Apparent involvement of protein kinase C in the central glucoregulatory action of insulin

We have studied the possible involvement of the calcium- and phospholipid/diacylglycerol-dependent enzyme, protein kinase C (PKC) in mediating insulin action in the central nervous system (CNS) by testing the effect of direct activation or blockade of the CNS PKC system on the plasma glucose responses to central insulin injection in mice. Insulin (0.1–1 μg), injected into the CNS, produced rapid transient hypoglycemia. This effect appeared to involve interaction of insulin with specific receptors, since insulin analogs exhibiting diminished receptor binding affinity and peripheral bioactivity compared to the native hormone were much less active (i.e., insulin >>> acetyl 3 insulin > proinsulin > IGF-I) or not active at all (i.e., insulin chain A and B). Central injection of the specific PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.01–0.5 μg), but not the inactive TPA analog, 4-orbol or the unstable synthetic diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), significantly enhanced the hypoglycemic response to co-administered insulin (0.5 μg) or the insulin derivative, acetyl 3 insulin (2.5 μg). Central TPA had no effect on basal glucose levels. Furthermore, central administration of the selective PKC blockers, polymyxin B (PMB, 1–25 μg) or 1-β-galactosylsphingosine (psychosine, 0.5–10 μg) but not their respective inactive analogs, polymyxin E and sphingomyelin, strongly inhibited the hypoglycemic response to insulin (1 μg) or acetyl 3 insulin (5 μg). PMB and psychosine, injected alone had no effect on basal glucose levels. These findings, of a significant enhancement or blockade of the hypoglycemic response to central insulin following direct, selective activation or inhibition, respectively, of the CNS PKC system are consistent with the view that PKC might play a role in the mediation of insulin action in the CNS.     

Effect of estrogen on the lordosis-inhibiting action of ketanserin and SB 206553

The effects of the 5-HT(2A/2C) receptor antagonist, ketanserin, and the 5-HT(2C) receptor antagonist, SB 206553, on lordosis behavior were investigated in ovariectomized rats hormonally primed with estradiol benzoate (EB) (0.5 or 25 microg) and progesterone (500 microg). Both ketanserin and SB 206553 inhibited lordosis behavior after infusion into the ventromedial nucleus of the hypothalamus (VMN), but ketanserin was slightly more effective than the 5-HT(2C) receptor antagonist. Either drug was more effective in rats primed with 0.5 microg EB than in rats hormonally primed with 25 microg EB. These findings support the suggestion that estrogen may enhance functioning of the 5-HT(2) receptor family and thereby protect against the 5-HT(2) receptor antagonists. These data are consistent with prior suggestions that estrogen modulates functioning of 5-HT(2) receptors within the VMN and that 5-HT(2) receptors play a facilitatory role in the modulation of female rat lordosis behavior.     

α1-Adrenergic agonists act on the ventromedial hypothalamus to cause neuronal excitation and lordosis facilitation: electrophysiological and behavioral evidence

To see if activation of central α₁-adrenergic receptors can cause facilitation of lordosis in rats, the behavioral effects of centrally administered α₁-agonist, methoxamine (MA) and phenylephrine (PhE), and related agents were studied. In ovariectomized rats treated with estrogen, infusion of MA, PhE, or a β-agonist isoproterenol, into the lateral ventricle, or bilateral infusions of MA or PhE into the ventromedial hypothalamus (VMH) facilitated lordosis. Conversely, intra-VMH infusion of the α₁-antagonist prazosin (PZ) inhibited lordosis. Intra-VMH infusion of isoproterenol or an α₂-agonist clonidine, had no effect. Neither was the intra-VMH infusion of MA effective if: (i) the rats were not primed with estrogen; (ii) the tips of the cannulae were outside the VMH; or (iii) it was preceded by an intra-VMH infusion of the α_1b-antagonist, chloroethylclonidine (CEC). These results not only verify implications from recent studies that α₁-receptors in the hypothalamus are important for lordosis facilitation, but further show that the adrenergic facilitatory effect are: (i) mediated specifically by α_1b-subtype of the α₁-receptor, (ii) estrogen-dependent, and (iii) site-specific to VMH. To investigate neural mechanisms potentially underlying the lordosis-facilitating effect of α₁-activation, the actions of MA and PhE on the electrical activity of single neurons of the ventromedial nucleus of the hypothalamus (VMN) in vitro were studied. As in the behavioral study, the neuronal actions of the agonists are similar to each other: (i) both are excitatory — MA affected 78% of the 65 units tested, all by excitation, and PhE affected 74% of 65 VMN units, predominantly with excitation; (ii) the excitatory actions of MA and PhE affected the same population of VMN neurons: and (iii) the excitatory actions of both agonists were blocked by both PZ and CEC, indicative of mediation by α_1b-receptors. Behavioral/electrophysiological parallels suggest that act α₁-agonists through α_1b-receptors to excite VMN neurons and thereby facilitate lordosis. Since α_1b-receptors are known to be coupled to phosphoinositide (PI) second messenger system, and since the behavioral effect is estrogen-dependent, the facilitatory effect of α₁-agonists may require estrogen modulation of α_1b-receptors and/or the coupled PI system in the VMN.     

