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.     

Effects of septal lesions and testosterone on lordosis behavior and luteinizing hormone release in female rats

The increased behavioral sensitivity to estrogen following a septal lesion in female rats has previously been found to be blocked by chronic treatment with testosterone propionate (TP) following the lesion. The effects of this chronic treatment on the ability of progesterone (P) to facilitate the secretion of luteinizing hormone (LH) in the spayed septal lesioned rat primed with estradiol benzoate (EB) was tested in the present study. EB was injected and followed 72 hr later with an injection of P. Blood samples for measurement of plasma LH were taken at 1700 hr, 29 and 53 hr after EB and 5 hr after P injection. Although there was no effect of the lesion, TP treatment significantly inhibited LH values after P administration. In a second experiment, spayed septal lesioned or sham operated rats were given either 0.0, 0.5 or 2.0 μg EB followed by 0.5 mg P just 24 hr later. The display of lordosis behavior, tested 4–6 hr after P injection, was significantly greater in septal lesioned than in sham operated rats following priming injections of 0.5 or 2.0 μ EB. This indicates that septal destruction significantly shortened the duration required for estrogen to prepare neural mechanisms for the effect of progesterone on lordosis behavior. Following a similar steroid regime, no differences were found between lesioned and sham operated rats in plasma LH levels in blood samples taken 5 hr after EB or 5 and 29 hr after P injection.     

Regulation of interleukin-1 receptors and hypothalamic-pituitary-adrenal axis by lipopolysaccharide treatment in the mouse

We measured iodine-125-labeled recombinant human interleukin-1α (¹²⁵I-IL-1α) binding in the hippocampus, pituitary, liver, spleen and testis, and plasma adrenocorticotropic hormone (ACTH) and corticosterone levels after i.p. injection of various dose and treatment regimens of the bacterial endotoxin, lipopolysaccharide (LPS) Plasma ACTH and corticosterone levels were significantly increased at 2 h after acute administration of LPS (60 or 300 μg/mouse).¹²⁵I-IL-1α binding in all peripheral tissues examined was significantly and comparably decreased at 2 h after a single injection of 30 μg or 300 μg LPS/mouse. On the other hand,¹²⁵I-IL-1α binding in hippocampus was significantly decreased only after high dose administration of LPS (300 μg/mouse). In order to evaluate if activation of IL-1 in brain resulting in the observed decrease in¹²⁵I-IL-1α binding may require more sustained exposure to endotoxin, we compared the effects of a single injection (60 μg/mouse) and two injections of LPS (30 μg/mouse each at 0 and 12 h). A single injection of LPS (60 μg/mouse) decreased¹²⁵I-IL-1α binding in the testis but not in the hippocampus, while two LPS injections (30 μg/mouse each at 0 and 12 h) caused dramatic reductions in¹²⁵I-IL-1α binding in both the hippocampus and testis. These data demonstrating differential effects of LPS treatment on the regulation of IL-1 receptors in the brain versus peripheral tissues and on ACTH and corticosterone secretion suggest that sites of action outside the blood-brain barrier may be important in regulating the effects of IL-1 on the hypothalamic-pituitary-adrenal axis.     

Inhibition of sexual behaviour and the luteinizing hormone surge by intracerebral progesterone implants in the female sheep

In female sheep, progesterone blocks the induction by oestradiol of both sexual behaviour and the pre-ovulatory surges of gonadotrophin releasing hormone (GnRH) and luteinising hormone (LH). However, the central sites of action of progesterone remain poorly defined, so we attempted to locate them by implanting progesterone intracerebrally in ovariectomised ewes treated with exogenous steroids to induce oestrous behaviour and the LH surge. Single bilateral implants or a double bilateral implants filled with progesterone or cholesterol were placed in the ventromedial hypothalamus (VMH) or the preoptic area (POA). Control ewes were not implanted. To determine the inhibitory capacity of the central progesterone implants, ewes received an injection (i.m.) of 8 μg or 16 μg of oestradiol. The single bilateral implants of progesterone failed to block oestrous behaviour and the LH surge induced by 8 μg of oestradiol. Double bilateral progesterone implants in the VMH blocked the sexual behaviour (P < 0.05) and the LH surge (P < 0.05), but implants in the POA blocked only sexual receptivity (P < 0.05). No changes were observed after central implantation of cholesterol. Our results support the hypothesis that progesterone acts centrally in the VMH and the POA to inhibit the induction of LH surge and sexual behaviour by oestradiol.     

