Conjugated estradiol increases female sexual receptivity in response to oxytocin infused into the medial preoptic area and medial basal hypothalamus

The ovarian steroid estradiol (E) has been found to increase both receptor affinity and release of the neuropeptide oxytocin (OT) in plasma membrane preparations. Therefore, we hypothesized that E conjugated to bovine serum albumin at position 6 (E-6-BSA) would increase behavioral responsiveness to OT. Preliminary results showed that 200 ng/microl of E-6-BSA increased sexual receptivity slightly, but not significantly. Therefore, this dose was used as a subthreshold dose to test whether it would increase sexual responsiveness when infused in combination with 100 ng/microl OT. After recovery from cannula implantation surgery animals were injected with 0.5 microg E benzoate daily for 3 days before testing. On the fourth day, after a baseline preinfusion test rats were infused bilaterally with E-6-BSA alone or with OT, OT with BSA, or conjugated progesterone, luteinizing hormone-releasing hormone equimolar to OT alone, or with E-6-BSA or conjugated progesterone alone. When infused into either the medial preoptic area-anterior hypothalamus or the medial basal hypothalamus the combination of OT and E-6-BSA significantly increased sexual receptivity over receptivity after artificial cerebrospinal fluid control infusions. Neither bilateral infusions of OT in combination with conjugated progesterone nor E-6-BSA in combination with luteinizing hormone-releasing hormone enhanced sexual receptivity. Results presented here strongly support the conclusion that some of the effects that E has in sensitizing brain systems to the facilitating effects of OT occur at the membrane level in the medial preoptic area-anterior hypothalamus and medial basal hypothalamus.     

Prolactin in CSF selectively increases dopamine turnover in the median eminence

The α-methyltyrosine-induced decline of dopamine was increased in the median eminence of rats at 16 but not at 2 hours after the start of intraventricular injections of 0.2–2 μg of rat prolactin. Intraventricular injections of prolactin did not alter the α-methyltyrosine-induced decline of dopamine in the striatum or olfactory tubercle. These results suggest that prolactin in the cerebrospinal fluid can selectively increase the activity of tuberoinfundibular dopaminergic neurons.     

Involvement of central GABAergic neurons in basal and diurnal changes of tuberoinfundibular dopaminergic neuronal activity and prolactin secretion

Central administration of gamma-aminobutyric acid (GABA) has been shown to stimulate the secretion of prolactin (PRL). Whether GABA acts via dopamine, the major PRL-inhibiting hormone, and which GABA receptor type(s) is involved have not been ascertained. Both GABA(A) and GABA(B) receptor agonists and/or antagonists were administered centrally in this study and their effects on both basal and diurnal changes of tuberoinfundibular dopaminergic (TIDA) neuronal activity were determined by measuring the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence (ME). Serum PRL level was determined by RIA. Ovariectomized, estrogen-primed Sprague-Dawley rats implanted with intracerebroventricular (icv) cannulae were used. Muscimol (1 ng/3 microl/rat, icv), a GABA(A) receptor agonist, but not baclofen (1-100 ng/3 microl/rat, icv), a GABA(B) receptor agonist, injected in the morning significantly lowered and elevated ME DOPAC and serum PRL levels, respectively at 15 and 30 min. Lower and higher doses of muscimol were not effective. The effects of muscimol could also be prevented by co-administration of bicuculline (0.1-10 ng/3 microl, icv), a GABA(A) receptor antagonist. When bicuculline (10-500 ng/3 microl, icv) was given in the afternoon (at 1500 h), it significantly reversed the lowered ME DOPAC level in the afternoon and prevented the concurrent PRL surge. We conclude that endogenous GABA acting through GABA(A) receptors may play a significant role in the control of basal and diurnal changes of TIDA neuronal activity, and in turn, PRL secretion.     

Sexual differences in the activity of periventricular-hypophysial dopaminergic neurons in rats.