Progesterone attenuates the effect of the 5-HT1A receptor agonist, 8-OH-DPAT,and of mild restraint on lordosis behavior

Ovariectomized, hormone-primed rats were used to test the hypothesis that progesterone treatment attenuated the effects of the 5-HT(1A) receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on female rat lordosis behavior. Based upon prior evidence that prepriming with estradiol benzoate (EB) reduced the ability of 8-OH-DPAT to inhibit lordosis behavior, rats were preprimed with 10 microg EB 7 days before a second priming with 10 microg EB followed 48 h later with 500 microg progesterone or vehicle. Independent of the presence of progesterone, prepriming with EB attenuated the lordosis-inhibiting effects of systemic treatment with 8-OH-DPAT. However, progesterone also reduced the effects of 8-OH-DPAT and this effect was also seen in females primed only once with EB. In contrast, progesterone was relatively ineffective in attenuating the effects of bilateral infusion with 8-OH-DPAT into the ventromedial nucleus of the hypothalamus (VMN). The failure of progesterone to substantially reduce the effects of VMN infusion with 8-OH-DPAT contrasts with prior studies in which estrogen's protective action against the drug did include the VMN. Thus, while both estrogen and progesterone reduce the lordosis-inhibiting effect of 8-OH-DPAT, the mechanisms responsible for the effects of the two gonadal hormones may be different. Priming with progesterone also prevented the effects of 5 min of restraint. When rats were hormonally primed with EB and oil, rats showed a transient, but significant, decline in lordosis behavior 5 and 10 min after restraint. Rats primed with EB and progesterone were unaffected by the restraint. These results are discussed in terms of their implications for the role of progesterone in altering the 5-HT(1A) receptor modulation of lordosis behavior.     

Steroid priming promotes oxytocin-induced norepinephrine release in the ventromedial hypothalamus of female rats

In vivo microdialysis was used to detect norepinephrine (NE) release in the ventromedial hypothalamus of estradiol (E₂)- or E₂ plus progesterone (P)-treated female rats injected with 1.0 IU of oxytocin (OXY). Dialysates were collected before and after OXY administration on 3 consecutive days and analyzed for NE content by high performance liquid chromatography with electrochemical detection. After the last sample was collected on day 1, animals were injected with 3 μg E₂ benzoate or oil. On day 3, E₂-primed animals received 200 μg of P and control females received oil prior to OXY administration. OXY administration did not induce NE release on day 1. When OXY was administered to animals that received E₂ approximately 20 h earlier, increased release of NE was not consistently seen. In contrast, E₂-primed animals that received P on day 3 displayed significant increases in the release of NE after OXY administration compared to their own basal levels and to NE levels in control animals. To distinguish whether E₂ priming is sufficient to promote OXY-induced release of NE without the addition of P, NE content of VMH dialysates in a second group of animals was examined following exposure to vehicle or E₂ alone. When OXY was administered 24 or 48 h after estrogen priming, only 1 of 4 E₂-primed females had modestly elevated dialysate NE levels. To evaluate the interactions between OXY and NE in the regulation of reproductive behavior, lordosis responses were observed in hormone-primed female rats receiving systemic injections of OXY, the ₁-adrenoceptor antagonist prazosin, or both OXY and prazosin. OXY enhanced lordosis behavior in females primed with subthreshold doses of E₂ and P. Prazosin abolished lordosis behavior in rats primed with behaviorally effective doses of E₂ and P and significantly inhibited lordosis in steroid-primed females given OXY. These data suggest that after priming with both E₂ and P together, but not with E₂ alone, OXY may facilitate lordosis behavior through activation of NE transmission.     