Neuronal histamine in the hypothalamus suppresses food intake in rats

Using probes to manipulate hypothalamic neuronal histamine, we report here that changes in neuronal histamine modulate physiological feeding behavior in rats. Infusion of α-fluoromethylhistidine (FMH), a “suicide” inhibitor of histidine decar☐ylase (HDC), into the third cerebroventricular induced feeding in the early light phase when the histamine synthesis was most accelerated. FMH at an optimum 2.24 μmol dose elicited feeding in 100% of rats. Treatment of FMH specifically and selectively decreased concentration of histamine without affecting concentrations of catecholamines in the hypothalamus. Immediately before the dark phase, when the histamine synthesis was normally lower, FMH infusion did not affect feeding-related parameters such as meal size, meal duration or latency to eat. Conversely, thioperamide, which facilitates both synthesis and release of neuronal histamine by blocking presynaptic autoinhibitory H₃ receptors, significantly decreased food intake after infusion of a 100-nmol dose into the third cerebroventricle. The effect of thioperamide was abolished with i.p. injection of 26 μmol/kg chlorpheniramine, an H₁ antagonist. FMH at 224 nmol was microinfused bilaterally into the feeding-related nuclei in the hypothalamus. The ventromedial nucleus (VMH) and the paraventricular nucleus (PVN), but not the lateral hypothalamus, the dorsomedial hypothalamus or the preoptic anterior hypothalamus were identified as the active sites for the modulation. Neuronal histamine may convey suppressive signals of food intake through H₁ receptors in the VMH and the PVN with diurnal fluctuation.     

Neonatal defeminization of the luteinizing hormone release mechanism by catecholestrogens: different potencies of 2- and 4-hydroxyestradiol

Female rats were neonatally treated with estradiol-17β-benzoate or the long-acting dibenzoate esters of the isomeric catecholestrogens, 2-hydroxyestradiol-17β and 4-hydroxyestradiol-17β. Estrogen benzoates were administered subcutaneously from day 1 to 5 of life at doses of 0.05, 0.10, 0.50 and 1.00 μg/day. All rats were ovariectomized as adults and, 4 weeks later, the luteinizing hormone (LH) response to progesterone (2.5 mg) was tested after priming with estradiol-17β-benzoate (20 μg). At a dose of 0.5 μg/day, estradiol-17β-benzoate and 4-hydroxyestradiol-17β-dibenzoate were equally effective in neonatally defeminizing the LH surge mechanism. In contrast, up to a dose of 1.00 μg/day, 2-hydroxyestradiol-17β-dibenzoate did not interfere with the LH response in adult life. In the pituitary gland and uterus of the neonatally defeminized rats estrogen responsiveness of cytosolic progestin receptor induction was unimpaired. Moreover, in the uterus of these rats nuclear translocation of cytosolic progestin receptors was intact.     

Intranigral GR-113808, a selective 5-HT4 receptor antagonist, attenuates morphine-stimulated dopamine release in the rat striatum

