phorbol ester (12-myristate 13-acetate) was infused into the MCG of EB-primed rats. Forskolin and phorbol esters each facilitated lordosis maximally at 60-90 minutes after infusion. Combining both agents also facilitated lordosis, and vehicle had no effect. These results suggest that infusing agents which stimulate cAMP and protein kinase C into the MCG can facilitate lordosis in estrogen-primed female rats.

Serotonergic mediation of a negative feedback effect of estrogen on luteinizing hormone release in ovariectomized rats.

Effects of the serotonin receptor antagonists ketanserin, metergoline, and methysergide on LH release were tested in ovariectomized (OVX) rats pretreated with estradiol benzoate (EB) (5 micrograms/100 g BW) on each of the 2 days preceding the experiment. LH concentration measured by RIA in plasma samples obtained at 10 min intervals was analyzed using the PULSAR computer program to identify and characterize pulses observed in the LH profile of each rat individually. Multiple pulses were identified in seven of seven OVX rats but in only two of eight OVX rats primed with EB. Multiple pulses were identified in OVX EB-primed rats pretreated with ketanserin (six of seven), metergoline (three of seven), and methysergide (six of eight). Administration of the serotonin (5HT) agonist quipazine (2 mg/kg iv) to OVX rats inhibited pulsatile LH release and depressed mean plasma concentrations for approximately 45 min. This inhibitory response was antagonized by pretreatment of the OVX animals with ketanserin. These results suggest that 1) quipazine inhibits pulsatile LH release in OVX rats by a serotonin2 receptor mechanism; and 2) the inhibitory effect of estrogen on pulsatile LH release may also be mediated, at least in part, via 5HT2 systems.

Effect of progesterone on monoamine turnover in the brain of the estrogen-primed rat

The effect of progesterone (P) on monoamine levels and turnover was evaluated in 8 brain nuclei in estrogen-primed rats. Animals were subcutaneously (SC) injected with P or vehicle 21 hours after SC treatment with 5 μg of estradiol benzoate (EB). EB-primed animals treated with P showed high levels of lordosis behavior and an LH surge three hours later. Initial concentrations of norepinephrine (NE), dopamine (DA), serotonin (5HT) and 5-hydroxyindole acetic acid were determined in EB-saline treated controls 3 hours after P or vehicle. NE and DA turnover was estimated from the exponential decline of these amines 2 hours after IP injection of alpha-methyl-p-tyrosine (5 hours after P or vehicle). The accumulation of 5HT 20 min following IP injection of pargyline was used as an index of 5HT turnover. P did not affect the initial NE, 5HT or 5HIAA concentrations in any of the brain nuclei studied, but decreased DA content in the arcuate-median eminence region (Ar-ME). The DA rate constant was elevated in the nucleus of the diagonal band of Broca and the DA turnover rate was decreased in the Ar-ME. In the periventricular region (PVE, anterior hypothalamic level) the NE turnover rate (K, pg/μg protein/hr) and rate constant (k, hr −1) decreased following P treatment. Progesterone treatment decreased the accumulation of 5HT in the ventromedial hypothalamus (VMN, pars lateralis) and the dorsal midbrain central grey (MCG). Progesterone effects on monoamine turnover were not found in the lateral septal, medial preoptic, anterior hypothalamic or dorsal raphe nuclei. These results suggest that progesterone influences monoamine activity in a few discrete brain nuclei in EB-primed rats. Decreased 5HT turnover in the VMN and MCG after P is consistent with the hypothesis that 5HT exerts an inhibitory modulation of lordosis behavior and suggests that a component of the facilitatory action of progesterone is the suppression of 5HT activity at these brain sites. Changes in catecholamine turnover are discussed in relation to possible contributions in regulating gonadotropin secretion.

Role of central serotonin systems in the stimulatory effects of ovarian hormones and naloxone on luteinizing hormone release in female rats.