Hypoxia-induced vascular endothelial growth factor expression in normal rat astrocyte cultures

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, which also enhances vascular permeability. Because this angiogenic factor has been suggested to play a role in brain tumor biology, we have begun to investigate the regulation of VEGF expression in cultures of rat type I astrocytes. In this report, we have focused on the influence of hypoxia on VEGF expression. Under standard in vitro conditions (21% O₂) VEGF expression in astrocytes is barely detectable by northern analysis. However, after exposure to 0.2% O₂ for as little as 3 h VEGF mRNA levels are markedly increased reaching a maximum by approximately 8 h of exposure. Treatment of astrocytes with CoCl₂ or desferrioxamine results in a similar induction of VEGF, suggesting that the oxygen sensor regulating VEGF expression in astrocytes is a heme-containing molecule. Although acute treatment with TPA (6 h) induces VEGF expression, chronic exposure to TPA (24 h) to deplete PKC activity does not reduce the hypoxia-induced VEGF expression. These data indicate that VEGF induction in astrocytes can proceed through PKC-dependent and -independent pathways. Furthermore, chronic exposure to TPA or treatment with herbimycin A results in the enhancement of the hypoxia-mediated increase in VEGF mRNA levels. These results suggest that PKC and herbimycin-sensitive tyrosine kinase may serve as negative regulators of the hypoxia-activated signal transduction pathway that leads to the induction of VEGF expression. However, treatment of astrocytes with the nonspecific kinase inhibitors H7 and H8 reduced the level of VEGF induction by hypoxia, indicating that some type of kinase activity is required in this signaling pathway. © 1995 Wiley-Liss, Inc.     

Ultrastructural changes in the hypothalamic ventromedial nucleus of ovariectomized rats after estrogen treatment

The changes produced in the hypothalamic ventromedial nucleus (VMN) of ovariectomized rats after administration of 100 μg estradiol benzoate/kg body weight were studied using light and electron microscopy. Quantitative morphometric studies included number and size of VMN neurons and nuclei, size and density of terminals and synaptic contacts, spine-to-shaft ratio of postsynaptic elements and relative frequency of two types of synaptic vesicles. Evidence was obtained favoring the concept of heterogeneous composition of the VMN: in ovariectomized animals many cells appeared in a state of quiescence, but other neurons showed no major alterations. Estrogen administration to ovariectomized rats produced evidence of metabolic stimulation such as increase in rough surfaced endoplasmic reticulum, condensation of nucleolar material, enlarged Golgi and presence of pleomorphic mitochondria. The number of neurons in the VMN was not modified by estrogen treatment; however, neuron soma and nuclei were larger. In the ventrolateral division of the VMN terminals and synaptic contacts per unit area were increased after estrogen treatment, but synaptic contact length, terminal size and spine-to-shaft ratio were not modified. The possibility that the differences observed may be consequent to changes in synaptic organization of the VMN related to its estrogen-dependent functions is discussed.     

Modulation of lordosis behaviour in the female rat by corticotropin releasing factor, β-endorphin and gonadotropin releasing hormone in the mesencephalic central gray

