Effect of gonadal steroids and gamma-aminobutyric acid on LH release and dopamine expression and activity in the zona incerta in rats.

A dopaminergic system in the zona incerta stimulates LH release and may mediate the positive feedback effects of the gonadal steroids on LH release. In this study the mechanisms by which steroids might increase dopamine activity in the zona incerta were investigated. In addition, experiments were conducted to determine whether the inhibitory effects of gamma-aminobutyric acid (GABA) on LH release in the zona incerta are due to suppression of dopamine activity in this area or conversely whether the stimulatory effects of dopamine on LH release are due to suppression of a tonic inhibitory GABAergic system. Ovariectomized rats were treated s.c. with oil, 5 micrograms oestradiol benzoate or 5 micrograms oestradiol benzoate followed 48 h later by 0.5 mg progesterone, and killed 54 h after the oestradiol benzoate injection. At this time the LH concentrations were suppressed in the oestradiol benzoate group and increased in the group treated with oestradiol benzoate and progesterone. The ratio of tyrosine hydroxylase:beta-actin mRNA in the zona incerta was significantly increased by the oestradiol benzoate treatment, but the addition of progesterone resulted in values similar to those in the control group. At the same time, the progesterone treatment increased tyrosine hydroxylase activity in the zona incerta as indicated by an increase in L-dihydroxyphenylalanine (L-DOPA) accumulation after 100 mg 3-hydroxybenzylhydrazine hydrochloric acid (NSD1015) kg-1 and an increase in dopamine release as indicated by a increase in dihydroxyphenylacetic acid (DOPAC) concentrations (one of the major metabolites of dopamine). Ovariectomized rats treated with oestradiol benzoate plus progesterone were also injected i.p. with 75 mg gamma-acetylenic GABA kg-1 (a GABA transaminase inhibitor) to increase GABA concentrations in the brain. This treatment had no effect on the ratio of tyrosine hydroxylase:beta-actin mRNA but decreased L-DOPA accumulation and DOPAC concentrations in the zona incerta, indicating a post-translational inhibition of dopamine synthesis and release. Treatment of ovariectomized rats with oestradiol benzoate followed by 100 mg L-DOPA i.p. to increase dopamine concentrations in the whole brain had no effect on glutamic acid decarboxylase mRNA expression in the zona incerta, although it increased the glutamic acid decarboxylase:beta-actin mRNA ratio in other hypothalamic areas (that is, the medical preoptic area, ventromedial nucleus and arcuate nucleus). In conclusion, the steroids act to increase dopamine activity in different ways: oestrogen increases tyrosine hydroxylase mRNA expression and progesterone acts after translation to increase tyrosine hydroxylase activity and dopamine release (as indicated by increases in DOPAC concentrations). This latter effect may be due to progesterone removing a tonic GABAergic inhibition from the dopaminergic system.

Behavioral effects of neuropeptide E-I (NEI) in the female rat: interactions with alpha-MSH, MCH and dopamine.

The behavioral and neurochemical effects of NEI, and its interaction with alpha-MSH or MCH were investigated in the ventromedial nucleus (VMN) and medial preoptic area (MPOA) in female rats (bilateral administration, 100 ng in 0.5 microliter/side). NEI in the VMN (but not in the MPOA) stimulated exploratory behavior, increased anxiety and reduced dopamine and DOPAC release. The behavioral effects were antagonized by alpha-MSH. NEI stimulated female sexual receptivity in the MPOA. In the VMN, NEI did not have any effect on sexual activity, but partially antagonized the stimulatory effect of MCH. These results show that NEI in the hypothalamus participates in the regulation of behavior, possibly through dopaminergic mediation.

alpha-Melanocyte-stimulating hormone (alpha-MSH) and melanin-concentrating hormone (MCH) modify monoaminergic levels in the preoptic area of the rat.

The effect of perfusion of melanin-concentrating hormone (MCH) or alpha-melanocyte-stimulating hormone (alpha-MSH) (100 ng/microliter) in the ventromedial nucleus (VMN) or medial preoptic area (MPOA) on monoaminergic levels of female rats was measured using microdialysis and HPLC-electrochemical detection. In the MPOA, alpha-MSH raised 5-HIAA concentration, whereas MCH reduced both 5-HT and 5-HIAA. Neither peptide had any effect in the VMN. The opposite effects of the peptides on the serotonergic system might be responsible for their antagonistic or opposite actions previously reported on several CNS functions. Dopamine may mediate the similar effects of the two peptides, because alpha-MSH inhibits dopaminergic release in the MPOA (but not VMN) and MCH tends to follow the same pattern.

Interaction of oestradiol, alpha-melanotrophin and noradrenaline within the ventromedial nucleus in the control of female sexual behaviour.