GR-113808, a potent and selective 5-HT₄ receptor antagonist, was infused through a microdialysis probe into the striatum and nucleus accumbens of awake rats, and basal and morphine-stimulated extracellular concentrations of dopamine (DA) were measured in these regions. At 1 and 10 μM GR-113808 did not effect the extracellular concentrations of DA in either region and 100 μM significantly reduced dialysate DA only in the striatum. A subcutaneous dose of 5 mg/kg morphine significantly raised extracellular concentrations of DA in the striatum and nucleus accumbens from 60 to 120 min after injection and the effect was not modified by 10 μM GR-113808 infused through the probe 20 min before and for 60 min after morphine. Bilateral injections of GR-113808 (1, 2.5 and 10 μg/0.5 μl) in the substantia nigra pars compacta did not affect dialysate DA in the striatum, except for a significant increase 120 min after the injection of 10 μg but the highest dose of GR-113808 prevented the increase of striatal DA caused by 5 mg/kg morphine s.c. The results suggest that 5-HT₄ receptors in the substantia nigra modulate the activity of the dopaminergic nigrostriatal system only when the neurons are activated.     

Intracerebroventricular injection of choline increases plasma oxytocin levels in conscious rats

In the present study, we examined the effect of intracerebroventricularly (i.c.v.) injected choline on both basal and stimulated oxytocin release in conscious rats. I.c.v. injection of choline (50–150 μg) caused time- and dose-dependent increases in plasma oxytocin levels under normal conditions. The increase in plasma oxytocin levels in response to i.c.v. choline (150 μg) was greatly attenuated by the pretreatment of rats with atropine (10 μg; i.c.v.), muscarinic receptor antagonist. Mecamylamine (50 μg; i.c.v.), a nicotinic receptor antagonist, failed to suppress the effect of 150 μg choline on oxytocin levels. Pretreatment of rats with 20 μg of hemicholinium-3 (HC-3), a specific inhibitor of choline uptake into nerve terminals, greatly attenuated the increase in plasma oxytocin levels in response to i.c.v. choline injection. Osmotic stimuli induced by either oral administration of 1 ml hypertonic saline (3 M) following 24-h dehydration of rats (type 1) or an i.c.v. injection of hypertonic saline (1 M) (type 2) increased plasma oxytocin levels significantly, but hemorrhage did not alter basal oxytocin concentrations. The i.c.v. injection of choline (50, 150 μg) under these conditions caused an additional and significant increase in plasma oxytocin concentrations beyond that produced by choline in normal conditions. These data show that choline can increase plasma oxytocin concentrations through the stimulation of central cholinergic muscarinic receptors by presynaptic mechanisms and enhance the stimulated oxytocin release.     

Estrogenic regulation and sex dimorphism of growth-associated protein 43 kDa (GAP-43) messenger RNA in the rat.

In addition to effects on brain protein synthesis, neurotransmitter release, and electrophysiology, estrogens alter neurite outgrowth and synaptogenesis. This study examined in the adult rat the effects of estrogen and sex on the expression of the GAP-43 gene; encoding a phosphoprotein structurally and physiologically linked to these two processes in the rat CNS. Ovariectomized (OVX) rats were injected with vehicle or estrogen, or male and female rats were either gonadectomized or left intact. Brains were dissected to obtain ventromedial hypothalamus (VMH), posterior hypothalamus (PH), or frontal cortex (CTX). Total RNA from these areas were extracted, and slot-blots of equal masses of total RNA were hybridized to 32P-labeled cDNAs for GAP-43 and beta-actin, and also to synthetic poly-dT. Resultant autoradiograms were scanned by laser densitometry, quantitated, and ratios of the gray scale generated by each probe were compared between experimental groups. GAP-43 mRNA expression, when compared to expression of either beta-actin mRNA or total poly(A)-containing RNA (poly(A) RNA), was higher in VMH and PH as compared to CTX. Estrogen treatment of OVX rats resulted in a 48-74% increase in GAP-43 mRNA levels in the VMH--in one experiment, this increase was noted after 2 h of estradiol treatment, and in another after 3 days of estradiol benzoate treatment; but PH and CTX were unaffected by either estrogen regimen. Conversely, ovariectomy of intact rats decreased GAP-43 mRNA expression by 45% in the VMH, but not in the CTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of apomorphine-induced stereotypy by estrogen: Time course and dose response