The activities of periventricular-hypophysial dopaminergic (DA) neurons were compared in male and female rats by measuring dopamine synthesis (accumulation of 3,4-dihydroxyphenylalanine [DOPA] after inhibition of L-aromatic amino acid decarboxylase) and metabolism (concentrations of 3,4-dihydroxyphenylacetic acid [DOPAC]) in terminals of these neurons in the intermediate lobe of the pituitary. For comparison, the synthesis and metabolism of dopamine in the neural lobe of the pituitary and median eminence were also determined. The concentrations of DOPAC and accumulation of DOPA were higher in females than in males in both the intermediate lobe and median eminence, revealing a sexual difference in the basal activity of periventricular-hypophysial and tuberoinfundibular DA neurons. In contrast, there were no differences between male and female rats in activity of DA neurons terminating in the neural lobe. One week following gonadectomy, DOPA accumulation in the median eminence was decreased in females and increased in males, but remained unchanged in the intermediate lobe. These results indicate that sexual differences in the activity of periventricular-hypophysial DA neurons terminating in the intermediate lobe are not dependent upon the presence of circulating gonadal steroids, and in this respect, these neurons differ from tuberoinfundibular DA neurons.     

Distribution of D(5) dopamine receptor mRNA in rat ventromedial hypothalamic nucleus

Estrogen induces lordosis through, in part, estrogen receptor (ER)-mediated synthesis of progesterone receptors (PR) in the ventromedial nucleus (VMN). In vitro, PR is activated by the neurotransmitter dopamine through D1-like receptors (1). In vivo, lordosis is induced by dopamine, an effect mediated in part by PR and D(5) dopamine receptors. The purpose of the present study was to determine mRNA distribution of D1-like receptors in the female rat brain using RT-PCR combined with punchout microdissection techniques. Employing specific primers to D(5) and D(1) dopamine receptors, we found detectable expression levels of D(5) dopamine receptor mRNA in VMN as well as the arcuate nucleus/median eminence (ArcN/ME). In contrast, D(1) dopamine receptor mRNA was detected only in VMN. By using this highly sensitive and specific RT-PCR methodology, we have confirmed the presence of D(5) dopamine receptor mRNA in an area of the brain that regulates reproductive behavior through PR. The data support the previous observation that D(5) dopamine receptors in VMN contribute to facilitation of female reproductive behavior by D1-like agonists.     

Effects of prolactin-releasing peptide on tuberoinfundibular dopaminergic neuronal activity and prolactin secretion in estrogen-treated female rats

Both systemic and central effects of a newly discovered prolactin (PRL)-releasing factor (PRF), prolactin-releasing peptide (PrRP), were determined in this study. Systemic injection of PrRP (1 and 10 microg/rat, i.v.) stimulated PRL secretion in ovariectomized, estrogen-treated rats similar to the effect of another PRF, thyrotropin-releasing hormone (TRH). Pretreatment with a dopamine D2 receptor antagonist, sulpiride (1 microg/rat, i.v.), potentiated the stimulatory effect of both PrRP and TRH on PRL secretion. Using the double-labeling immunohistochemical method, PrRP-immunoreactive terminals were found in close contact with tyrosine-hydroxylase-immunoreactive neurons in the hypothalamic arcuate nucleus. Central administration of PrRP (0.1-1,000 ng/rat, i.c.v.) stimulated tuberoinfundibular but not nigrostriatal dopaminergic neuronal activity in 15 min. Levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence and striatum were used as indices for tuberoinfundibular dopaminergic (TIDA) and nigrostriatal dopaminergic neuronal activities, respectively. The serum PRL level, however, was not significantly changed. Similar treatment with TRH (10 ng/rat, i.c.v.) stimulated and inhibited TIDA neuronal activity and serum PRL, respectively, at 30 min. In summary, PrRP may play a role in both the central and peripheral control of PRL secretion.     

