Molecular mechanisms involved in interleukin 1-beta (IL-1ß)-induced memory impairment. Modulation by alpha-melanocyte-stimulating hormone (α-MSH).

Pro-inflammatory cytokines can affect cognitive processes such as learning and memory. Particularly, interleukin-1ß (IL-1ß) influences the consolidation of hippocampus-dependent memories. We previously reported that administration of IL-1ß in dorsal hippocampus impaired contextual fear memory consolidation. Different mechanisms have been implicated in the action of IL-1ß on long-term potentiation (LTP), but the processes by which this inhibition occurs in vivo remain to be elucidated. We herein report that intrahippocampal injection of IL-1ß induced a significant increase in p38 phosphorylation after contextual fear conditioning. Also, treatment with SB203580, an inhibitor of p38, reversed impairment induced by IL-1ß on conditioned fear behavior, indicating that this MAPK would be involved in the effect of the cytokine. We also showed that IL-1ß administration produced a decrease in glutamate release from dorsal hippocampus synaptosomes and that treatment with SB203580 partially reversed this effect. Our results indicated that IL-1ß-induced impairment in memory consolidation could be mediated by a decrease in glutamate release. This hypothesis is sustained by the fact that treatment with d-cycloserine (DCS), a partial agonist of the NMDA receptor, reversed the effect of IL-1ß on contextual fear memory. Furthermore, we demonstrated that IL-1ß produced a temporal delay in ERK phosphorylation and that DCS administration reversed this effect. We also observed that intrahippocampal injection of IL-1ß decreased BDNF expression after contextual fear conditioning. We previously demonstrated that α-MSH reversed the detrimental effect of IL-1ß on memory consolidation. The present results demonstrate that α-MSH administration did not modify the decrease in glutamate release induced by IL-1ß. However, intrahippocampal injection of α-MSH prevented the effect on ERK phosphorylation and BDNF expression induced by IL-1ß after contextual fear conditioning. Therefore, in the present study we determine possible molecular mechanisms involved in the impairment induced by IL-1ß on fear memory consolidation. We also established how this effect could be modulated by α-MSH.

The effect of melanotropic peptides on binding of [(3)H]dihydroalprenolol-hydrochloride to hypothalamic membranes.

In the present work we studied the interaction of alpha-melanocyte-stimulating hormone (alpha-MSH) and ACTH-(1-24) with beta-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [(3)H]dihydroalprenolol-hydrochloride ([(3)H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by alpha-MSH or ACTH-(1-24). These data indicate a non competitive interaction between these melanocortin peptides and [(3)H]DHA on beta-adrenergic receptors in hypothalamic membranes.

The role of melanocortin receptors in sexual behavior in female rats.

Earlier data have indicated that alpha-MSH may play a role for sexual behavior in rats. In this study we investigated the effects of MSH peptides on sexual receptivity in ovariectomized-adrenalectomized female rats, pre-treated with benzoate of estradiol, in presence of vigorous male rats. The results show that alpha-MSH significantly increases lordosis behavior in female rats after injections into the ventromedial nucleus. Interestingly, we have for the first time shown that gamma-MSH also causes significant increase in lordosis in female rats. Furthermore, we show that HS014, an antagonist for the central MC receptors, in dose dependent manner blocks the effect of alpha-MSH on lordosis. The results indicate that the effects of MSH peptides on female sexual behaviour are mediated through a specific MC receptor, which could be the MC3 receptor.

Interaction of alpha-melanotropin (alpha-MSH) and noradrenaline in the median eminence in the control of female sexual behavior.