A possible functional relationship between corticotropin-releasing factor (CRF) and opiate peptide neuronal systems (β-endorphin, dynorphin_1–17 and Met-enkephalin) and their interactions with gonadotropin releasing hormone (GnRH) in the mesencephalic central gray (MCG) for the regulation of lordosis behaviour was assessed in ovariectomized, oestrogen-treated and oestrogen-progesterone-treated female rats. Lordosis behaviour triggered by male mounting was inhibited in a dose-dependent fashion by CRF microinfused into the MCG in both oestrogen-treated and oestrogen-progesterone-treated female rats. This CRF-induced inhibition of lordosis could be overcome by a pre-infusion of naloxone or anti-β-endorphin-globulin (anti-β-end-G) directly into the MCG but not by anti-Met-enkephalin globulin (anti-enk-G) or anti-dynorphin_1–17 globulin (anti-dynor-G). Supporting data indicate that the facilitation of lordosis behaviour induced by treatment with naloxone or anti-β-end-G alone but not with anti-enk-G or anti-dynor-G may be due to enhanced GnRH release. This results from the action of these substances in overcoming the inhibition of GnRH secretion mediated specifically by β-endorphin but not by Met-enkephalin or dynorphin_1–17 in the MCG. These studies together with previous data showing that GnRH can overcome the abolition of lordosis by β-endorphin in the MCG, indicate a close relationship between β-endorphin (but not Met-enkephalin or dynorphin) and GnRH systems in the MCG in the control of lordosis behaviour. Thus, the inhibition of lordosis by CRF and the complete reversal of this blockade by naloxone or anti-β-end-G may suggest that CRF could enhance the release of β-endorphin from fibres in the MCG; β-endorphin then inhibits lordosis by inhibiting the release of GnRH. However, a direct inhibitory effect of CRF on GnRH release is also likely since anti-CRF-γ-globulin (anti-CRF-G) infused into the MCG produced a long-lasting facilitation of lordosis which can be blocked by an antagonist analogue of GnRH; in addition, previous studies have shown that GnRH infused into the MCG completely overcame the CRF-induced abolition of lordosis and potentiated lordosis to high levels. These results suggest that there may be functional neuroanatomical relationships between CRF, β-endorphin and GnRH neuronal systems in the MCG in the control of female sexual behaviour. Neither Met-enkephalin nor dynorphin_1–17 appear to participate in such mechanisms.     

Lordosis-inhibiting effect of progesterone in male and female rats with septal lesions

The inhibitory role of progesterone (P) in regulating lordosis was investigated in male and female rats with septal lesions (SL). Male rats with SL showed lordosis quotients (LQ) as high as female rats with SL and female control rats without brain surgery after injection of 50 microg/kg estradiol benzoate (EB) followed by 0.5 mg P 44 h later. Even when primed with 5 mg P 1 h prior to the 50 microg EB-injection, the mean LQs were still high in all groups. When the dose of EB was decreased to 5 microg/kg, all rats showed high-score LQs. In contrast, all animals in both male and female in which 5 mg P was injected 1 h before 5 microg EB, showed low LQs. These results suggest that P is effective in suppressing lordosis enhanced by estrogen in either male rats or females. Furthermore, the high dose of estrogen overcomes the inhibitory action of P on lordosis in both sexes.     

Acute estrogen potentiates excitatory responses of neurons in rat hypothalamic ventromedial nucleus

In a previous behavioral study, brief application of a membrane-limited estrogen to neurons in rat hypothalamic ventromedial nucleus (VMN) facilitated lordosis behavior-inducing genomic actions of estrogen. Here, electrophysiological recordings from single neurons were employed to characterize these membrane-initiated actions. From rat hypothalamic slices, electrical activity was recorded from neurons in the ventrolateral VMN, the cell group crucial for estrogen induction of lordosis. In addition to the resting activity, neuronal responses to histamine (HA) and N-methyl-d-aspartate (NMDA) were also recorded before, during, and after a brief (10-15 min) application of estradiol (E, 10 nM). These two transmitters were chosen because their actions are mediated by different mechanisms: HA through G protein-coupled receptors and NMDA by ligand-activated ion channels. Vehicle applications did not affect either resting activity or neuronal responses. In contrast, acute E exposure modulated neuronal responses to transmitters, with no significant effect on the resting activity. It potentiated excitatory responses to HAs (20 out of 48 cells tested) and to NMDA (10 out of 19 cells), but attenuated inhibitory responses to HA (3 out of 6 units). Both of these hormonal actions would increase VMN neuronal excitation. In separate experiments, neuronal excitation was found to be suppressed by anesthetics, which would block E's induction of lordosis when administered at the time of estrogen application. These data are consistent with the notion that increasing electrical excitation of VMN neurons can be a mechanism by which acute E exposure facilitates the lordosis-inducing genomic actions of estrogens.     