Previous data have indicated that estrogen may either suppress or enhance the potency of dopamine and/or dopamine agonists. The effects of estrogen (estradiol benzoate; EB) in ovariectomized rats were indicative of a dose related suppression of apomorphine-induced stereotypy at 24 hours after the last dose of EB. However, at 48 hours after the last dose of EB, the apomorphine-induced stereotypy was enhanced in those animals that received EB at 100 μg/kg × 3 days. Ninety-six hours after the last injection of EB, all animals that received either the 100 or 50 μg/kg dose of EB displayed an enhanced behavioral response to apomorphine. Animals pretreated with a low dose of EB (10 μg/kg × 3 days) displayed a suppressed apomorphine stereotypy score at 24 hours, after the last dose, but were indistinguishable from Oil injected ovariectomized controls in all subsequent behavioral tests. These data indicate that estrogen can suppress dopamine and/or dopamine agonist potency 24 hours after the last dose of estrogen. However, an enhanced behavioral response to dopamine agonists can be seen at 48 hours (or more) after the last dose of estrogen. The time delay and the dose dependence of the enhanced response to dopamine agonists are indicative of a “withdrawal” phenomenon, suggesting the possibility that the role of endogenous estrogen is to suppress dopaminergic function.     

Inhibitory effect of 17β-estradiol in the parabrachial nucleus is mediated by GABA

In the present investigation, electrophysiological recordings of thalamic relay neurons were used to investigate the role of estrogen as a modulator of visceral afferent information through the PBN to forebrain structures. Experiments were done in anaesthetized (sodium thiobutabarbitol; 100 mg/kg) male and ovariectomized female rats supplemented for 7 days prior with either 17β-estradiol (OVX-E₂) or saline (OVX-S). A portion of the right cervical vagus was isolated for the electrical activation (0.8 Hz, 2 ms duration) of visceral afferents. The evoked single and multi-unit activity was recorded via a recording electrode in the ventrobasal thalamus. Exogenous microinjection of 17β-estradiol (0.1, 0.25 and 0.5 μM; 200 nl) into the parabrachial nucleus (PBN) produced a significant, dose-dependent attenuation in the magnitude of visceral afferent activation-evoked responses of neurons recorded in the thalamus in both male and OVX-E₂ groups. No effect on evoked thalamic activity was observed following injection of estrogen into the PBN of OVX-S animals. Co-injection of estrogen with the GABA_A receptor antagonist, bicuculine (0.1 μM; 200 nl) but not phaclofen (GABA_B; 0.1, 0.5 or 1 μM; 200 nl) resulted in an increase in the evoked thalamic response in males (55±11%) and OVX-E₂ female (68±15%) rats. These studies suggest that estrogen inhibits neurotransmission in the PBN via an interaction with the GABA_A receptor to modulate the flow of visceral information to the thalamus.     

Glutamate injection into the suprachiasmatic nucleus stimulates brown fat thermogenesis in the rat

Injection of glutamate (100 mM to 1 M, in 0.25 μl saline) into the hypothalamic suprachiasmatic nucleus (SCN) dose-dependently increased interscapular brown adipose tissue (IBAT) and core temperatures in the urethane-anaesthetized rat. This effect was more pronounced in rats tested during the light-off period than in animals tested during the light-on period. Prior injection of the local anaesthetic, procaine (5% in 0.5 μl saline), into the ipsilateral ventromedial hypothalamic nucleus (VMH) attenuated the increases in IBAT and core temperatures induced by intra-SCN glutamate. The VMH has previously been implicated in the central regulation of BAT thermogenesis; the present results suggest the pathway arising in the SCN exerts an excitatory influence on VMH neurons involved in the control of BAT function.     