A possible inter-relationship between oestrogen, alpha-melanotrophin (alpha MSH) and NA in the ventromedial nucleus (VMN) has been studied in ovariectomised-adrenalectomised rats primed with a low dose of oestradiol benzoate (2 or 5 micrograms OB), which induces receptivity in approximately half the rats. Priming with OB increased NA turnover in the VMN (as assessed by the decline in NA concentration 1 h after 250 mg/kg alpha-methyl tyrosine alpha MT) and also enhanced the release of NA from basal medial hypothalamic fragments in vitro. This occurred whether the rats were receptive or non-receptive. Injection of 20 micrograms/rat alpha MSH, 4 h before autopsy in OB-primed rats, reduced NA turnover in the VMN but only in the receptive animals. alpha MSH had no effect on NA content in the VMN, but prevented the decline normally induced by the alpha MT, indicating an inhibition of release. Application of 1 microgram/ml alpha MSH to incubated hypothalamic fragments enhanced release of NA in the tissue obtained from untreated controls and the OB-non-receptive group, but had no effect on the tissue of the OB-receptive animals. Perhaps NA release had already occurred in vivo in the latter group. alpha MSH (100 ng/side/rat) and NA (20, 200 and 2,000 ng/side/rat) were injected into the VMN of ovariectomised-adrenalectomised rats primed with 1 microgram OB. Both agents stimulated lordosis in non-receptive animals with a peak activity at 60 min.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Involvement of catecholaminergic systems in the zona incerta in the steroidal control of gonadotrophin release and female sexual behaviour.

Previous reports have shown that dopamine (DA) in the zona incerta (ZI) has a stimulatory effect on gonadotrophin release. We have now investigated the possibility that steroids exert their feedback effects on the release of luteinizing hormone (LH) via catecholamine systems in the ZI. Since the same steroid regimes also stimulate female sexual behaviour, the possibility that the ZI is also involved in the control of sexual activity was investigated. Lesions in the ZI increased proceptive and receptive behaviour in oestrogen-primed ovariectomised rats that exhibited a low level of lordosis in a pre-lesion test and had no effect in receptive animals. Turnover rates (TR) of DA, noradrenaline (NA) and adrenaline were measured in the ZI, preoptic area (POA), arcuate nucleus (ARC), median eminence (ME) and ventromedial nucleus in ovariectomised rats treated 54 h before with either oil, oestradiol benzoate (OB) at 5, 10 or 50 micrograms/rat, or 5 micrograms/rat OB followed by 0.5 mg/rat progesterone (P) 48 h later. The TR as measured from the decline in concentration after alpha-methyltyrosine and changes in DOPAC concentration were correlated with the effect of the steroids on plasma LH and lordotic activity. Confirming previous reports, NA turnover in the POA and ME was increased, and DA turnover in the ME was decreased by steroids when they enhanced LH release. DA turnover in the ARC and ME was reduced when lordosis behaviour increased. Treatment with OB plus P stimulated LH release and sexual receptivity and at the same time significantly increased DA and NA turnover in the ZI. There was no correlation between these parameters after OB alone. This report shows that the DA system in the ZI may mediate the stimulatory effect of P on LH release in OB-primed rats but is not involved in the feedback effects of oestradiol alone. NA activity in the ZI is increased after OB plus P and may therefore also be concerned with stimulating LH release. The ZI is involved in the control of sexual behaviour, as electrolytic lesions in this area enhance receptivity and proceptivity, but the catecholamine systems in the ZI do not appear to mediate this control.

Differential involvement of 5-hydroxytryptamine (5HT) in specific hypothalamic areas in the mediation of steroid-induced changes in gonadotrophin release and sexual behaviour in female rats.

Ovariectomised rats were treated with either oestradiol benzoate (OB) alone or OB followed by progesterone (P). The effects of the steroid treatments were noted on plasma LH concentration, lordosis quotient and concentration of 5-hydroxytryptamine (5HT) and 5-hydroxyindole acetic acid (5HIAA) in microdissected hypothalamic areas, i.e. the preoptic area (POA), arcuate nucleus (ARC), median eminence (ME), ventromedial nucleus (VMN), suprachiasmatic nucleus (SCN) and zona incerta (ZI). The ratio of 5HIAA:5HT or parallel changes in the concentrations of 5HT and 5HIAA were taken as indices of 5HT neuronal activity. 5 micrograms OB alone reduced LH release and had no effect on receptivity. This treatment reduced 5HT activity in the SCN and raised it in the ZI. After 5 micrograms OB plus 0.5 mg P, all the rats exhibited an LH surge and full sexual receptivity. 5HT activity was significantly raised in the ME and reduced in the ARC, VMN and ZI. By using submaximal steroid regimes (2 micrograms OB plus 0.05 mg P, or 50 micrograms OB alone) correlations could be made between sexual behaviour, LH release and 5HT activity. There was no correlation between LH release and receptivity indicating that the two functions are controlled by different systems. Correlating 5HT activity in the various sites with the two physiological parameters indicated whether the changes seen after the maximal steroid treatment were concerned with behaviour, LH release or both. The results indicate that the changes seen in the VMN, ARC and ME are correlated with sex behaviour and those in the VMN and ZI with increased LH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioral effects in adulthood of serotonin depletion by P-chlorophenylalanine given neonatally to male rats.