In vitro electro-pharmacological and autoradiographic analyses of muscarinic receptor subtypes in rat hypothalamic ventromedial nucleus: implications for cholinergic regulation of lordosis

Muscarinic agonists can act through the hypothalamic ventromedial nucleus (VMN) to facilitate lordosis. To elucidate the neuronal mechanism(s) underlying this muscarinic facilitation, effects of muscarinic agents on the single-unit activity of VMN neurons recorded in brain tissue slices of estrogen-primed female rats were analyzed. All the agonists tested, including acetylcholine (ACh), oxotremorine-M (OM), carbachol (CCh) and McN-A-343 (McN), evoked primarily excitation (80–100%), some inhibition (0–20%) and occasional biphasic responses (0–8%). By comparing the response magnitude and the effectiveness in evoking a response, the rank order for evoking excitation, the primary response, was found to be: OM > CCh > ACh McN, which is consistent with that (OM > CCh McN) for facilitating lordosis reported by others. This consistency and the frequency of its occurrence suggest that the excitatory electric action of the muscarinic agonists is related to their facilitatory behavioral effect. Experiments with antagonists selective for M1 (pirenzepine), M2 (AF-DX 116) and M3 (4-DAMP and p-F-HHSiD) indicate that muscarinic excitations are mediated by M1 and/or M3, but not M2. Since M1 receptors have been shown to be neither sufficient nor necessary to mediate the muscarinic facilitation, M3 receptor may be crucially involved in this behavioral effect. Autoradiographic assays of binding to [³H]4-DAMP with or without pirenzepine and AF-DX 116, also indicate the presence of M3 receptors in the VMN. Quantitative analyses show that the M3 binding was not affected by the in vivo estrogen priming required to permit muscarinic agonists to facilitate lordosis. Thus, while the excitation mediated by M3 is likely to be involved in muscarinic facilitation of lordosis, the regulation of M3 receptor density does not seem to be involved in the permissive action of estrogen.     

Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulate reproductive behavior in the female rat

Increased GABA activity in the medial hypothalamus (HYP) and midbrain central gray (MCG), but not the preoptic area (POA), facilitates sexual receptivity in the female rat [40]. In the current experiments, ovariectomized females were chronically treated with estrogen (via silastic capsules) to maintain a continuously high level of lordosis response. Administration of crystalline antisense oligodeoxynucleotide to the GABA synthetic enzyme, GAD₆₇, into the HYP and MCG significantly and reversibly reduced lordosis response for 1–2 days, but did not inhibit lordosis when administered into the POA. Administration of a control oligonucleotide, consisting of the same nucleotide bases but in a scrambled sequence, did not significantly modulate behavior when infused into any brain areas. When oligodeoxynucleotide antisense to GAD₆₇ was suspended in oil and then infused into the HYP or MCG it was more effective and resulted in less inter-animal variability. Subsequent experiments involving infusions into the MCG compared the effectiveness of antisense oligonucleotides to the two different forms of GAD, known as GAD₆₅ and GAD₆₇. Oligodeoxynucleotides antisense to the mRNA for either gene were effective at reducing lordosis behavior but with a different time course. Oligonucleotide antisense to GAD₆₇ significantly reduced behavior within 24 h of infusion and there was full recovery by 4 days post-infusion. GAD₆₅ antisense oligonucleotide did not significantly reduce behavior until 48 h post infusion and animals did not fully recover to pretest levels of lordosis until 5 days post-infusion. When antisense oligonucleotide for the two genes was administered simultaneously, the inhibition of lordosis was maximal at 24 h and stayed depressed for 4 days. There did not appear to be an additive effect of the two different antisense oligonucleotides when administered together. Tissue GABA levels in HYP and MCG of individual rats assayed by HPLC were no longer correlated with lordosis score after antisense oligonucleotide infusion but were after infusions of scrambled control oligos. Immunoblotting for the two forms of GAD revealed that GAD₆₇ antisense oligonucleotide infusion led to significant decreases in both GAD₆₇ and GAD₆₅ protein levels as compared to infusions of scrambled control oligo. In addition, the levels of a neuronal marker, neuron-specific enolase, also decreased (although nonsignificantly) suggesting either a temporary shutdown of protein synthesis or a degeneration of GABAergic neurons after GAD₆₇ antisense oligonucleotide infusion.     