The present experiments tested whether serotonergic neurons that innervate discrete areas of the hypothalamus are involved in the stimulation of LH release by ovarian hormones or the opiate antagonist naloxone in female rats. Ovariectomized rats received oil vehicle, estradiol benzoate (EB) alone, or EB followed by progesterone (P) 2 days later. Serotonin (5-HT) activity was assessed from the accumulation observed after the administration of the monoamine oxidase inhibitor pargyline. Two days after EB treatment, LH concentrations were reduced in the morning and rose by later afternoon. Administration of P to EB-primed rats stimulated a LH surge. This latter treatment also enhanced pargyline-induced 5-HT accumulation, suggesting increased 5-HT activity, in the medial preoptic nucleus and interstitial nucleus of the stria terminalis, but reduced 5-HT accumulation, suggesting decreased 5-HT activity, in the ventromedial nucleus. 5-HT activity was unaffected after EB alone, either in the morning or afternoon, or by the administration of naloxone to EB-primed rats. Specific depletion of 5-HT in the medial preoptic/stria terminalis area, achieved by microinjection of the neurotoxic indoleamine 5,7-dihydroxytryptamine resulted in a blockade of the LH surge induced by EB plus P. The present results suggest that central 5-HT neurons innervating the preoptic area are involved in the LH surge induced by progesterone, but not in the increases in LH occurring after treatment with estradiol alone or after blockade of opiate receptors.

Effects of ovarian hormones on the concentrations of immunoreactive neuropeptide Y in discrete brain regions of the female rat: correlation with serum luteinizing hormone (LH) and median eminence LH-releasing hormone.

Recent pharmacological studies have suggested a role for neuropeptide Y (NPY) in control of LH secretion. The present study examined the effects of estradiol benzoate (EB) given alone or in combination with progesterone (P), on the concentrations of immunoreactive NPY in microdissected nuclei of the rat brain, in association with changes in LHRH concentrations and LH release. Forty-eight hours after the administration of EB to ovariectomized rats, serum LH was significantly reduced. Concentrations of NPY, measured by a sensitive and specific RIA, were also reduced in the median eminence, arcuate nucleus, and interstitial nucleus of the stria terminalis. As expected, administration of P to these EB-primed rats induced a sequential rise and fall of LHRH in the median eminence, followed by a marked LH surge in the afternoon. Interestingly, NPY concentrations in the median eminence also increased and then decreased after P, with a time course similar to that shown by LHRH. In the arcuate nucleus and interstitial nucleus of the stria terminalis, P treatment did not affect NPY concentrations further. Sequential EB plus P treatment significantly reduced NPY levels in the medial preoptic nucleus compared to oil vehicle controls. Levels of immunoreactive NPY in the ventromedial nucleus and periventricular nucleus were largely unaffected by ovarian hormone treatments. These results indicate that ovarian steroids, which modulate LH secretion, affect NPY concentrations in those specific areas of the brain which are known to be innervated by the LHRH neurons. These observations support the hypothesis that NPY may participate in the neural regulation of LHRH and LH secretion.

A facilitatory role for serotonin in the sexual behavior of the female rat

The peripheral administration of the serotonin type 2 receptor (5-HT 2) antagonist pirenperone inhibited sexual receptivity in ovariectomized female rats primed either chronically with estradiol benzoate (EB), or acutely with EB plus varying doses of progesterone. An inhibition occurred at 50, 100 and 150 but not 25 μg/kg pirenperone. Increasing the dose of progesterone did not attenuate the inhibitory effect of pirenperone. Two other 5-HT 2 antagonists, ketanserin (2.5 mg/kg) and spiperone (250 μg/kg), also inhibited receptivity in females primed with EB and progesterone. The inhibitory effect of pirenperone on receptivity was attenuated by the 5-HT agonist quipazine (3 mg/kg), though quipazine alone had no effect on receptivity. Whereas the 5-HT antagonist methysergide (3 mg/kg) failed to have an effect on receptivity in EB-primed females, methysergide co-administered with quipazine facilitated receptivity. Pirenperone also inhibited proceptivity in females primed with EB and progesterone. Although quipazine did not attenuate the pirenperone-induced inhibition of proceptivity, quipazine alone increased proceptivity. Moreover, quipazine facilitated proceptivity in EB-primed rats whether progesterone was present or absent. The results suggest that 5-HT may serve both a facilitatory and inhibitory role in female sexual behavior, perhaps reflecting 5-HT 2 and 5-HT 1 receptor activity, respectively.