Effects of cerebrospinal fluid preparation from male and female patients with opiate dependence on rat open-field behavior

The effects of the intranasal administration of preparations made from the cerebrospinal fluid of male and female opiate users on the open-field rat behavior were studied. Behavioral differences were demonstrated in the effects of preparations from female and male cerebrospinal fluid. The administration of the "male" preparation produced a significant decrease in the locomotor activity and increase in the immobilization time and grooming duration, while the "female" preparation had the opposite effects. These differences may result from different content of endogenous and exogenous opiates and dopamine (and its metabolites) in the cerebrospinal fluid of male and female opiate users.     

Effects of centrally injected amylin on sexually behavior of male rats.

Amylin (AMY) is a peptide of pancreatic origin principally involved in the carbohydrate metabolism, but that may interfere with central and peripheral dopamine (DA) pathways. The peptide, injected intracerebroventricularly (ICV) at the dose of 2.5 microg/rat, induced a decrease of copulatory activity in good copulators (GCO) and sluggish (SLU) male rats. The dose of 0.1 microg/rat did not affect significantly the sexual behavior of GCO rats, whereas AMY 0.5 microg/rat increased only the latency and reduced the frequency of ejaculation. At the dose of 2.5 microg/rat AMY antagonized the activation of sexual behavior induced by the DA receptor agonist, apomorphine administered subcutaneously (SC) at the dose of 100 microg/kg. Moreover, this inhibitory effect was blocked by the calcitonin gene-related peptide and AMY receptor antagonist, CGRP (8-37) fragment (injected ICV at the dose of 1 microg/rat). These data suggest that AMY may exert inhibitory effects on male sexual behavior in rats, probably interfering with central DA neurotransmission and with CGRP receptors.     

Single-step procedure for gas chromatography-mass spectrometry screening and quantitative determination of amphetamine-type stimulants and related drugs in blood, serum, oral fluid and urine samples

We describe a rapid GC/MS assay for amphetamine-type stimulant drugs (ATSs) and structurally related common medicaments in blood, serum, oral fluid and urine samples. The drugs were extracted from their matrices and derivatized with heptafluorobutyric anhydride (HFBA) in a single step, using the following procedure: 100 microl (oral fluid) or 200 microl (blood, serum, urine) of the sample were mixed with 50 microl of alkaline buffer and 500 microl of extraction-derivatization reagent (toluene + HFBA + internal standard), centrifuged, and injected into a GC/MS apparatus. As revealed by the validation data this procedure, with its limit of quantitation being set at 20 ng/ml for oral fluid, 25 ng/ml for blood or 200 ng/ml for urine, is suitable for screening, identification and quantitative determination of the ATSs and related drugs in all the matrices examined. Thus, time-consuming and expensive multiple analyses are not needed, unless specifically required.     

Interactions between prolactin and dopaminergic neurons

The secretion of prolactin from the adenohypophysis is tonically inhibited by dopamine that is released into the hypophysial portal blood from terminals of tuberoinfundibular neurons located in the external layer of the median eminence. These tuberoinfundibular neurons are unique among other dopaminergic neurons in the brain (including the well-characterized nigrostriatal neurons) in that they are not directly regulated by dopaminergic receptor-mediated mechanisms, but instead are selectively responsive to changes in prolactin concentrations in blood and cerebrospinal fluid. In the rat, the activity of the tuberoinfundibular dopaminergic neurons is higher in the female than in the male, exhibits a characteristic cyclical pattern during the first half of pregnancy and is constantly high as a result of stimulation by placental lactogen during the last 9 days of pregnancy, and is reduced in lactating animals and acutely inhibited during suckling.     