Modulation of lordosis behaviour in the female rat by corticotropin releasing factor, beta-endorphin and gonadotropin releasing hormone in the mesencephalic central gray

A possible functional relationship between corticotropin-releasing factor (CRF) and opiate peptide neuronal systems (beta-endorphin, dynorphin1-17 and Met-enkephalin) and their interactions with gonadotropin releasing hormone (GnRH) in the mesencephalic central gray (MCG) for the regulation of lordosis behaviour was assessed in ovariectomized, oestrogen-treated and oestrogen-progesterone-treated female rats. Lordosis behaviour triggered by male mounting was inhibited in a dose-dependent fashion by CRF microinfused into the MCG in both oestrogen-treated and oestrogen-progesterone-treated female rats. This CRF-induced inhibition of lordosis could be overcome by a pre-infusion of naloxone or anti-beta-endorphin-globulin (anti-beta-end-G) directly into the MCG but not by anti-Met-enkephalin globulin (anti-enk-G) or anti-dynorphin1-17 globulin (anti-dynor-G). Supporting data indicate that the facilitation of lordosis behaviour induced by treatment with naloxone or anti-beta-end-G alone but not with anti-enk-G or anti-dynor-G may be due to enhanced GnRH release. This results from the action of these substances in overcoming the inhibition of GnRH secretion mediated specifically by beta-endorphin but not by Met-enkephalin or dynorphin1-17 in the MCG. These studies together with previous data showing that GnRH can overcome the abolition of lordosis by beta-endorphin in the MCG, indicate a close relationship between beta-endorphin (but not Met-enkephalin or dynorphin) and GnRH systems in the MCG in the control of lordosis behaviour. Thus, the inhibition of lordosis by CRF and the complete reversal of this blockade by naloxone or anti-beta-end-G may suggest that CRF could enhance the release of beta-endorphin from fibres in the MCG; beta-endorphin then inhibits lordosis by inhibiting the release of GnRH. However, a direct inhibitory effect of CRF on GnRH release is also likely since anti-CRF-gamma-globulin (anti-CRF-G) infused into the MCG produced a long-lasting facilitation of lordosis which can be blocked by an antagonist analogue of GnRH; in addition, previous studies have shown that GnRH infused into the MCG completely overcame the CRF-induced abolition of lordosis and potentiated lordosis to high levels. These results suggest that there may be functional neuroanatomical relationships between CRF, beta-endorphin and GnRH neuronal systems in the MCG in the control of female sexual behaviour. Neither Met-enkephalin nor dynorphin1-17 appear to participate in such mechanisms.     

Dopamine Agonist Signalling in the Hypothalamus of Female Rats is Independent of Calcium-Dependent Kinases

We have previously demonstrated that dopamine agonist, SKF38396 (SKF), can substitute for progesterone in the facilitation of female reproductive behaviour in oestradiol benzoate-primed female rats and mice. We also reported that both progesterone- and SKF-initiated signalling were mediated by the cAMP-dependent protein kinase A signal transduction cascade. As the rapid effects of progesterone are also mediated by calcium-dependent kinases, calcium- and calmodulin-dependent kinase (CaMKII) and protein kinase (PKC), we sought to determine whether SKF-initiated signalling also recruited calcium as a second messenger. We measured the changes in the activation of CaMKII and PKC in the ventromedial nucleus (VMN) of the hypothalamus and preoptic area (POA) of the rat brain, which are the two regions implicated in the regulation of female reproductive behaviour in rodents. We measured the basal activities representing the activation of the kinases by in vivo treatments, as well as the total kinase activities assayed in the presence of exogenous cofactors in vitro . We report that, in contrast to progesterone-initiated signalling, there was no recruitment of calcium by SKF in the hypothalamus, as shown by the absence of changes in CaMKII activities in the VMN and POA. Furthermore, SKF-treatment resulted in a rapid increase in calcium-independent basal PKC activity in the VMN but not the POA. These rapid changes were not the result of changes in PKC protein levels or phosphorylation status. These data indicate that progesterone- and SKF-recruit distinct signalling molecules within the same regions of the brain to activate region-specific signal transduction pathways.     