The effect in male rats of depletion of brain 5HT over days 1-7 or 8-16 after birth, by daily administration of 100 mg/kg PCPA was noted on various behaviors after the animals had reached adulthood. Treatment over days 1-7 had little or no effect, but over days 8-16 induced the following behavioral changes: In the elevated plus-maze, the treated group spent longer on the open arms, indicating a lower level of anxiety. In a test for sexual orientation, the treated animals were more sexually oriented toward the female stimulus and they showed a reduction in pendulum movements (a fear-related parameter); however their sexual performance with a receptive female was not different from the controls. The animals were also tested as residents in a resident-intruder paradigm for their social and agonistic activity. The treatment induced an increase in offensive activity confined to the high intensity elements and a parallel decrease in defensive activity. These results can be interpreted in terms of an increased reactivity to environmental and social cues and a decreased anxiety.

Studies into the dual effects of serotonergic pharmacological agents on female sexual behaviour in the rat: preliminary evidence that endogenous 5HT is stimulatory.

The potential stimulatory and inhibitory effects on female sexual behaviour of five 5HT antagonists and five agents that increase 5HT activity, were noted in ovariectomised rats primed with various steroid regimes such that they were either "receptive" (LQ greater than 50%) or "non-receptive" (LQ less than 50%). The 5HT antagonists cinanserin, mianserin, ketanserin and metergoline all inhibited behaviour in receptive rats. Methysergide and cinanserin stimulated behaviour in non-receptive rats. All the drugs which increased 5HTP activity, i.e., 5HTP, zimelidine, alaproclate, WY 26002 and quipazine stimulated sex behaviour in non-receptive rats. In rats that had been ovariectomised only, part of this effect was probably due to stimulation of adrenal progesterone, but a significant stimulatory effect could still be observed in ovariectomised-adrenalectomised rats. 5HT also had a significant inhibitory effect on receptive rats, and the other agonists showed a similar but non-significant tendency. In view of the fact that 4 out of 5 of the 5HT antagonists inhibited sexual behaviour, we hypothesise that 5HT has a stimulatory role in the control of female sexual behaviour. The possible mechanisms mediating the dual action of 5HTP on female sexual behaviour are discussed.

The role of dietary lipid in the regulation of unconjugated hyperbilirubinaemia in Gunn rats.

1. The purpose of this study was to characterize the mechanisms of diet-induced hyperbilirubinaemia in Gunn rats with emphasis on the role of lipids, and to examine their relationship with regard to fasting hyperbilirubinaemia. 2. A lipid-free normocaloric diet produced a threefold increase in plasma bilirubin concentration (baseline 109.4 mumol/l), which was maximal by 10 days and thereafter remained constant. The level of hyperbilirubinaemia attained was not influenced by fasting or phenobarbitone, and returned to baseline concentration within 10 days of resuming a normal diet. 3. Determination of hepatic bilirubin showed that the magnitude of the hepatic bilirubin pool was increased by the lipid-free diet but was unchanged by fasting. Hepatic ligandin concentrations were comparable in fasted Gunn rats and those fed normal or lipid-free diets, although total hepatic ligandin was reduced in the fasted animals. 4. The hyperbilirubinaemic effect of the lipid-free diet was largely reversed by the inclusion of 10% lipid in the diet and was affected to a lesser extent by 5% lipid. Similar reductions in plasma bilirubin concentration were observed with a variety of other lipids (10%), regardless of their fatty acid chain length or degree of saturation. 5. In fasting animals a direct correlation was observed between plasma bilirubin and free fatty acid concentrations and insulin levels were greatly depressed, whereas in those fed on the lipid-free diet no significant changes were evident in plasma concentrations of free fatty acids or insulin. 6. Plasma bilirubin concentration was unrelated to alterations in plasma triglycerides produced by the administration of clofibrate. However, an unexplained decrease in plasma bilirubin (40%) without a significant change in triglycerides was noted when clofibrate was added to the lipid-free diet. 7. Analysis of kinetic data obtained from [14C]bilirubin clearance studies revealed that hyperbilirubinaemia associated with the lipid-free diet reflected a marked reduction (60%) in plasma clearance with no change in bilirubin turnover. This was accompanied by a relative redistribution of bilirubin from the extravascular pool to the plasma pool. 8. Although these studies indicate that fasting and the withdrawal of dietary lipid have some similar effects on bilirubin metabolism, it seems likely that different mechanisms are responsible for the hyperbilirubinaemia.

Trial of combination of guanethidine and oxprenolol in hypertension.

Thirteen hypertensive patients entered a double-blind crossover trial of guanethidine and oxprenolol in combination. In nine patients who completed the trial there was an additive effect on blood pressure, but the combination had a smaller effect on heart rate than was expected from the individual effects, and side effects were not increased. During treatment with oxprenolol the plasma potassium concentration rose from 3.6 mmol (mEq)/1 to 3.9 mmol (mEq)/1. No correlation was found between the plasma oxprenolol concentration and changes in blood pressure or response to injected isoprenaline, but measurements of plasma oxprenolol concentrations were of value in determining compliance with the protocol.