Differential effects of colchicine on central dopaminergic neuronal activity and prolactin secretion in estrogen-primed ovariectomized rats

Colchicine is a potent chemical that disrupts the assembly of microtubulin and affects the integrity of cytoskeleton. It is commonly used to block the axonal transport in neurons. Central administration of colchicine (48 microg/3 microl/rat) two days earlier significantly lowered 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum and nucleus accumbens, both in the morning and in the afternoon. Median eminence DOPAC levels exhibit a diurnal change between morning and afternoon as previously shown. Colchicine treatment lowered and elevated median eminence DOPAC levels in the morning and afternoon, respectively. The estrogen-induced prolactin surge was also blocked. The findings indicate that neuronal inputs are necessary for maintaining basal activities in all dopaminergic neurons, while an inhibitory one predominates in the afternoon for TIDA neurons.     

Regional effect of naltrexone in the nucleus of the solitary tract in blockade of NPY-induced feeding

Naltrexone (NLTX) in the nucleus of the solitary tract (NTS) decreases feeding induced by neuropeptide Y (NPY) in the paraventricular nucleus (PVN). We sought to determine the NTS region most sensitive to NLTX blockade of PVN NPY-induced feeding. Male Sprague-Dawley rats were fitted with two cannulas; one in the PVN and one in a hindbrain region: caudal, medial, or rostral NTS or 1 mm outside the NTS. Animals received NLTX (0, 1, 3, 10, and 30 microg in 0.3 microl) into the hindbrain region just prior to PVN NPY (0.5 microg, 0.3 microl) or artificial cerebrospinal fluid (0.3 microl). Food intake was measured at 2 h following injection. PVN NPY stimulated feeding, and NLTX in the medial NTS significantly decreased NPY-induced feeding at 2 h, whereas administration of NLTX in the other hindbrain regions did not significantly influence PVN NPY induced feeding. These data suggest that opioid receptors in the medial NTS are most responsive to feeding signals originating in the PVN after NPY stimulation.     

AT(1) receptor blockers prevent sympathetic hyperactivity and hypertension by chronic ouabain and hypertonic saline

Sympathetic hyperactivity and hypertension caused by chronic treatment with ouabain or sodium-rich artificial cerebrospinal fluid (aCSF) can be prevented by central administration of an angiotensin type 1 (AT(1)) receptor blocker. In the present study, we assessed whether, in Wistar rats, chronic peripheral treatment with the AT(1) receptor blockers losartan and embusartan can exert sufficient central effects to prevent these central effects of ouabain and sodium. Losartan or embusartan (both at 100 mg x kg(-1) x day(-1)) were given subcutaneously once daily. Ouabain (50 microg/day) was infused subcutaneously, and sodium-rich aCSF (1.2 M Na(+), 5 microl/h) was infused intracerebroventricularly, both by osmotic minipump for 13-14 days. The mean arterial pressure (MAP) at rest and in response to air stress and intracerebroventricularly injection of guanabenz (75 microg/7.5 microl), ANG II (30 ng/3 microl), and ouabain (0.5 microg/2 microl) were then measured. In control rats, chronic treatment with ouabain subcutaneously and hypertonic saline intracerebroventricularly both increased baseline MAP by 20-25 mmHg and enhanced twofold the pressor responses to air stress and depressor responses to the alpha(2)-adrenoceptor agonist guanabenz. Simultaneous treatment with losartan or embusartan fully prevented hypertension, maintained normal responses to air stress and guanabenz, and attenuated pressor responses to acute intracerebroventricular injection of ANG II and ouabain. We concluded that peripheral administration of losartan as well as embusartan can cause sufficient central effects to prevent the sympathetic hyperactivity and hypertension induced by chronic peripheral ouabain and central sodium.     