In vivo evidence for progesterone dependent decreases in serotonin release in the hypothalamus and midbrain central grey: relation to the induction of lordosis

The effects of progesterone (P) on serotonin (5HT) overflow in the ventromedial hypothalamus (VMH), preoptic area (POA) and midbrain central grey (MCG) were studied using in vivo microdialysis. Ovariectomized rats, pretreated with 5 μg estradiol, were anesthetized with chloral hydrate and stereotaxically implanted with dialysis probes directed towards one of the respective brain sites. Extracellular 5HT levels stabilized 3 to 5 h following probe implantation. Under stable baseline conditions, perfusion of 1 μM tetrodotoxin through the dialysis probe resulted in a 60–65% reduction in 5HT overflow in the brain areas studied. In experiments testing the effect of P on 5HT overflow, rats were subcutaneously injected with 0.5 mg P or propylene glycol vehicle. Samples were analyzed for 5HT at 20 min intervals for 4 h after treatment. Perfusate levels of 5HT were not significantly changed in the VMH, POA or MCG in vehicle-treated rats. Similarly, P treatment failed to significantly alter 5HT overflow in the POA. In the VMH, perfusate levels of 5HT were significantly reduced 60 min after P treatment. Decreases in perfusate 5HT levels were detected 20 min after P in the MCG. The decreases in 5HT overflow measured in the VMH and MCG following P treatment persisted for the remainder of the sampling period with the exception of 1 time point in the VMH. The results provide in vivo evidence for P-influenced decreases in 5HT release in the VMH and MCG. The rapid decrease in extracellular 5HT in the MCG suggests that this effect may represent a non-genomic action of P. These results are discussed in relation to the role of 5HT in the regulation of lordosis behavior.     

Lordosis-enhancing medial preoptic area lesions do not alter hypothalamic estrogen receptor- or progestin receptor-immunoreactivity in prepubertal female guinea pigs

Female guinea pigs rarely display adult-typical lordosis responses to ovarian steroid hormones until 40–50 days of age. Behavioral hyporesponsiveness in prepubertal females may be due, in part, to deficiencies in hypothalamic estrogen receptors and/or estradiol-induced progestin receptors. This study was designed to test the hypothesis that bilateral medial preoptic area (MPOA) lesions, which enhance the display of progesterone-facilitated lordosis in juvenile females, increase levels of hypothalamic estrogen receptors and/or estradiol-induced progestin receptors. Hartley guinea pigs were ovariectomized at 11–12 days of age and at 14–15 days of age received bilateral electrolytic or sham lesions aimed at the MPOA. At approximately 3 weeks of age, lesioned and sham-lesioned animals were either tested for the display of progesterone-facilitated lordosis or perfused, and their hypothalamic tissue processed for estrogen receptor- or estradiol-induced progestin receptor-immunostaining. Although a significantly higher percentage of MPOA-lesioned than sham-lesioned guinea pigs displayed progesterone-facilitated lordosis (85.7% vs. 5.8%, respectively, p<0.05), there were no significant lesion-related differences in the number or staining intensity of cells containing estrogen receptor- or estradiol-induced progestin receptor-immunoreactivity in the ventrolateral hypothalamus or arcuate nucleus. These data do not support the hypothesis that the enhanced display of progesterone-facilitated lordosis in prepubertal guinea pigs following MPOA lesions is due to increased hypothalamic concentrations of estrogen receptors or estradiol-induced progestin receptors.     