Diethylstilbestrol regulates the number of α- and β-adrenergic binding sites in incubated hypothalamus and amygdala

Previous work has identified an effect of circulating estrogens on the number of central adrenergic binding sites. We have further characterized this effect by performing the experiments in vitro and have taken advantage of a well-described hypothalamic preparation in which diethylstilbestrol (DES), is known to elevate cAMP levels through a pathway which involves adrenoreceptors. We show that DES induces a reciprocal change in the numbers of α- and β-adrenergic binding sites in incubated hypothalami obtained from intact immature female rats as well as from ovariectomized adult rats. The α-adrenergic binding sites are reduced by 25–30% whereas the β-adrenergic sites are increased by 60–100%. The effect is maximal at 3 h in vitro (20 μM DES) and largely reversible following a 2 h wash in the absence of DES. Using the change in β-adrenergic binding sites as a probe, we were further able to show that estradiol (100 μM) and 2-hydroxyestradiol (50 μM) had no effect. Further, the effect of DES was not blocked by the anti-estrogens clomiphene or tamoxifen. Since DES is able to elevate β-adrenergic binding sites in hypothalamus and amygdala (brain areas known to contain high levels of estrogen receptors) but has no effect in cerebellum, we conclude that we have observed an effect of DES not shared by estradiol but which may be confined to estrogen target areas of the brain.     

Attenuation by chronic imipramine treatment of [H]clonidine binding to cortical membranes and of clonidine-induced hypothermia: the influence of central chemosympathectomy

Central chemosympathectomy with intraventricular injection of 250 μg of 6-hydroxydopamine, resulting in depression of cerebral noradrenaline level by 75%, led to an increase in specific binding of [³H]clonidine to cerebral cortical membranes, but did not affect the hypothermic response to clonidine. Chronic imipramine treatment depressed [³H]clonidine binding and attenuated clonidine-induced hypothermia similarly in non-chemosympathectomized and chemosympathectomized rats.     

Excitatory effect of dopamine on oxytocin and vasopressin reflex releases in the rat

The involvement of dopamine in the release of oxytocin and vasopressin was investigated in lactating rats during suckling or after changes in plasma osmolality. The effects of intraventricular injections of dopamine, agonists and antagonists, were tested on electrical unit activity of oxytocinergic or vasopressinergic cells in the paraventricular nucleus, on intramammary pressure (index of oxytocin release) and diuresis (index of vasopressin release).In urethane-anaesthetized lactating suckled rats, dopamine (1 μg), apomorphine (2.5 and 5 μg) facilitated the established milk-ejection reflex, increasing the frequency and the amplitude of neurosecretory bursts of oxytocinergic cells. They also triggered the reflex in lactating rats without milk-ejections during suckling. The small doses injected were in no way such as to induce an acceleration in firing rate of oxytocinergic cells or an increase in mammary pressure.In alcohol-loaded rats, during water diuresis, dopamine (2 μg) and apomorphine (5 μg) activated the depressed vasopressinergic cells and inhibited diuresis. These facilitatory effects were progressive, reaching a maximum 10–15 min after injection.Haloperidol (5 μg) and α-flupentixol (10 μg) had an inhibitory effect on both types of neurosecretory cells in urethane-anaesthetized rats. They prevented the reflex activation of oxytocinergic cells induced by suckling and of vasopressinergic cells after a hyperosmotic stimulus (1 ml i.p 9% NaCl solution). These inhibitory effects were not of the ‘all-or-none’ type.So, we can postulate that dopamine regulates the reflex release of oxytocin and vasopressin in the hypothalamus. On the one hand, dopamine permits and controls the periodic activation of oxytocinergic cells as long as the mothers are being suckled. On the other hand, it modulates the activity of vasopressinergic cells whenever the plasma osmolality changes.     