Stimulation of sexual behavior in the male rat by galanin-like peptide

Galanin-like peptide (GALP) is a recently described neuropeptide, which shares a partial sequence identity with galanin but is derived from a separate gene. Central injections of GALP stimulate the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) and induce the expression of Fos in several brain areas known to regulate male sexual behavior in the rat. We postulated that GALP may also stimulate sexual behavior in concert with its stimulatory effect on the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we administered GALP, galanin, or the vehicle (artificial cerebrospinal fluid, aCSF) alone to sexually experienced male rats and assessed the effects of these agents on sexual behavior. We observed that compared to aCSF alone, GALP significantly increased all aspects of male-typical sexual behavior, whereas galanin inhibited all of these same behaviors. To examine whether the stimulatory effects of GALP on sexual behavior were mediated by GALP's stimulatory effects on the HPG axis, we castrated the same male rats and repeated the behavioral experiment. We found that GALP maintained its inductive action on male-typical sexual behaviors in the castrated animals, suggesting that the effects of GALP on sexual behavior are not the result of GALP's ability to stimulate testosterone secretion. These observations suggest that GALP neurons are part of the hypothalamic circuitry controlling sexual behavior in the male rat.     

Effect of hypothalamic implants changing monoamine levels on continuous estrus in rats induced by androgen treatment in the neonatal period]

The constant estrus induced by neonatal androgenization in the female rat may be disrupted following a drug-induced change of the monoamine levels in the preoptic area and arcuate nucleus of the median eminence. The most precise effect is obtained with 5-HT. This is in favour of a predominant alteration of the serotonergic system which seems to be involved in the sexual differentiation of the brain.     

The regulation of the corticomelanotropic cell activity in aves. III--Effect of various peptides on the release of MSH from dispersed, perfused duck pituitary cells. Cosecretion of ACTH with MSH

1. The melanotropin-releasing activity of arginine-vasopressin (AVP), arginine-vasotocin (AVT), oxitocin (OT), mesotocin (MT) and corticotropin-releasing factor (CRF) was studied in the duck using dispersed, perfused pituitary cells and a specific alpha-MSH RIA. 2. Log dose-response curves were obtained for all the peptides ranging from 5 to 100 ng/ml. All peptides behaved as partial agonists compared to duck median eminence extracts (DME). 3. AVT and MT displayed an alpha-MSH releasing capacity of 60% relative to DME whereas all other peptides behaved as weak agonists with less than 15% capacity relative to DME. 4. AVT and CRF when perfused together acted synergistically on alpha-MSH release yielding a dose response line whose slope approximated that of DME. 5. ACTH was cosecreted together with alpha-MSH in all situations studied with an ACTH to alpha-MSH molar ratio of about 10. 6. It is concluded that CRF and neurohypophyseal peptides may be physiological stimulators of both alpha-MSH and ACTH release in aves.     

Medianosomes as integrative units in the external layer of the median eminence. Studies on grf/catecholamine and somatostatin/catecholamine interactions in the hypothalamus of the male rat

Somatostatin/catecholamine as well as growth hormone releasing factor/catecholamine interactions have been characterized in the hypothalamus and the preoptic area using morphometrical and quantitative histofluorimetrical analyses.

• 1.(1) The morphometrical analysis of adjacent coronal sections of the rat median eminence demonstrated a marked overlap of somatostatin and tyrosine hydroxylase immunoreactive nerve terminals as well as of growth hormone releasing factor and tyrosine hydroxylase immunoreactive nerve terminals in the medial and lateral palisade zones of the rostral and central parts. Furthermore, the studies on codistribution of growth hormone releasing factor and tyrosine hydroxylase immunoreactivity indicate that only a limited proportion of the growth hormone releasing factor and the dopamine nerve terminals may costore dopamine and growth hormone releasing factor respectively in the medial and lateral palisade zones (see Meister et al., 1985).
• 2.(2) Intravenous injections of somatostatin 1–14 (100 μg/kg, 2 h) into the hypophysectomized male rat produced an increase in dopamine utilization in the medial and lateral palisade zones of the median eminence.
• 3.(3) Intravenous injections of rat hypothalamic growth hormone releasing factor (80 μg/kg, 2 h) in the hypophysectomized male rat did not change dopamine utilization in the median eminence but increased noradrenaline utilization in the ventral zone of the hypothalamus and produced a depletion of noradrenaline stores in the paraventricular hypothalamic nucleus.
• 4.(4) Intravenous injections of human pancreatic growth hormone releasing factor 1–44 (80 μg/kg, 2 h) in the hypophysectomized male rat did not change dopamine utilization in the median eminence, but reduced noradrenaline utilization in the subependymal layer and increased noradrenaline utilization in the suprachiasmatic preoptic nucleus.