Reduction of insulin binding in the arcuate nucleus of the rat hypothalamus after 6-hydroxydopamine treatment

Insulin receptors are present in the hypothalamus, but the cell types bearing them are unknown. In order to test the hypothesis that some insulin receptors in the hypothalamus are associated with catecholamine terminals, rats were injected with 50 μg or 75 μg doses (intracerebroventricular) of 6-hydroxydopamine (6-OHDA). Control rats received vehicle only. The animals were sacrificed 7 days after injection, and catecholamine and indolamine levels in the hypothalamus were measured by high performance liquid chromatography with electrochemical detection. Localization of specific binding sites for [¹²⁵I]-insulin in the arcuate (ARC), dorsomedial (DMN) and ventromedial (VMN) nuclei were determined by quantitative film autoradiography. Treatment with 6-OHDA resulted in a 70% reduction in hypothalamic norepinephrine content as compared to vehicle-treated controls (P < 0.01). A slight depletion of epinephrine, dopamine and indolamines was also detected. Computerized image analysis of the autoradiograms was used to determine radioactivity bound (DPM/mm²) in each nucleus. Highest binding was in the ARC and DMN, with much lower binding in the VMN. Insulin binding in the ARC of the 6-OHDA-treated group was decreased by 25% compared to controls (P < 0.01). No significant change in insulin binding was observed in the DMN or VMN. The 6-OHDA treatment had no significant effect on weight gain or on plasma insulin levels. The reduction of insulin binding in the ARC after 6-OHDA treatment supports the hypothesis that some insulin binding sites are located on catecholamine terminals in the arcuate nucleus.     

Progestin receptors in the ventromedial nucleus of the hypothalamus and arcuate nucleus-median eminence are decreased by idazoxan

Steroid-dependent lordosis behavior in ovariectomized (OVX) guinea pigs is attenuated by alpha 1- and/or alpha 2-noradrenergic (NE) receptor antagonists. Correlated with the decrease in lordosis after alpha 1-NE receptor blockade by prazosin is a decrease in 'cytosol' progestin receptors in the ventromedial hypothalamic nucleus (VMN). We examined whether a presumed alpha 2-NE receptor blocker (idazoxan, IDA) also affects progestin receptors. A decrease in 'cytosol' progestin receptors was found after IDA treatment of OVX, estrogen-treated guinea pigs in the VMN and the arcuate nucleus-median eminence (ARC-ME). Apparently, either prazosin or IDA can inhibit lordosis behavior and decrease 'cytosol' progestin receptors in the VMN. In contrast, idazoxan but not prazosin, decrease 'cytosol' progestin receptors in the ARC-ME.     

Recovery of sexual receptivity in female rats with lesions of the ventromedial hypothalamus

Bilateral lesions of the hypothalamic ventromedial nucleus (VMN) effectively eliminated the induction by estrogen-progesterone of lordosis behaviors 4 to 6 weeks after the brain surgery. However, recovery of the lordosis response was clearly evident in the rats with effective VMN lesions 2 weeks after the first test. This suggests that the VMN may not be essential to the expression of hormonally induced lordosis behavior, but may be involved in a supplemental lordosis mediating mechanism.     

The role of delta-opioid receptors in estrogen facilitation of lordosis behavior

The present study investigated the role of delta-opioid receptors (ORs) in estrogen facilitation of female rat reproductive behavior (lordosis). Infusion of 2 microg of the selective delta-OR agonist [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE), into the third ventricle facilitated lordosis behavior in ovariectomized (OVX) rats injected with estrogen (E) 48 and 24 h before behavioral testing. Pretreatment with the selective delta-OR antagonist naltrindole (NTDL) blocked DPDPE effects on lordosis behavior. Ventricular infusion of NTDL (40 microg) also suppressed lordosis behavior in fully receptive OVX rats primed with both E and progesterone (P). In addition, NTDL blocked lordosis behavior when infused into the ventromedial nucleus of the hypothalamus (VMH) but not into the medial preoptic area (mPOA). Site-specific infusion of DPDPE into the VMH had dose-dependent, dual effects on lordosis behavior. While a very low dose of DPDPE (0.01 microg) facilitated lordosis behavior, a higher dose (1.0 microg) inhibited receptivity in OVX rats primed with E and a low dose (50 microg) of P. We used 3H-DPDPE to measure the density of delta-ORs in OVX rats treated with vehicle or with E by receptor autoradiography. E treatment did not have any effect on the density of DPDPE binding sites in the VMH, mPOA, medial amygdala, or caudate putamen. The behavioral effects of the ligands used in this study suggest that activation of delta-OR in the VMH by endogenous opioids facilitates estrogen-dependent lordosis behavior.