Sex differences in the amphetamine stimulated release of catecholamines from rat striatal tissue in vitro

Sex and estrous cycle-related differences in the amphetamine (AMT)-stimulated release (pg/mg/min) of catecholamines (CA) from rat striatal and mediobasal hypothalamus (MBH) fragments were measured in an in vitro perifusion system. In striatal tissue from intact males, AMT stimulated the release of both norepinephrine (NE) and dopamine (DA). The AMT-stimulated release of DA from striatal tissue obtained from intact females varied with the stage of the estrous cycle. This increase in DA release was lower in striatal tissue from proestrous females than from females in estrus(P < 0.05) or diestrus1 (P < 0.01). The NE release stimulated by AMT was greater than basal release only on estrus and diestrus 2. Following castration (CAST) or CAST plus 500 μg testosterone propionate (TP), daily for 4 days, striatal tissue fragments from male rats continued to release CA in response to AMT stimulation. In contrast, ovariectomy (OVX) severely attenuated the AMT-stimulated release of both CA. Treatment of OVX females with 5 μg estradiol benzoate (EB), daily for 4 days, or 1.2 mg progesterone (P) slightly increased the AMT-stimulated release of DA but not NE. Treatment of OVX females with 5 μg EB, daily for 4 days, plus 1.2 mg P completely restored the AMT-stimulated release of both CA. Interestingly, MBH fragments from intact of gonadectomized rats, with or without hormonal treatment, demonstrated a consistent AMT-stimulated release of DA regardless of the sex of the animal. The AMT-stimulated NE release from these MBH fragments was less consistent, but there were no significant differences between the groups. These results demonstrate that the AMT-stimulated release of DA from striatal tissue in vitro is sex, hormonal, and tissue dependent.     

Somatostatin receptor-GTP binding regulatory protein-adenylyl cyclase system in hippocampal membranes of strychnine-treated rats

Wistar rats were injected with either a non-convulsive dose (37.5 μg/100 g body weight (b.wt.), intravenously (i.v.)) or a convulsive dose (50 or 80 μg/100 g b.wt., i.v.) of strychnine. Binding of¹²⁵I-Tyr¹¹-somatostatin (¹²⁵I-Tyr¹¹-SS) to its specific receptors was measured in hippocampal membranes 15 min after strychnine injection at these three doses. The non-convulsive dose of strychnine did not affect binding of SS in the hippocampus whereas both convulsive doses decreased the number of specific SS receptors without influencing their apparent affinity. Somatostatin-like immunoreactivity (SSLI), SS-modulated adenylel cyclase (AC) activity and the inhibitory guanine-nucleotide binding regulatory protein were also measured in rats treated with 80 μg/100 g b.wt. of strychnine. SSLI content remained stable. No significant differences were seen for the basal and forskolin (FK)-stimulated AC enzyme activities in the hippocampus of strychnine-treated rats when compared to the control group. The capacity of SS to inhibit basal and FK-stimulated AC activity in the hippocampus was significantly lower in the strychnine group than in the control group. The ability of the stable GTP analogue 5′-guanylylimidodiphosphate [Gpp(NH)p] to inhibit FK-stimulated AC activity was also decreased in hippocampal membranes from strychnine-treated rats. These results suggest that the attenuated inhibition of AC by SS in hippocampal membranes from strychnine-treated rats may be caused by decreases in both G_i activity and in the number of SS receptors. Alternatively, an uncoupling of SS receptors from G_i and/or a decrease in the level of functional G_i may result in both a decrease in apparentB_max for SS binding and in SS-mediated inhibition of AC. Since recent studies of other authors support the view that SS is predominantly an inhibitory transmitter in the hippocampus, it is possible that the decreased inhibition of AC activity by SS as well as the decrease in the number of SS receptors found in the hippocampus of strychnine-treated rats may be a mechanism involved in the development of increased seizure susceptibility.     

Long-term observation of rats after unilateral intra-amygdaloid injection of kainic acid

Electroencephalographic and clinical observations of the rats were done for 3 months after microinjection of kainic acid (KA, 0.2–1.2 μg)into the unilateral amygdala. With high doses of KA (0.6–1.2μg) injection, 64% of rats developed spontaneous limbic siezures 14–25 days after KA injection. Among those which developed spontaneous limbic seizures, 33% of rats developed secondarily generalized seizures 26–71 days after KA injection. This is one of the best chronic models of spontaneous complex partial seizure secondarily generalized in rats, which is economical and easy to prepare.