The combined results of the present and previous studies have led us to put forward the medianosome concept. The medianosome is defined as an integrative unit, which consists of well defined aggregates of transmitter identified nerve terminals interacting with one another in the external layer of the median eminence. Our present data indicate the existence of putative medianosomes consisting predominantly of growth hormone releasing factor nerve terminals costoring dopamine as well as of somatostatin and dopamine nerve terminals, which interact locally to control growth hormone secretion. A complementary control of growth hormone secretion may be exerted by noradrenaline mechanisms in the subependymal layer, in the ventral zone and/or in the suprachiasmatic preoptic nucleus. However, further analyses in view of the differential effects seen with the present doses of rat hypothalamic and human pancreatic growth hormone releasing factor have to be done. The results also indicate the possible existence of growth hormone releasing factor receptors in the median eminence which may participate in the feedback control of the growth hormone releasing factor immunoreactive neurons in the ventral zone of the hypothalamus.     

alpha-melanocyte-stimulating hormone and habituation of prey-catching behavior in the Texas toad, Bufo speciosus.

We investigated dose-dependent effects of alpha-melanocyte-stimulating hormone (alpha-MSH) on habituation in the Texas toad, Bufo speciosus. Additionally, we determined changes in plasma and brain levels of alpha-MSH following peripheral administration of the peptide or following exposure to an ether stressor. The ability of alpha-MSH to facilitate acquisition of habituation was dose dependent. Plasma alpha-MSH concentrations were elevated within 5 min of dorsal lymph sac injection and remained elevated up to 600% over controls after 30 min. Administration of 50 microgram alpha-MSH had no effect on plasma corticosterone levels. Radiolabeled alpha-MSH was detected in cerebrospinal fluid microdialysates within minutes of peripheral injection. Concentrations of alpha-MSH in the telencephalon and preoptic area were significantly lowered after ether exposure, whereas levels in the optic tectum, thalamus/hypothalamus, brainstem, and plasma were unchanged. We conclude that alpha-MSH administered peripherally facilitates habituation in a dose-dependent fashion. Our results confirm that the effects of alpha-MSH are independent of corticosterone secretion. The peptide is cleared rapidly into the bloodstream and enters the cerebrospinal fluid after dorsal lymph sac injection. Neuronal alpha-MSH may help toads gather information about their environment when exposed to certain stressors.     

Rapid, concurrent analysis of dopamine, 5-hydroxytryptamine, their precursors and metabolites utilizing high performance liquid chromatography with electrochemical detection: analysis of brain tissue and cerebrospinal fluid

Assays capable of measuring picomole quantities of dopamine (DA), 5-hydroxytryptamine (5-HT), several of their precursors and metabolites concurrently within 25 minutes were developed utilizing high performance liquid chromatography with electrochemical detection (LCEC). Several parameters of the LCEC were altered in order to separate the compounds while maintaining a short assay time. The final LCEC systems demonstrated biological utility in that the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the 5-HT metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were detected in rat cerebrospinal fluid; in addition to these compounds, DA and 5-HT were measurable in the striatum, hypothalamus and median eminence of the rat brain. Pargyline decreased the concentrations of DOPAC, HVA and 5-HIAA and increased the 5-HT concentration in all three brain regions, and increased the DA concentration in the striatum. Probenecid increased all three acid metabolite concentrations in the hypothalamus and median eminence, while only the HVA and 5-HIAA concentrations were increased in the striatum. The DA and 5-HT concentrations were unaltered. The LCEC methods described in this paper should be useful in elucidating the mechanisms and roles of 5-HT and DA neurons in experimental paradigms of biological interest.