In the present study, we examined the effects of the injection of alpha-melanotropin (alpha-MSH), noradrenaline (NA), and dopamine in the median eminence of ovariectomized-adrenalectomized rats on female sexual behavior. The animals were primed with l0 microg of estradiol benzoate, and 52-54 h later they were injected into the median eminence with either 1 microl of artificial cerebrospinal fluid, 1 microg/rat alpha-MSH, 200 ng/rat NA, 200 ng or 2 microg/rat dopamine, in 1 microl of artificial cerebrospinal fluid. Both alpha-MSH and NA significantly stimulated sexual behavior. This effect was antagonized by two beta-adrenergic antagonists: propranolol (500 ng/rat) and metoprolol (400 ng/rat) applied 15 min before the alpha-MSH or NA. The alpha-adrenergic antagonist prazosine (500 ng/rat) was ineffective in reducing the effect of alpha-MSH. The vehicle and dopamine at both doses had no effect on sexual activity. These results indicate that alpha-MSH and NA in the median eminence stimulate female sexual behavior and that NA mediates the action of alpha-MSH via beta-receptors.

Age-related changes in grooming behavior and motor activity in female rats.

The influence of hormonal status and the age of the rat on the expression of grooming behavior and motor activity were studied. Grooming, locomotion, and rearing were measured in young (4-months-old), adult (6-8-months-old), and old (18-months-old) female rats, during the estrous cycle. These behavioral performances were influenced by the hormonal changes that occur in young and adult female rats during the estrous cycle. In old rats there were no significant differences among the different days of the estrous cycle. A significant age-related decrease in grooming behavior and motor activity was also found. Locomotion and rearing were the parameters most affected by age. These findings could be related to: (a) the gonadal hormonal status, which appears to be able to modulate behavioral responses; and (b) the age-related changes, which may affect the normal display of these behaviors. The possible role of central peptidergic, cholinergic, and dopaminergic neural systems is discussed.

The inter-relationship between gonadal steroids and POMC peptides, beta-endorphin and alpha-MSH, in the control of sexual behavior in the female rat.

Estradiol benzoate (10 microg EB) given to ovariectomized-adrenalectomized rats induced sexual receptivity in half the animals and increased alpha-MSH in the preoptic area, ventromedial nucleus (VMN) and arcuate nucleus (ARC), in all the animals, although levels were significantly higher in the VMN and ARC of the receptive (R) subgroup. EB also raised levels of beta-endorphin in the VMN and ARC in the R rats only. POMC expression was not altered. EB did not affect alphaMSH in extra-hypothalamic areas, but addition of progesterone, raised levels in the septum, amygdala, hippocampus and caudate putamen. Only in the VMN, ARC and septum were the steroid-induced increases correlated with onset of sexual behavior.

Evidence for catecholaminergic control of alpha-melanotropin (alpha-MSH) content in hypothalamic areas.

A possible catecholaminergic regulation of hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) has been investigated in male rats by an in vivo approach. The hormone was measured by radioimmunoassay in three hypothalamic regions: medial basal hypothalamus, preoptic hypothalamic area and dorsolateral hypothalamus. The tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (300mg/kg) increased the hypothalamic alpha-MSH content in medial basal hypothalamus and preoptic hypothalamic area when it was measured at 22:00 h. Diethyldithiocarbamate (600mg/kg), which inhibits dopamine beta-hydroxylase, as well as 2-3-dichloromethylbenzylamide (25mg/kg), which acts on the phenylethanolamine-NCH3 transferase also increased the alpha-MSH content in the above mentioned discrete areas. The alpha-adrenoceptor antagonist phenoxybenzamine (15mg/kg), as well as the alpha 1-adrenoceptor antagonist prazosin (1.0mg/kg), also increased the hypothalamic alpha-MSH content in medial basal hypothalamus and preoptic hypothalamic area. None of these agents modified alpha-MSH content in dorsolateral hypothalamus. Haloperidol (1.2mg/kg), a dopaminergic receptor antagonist, propranolol (6.0mg/kg) and yohimbine (10mg/kg) (non selective beta- and alpha 2-adrenergic antagonist drugs respectively) had no effect on the alpha-MSH in any of the hypothalamic areas studied. These results indicate that the catecholaminergic system is involved in the control of proopiomelanocortin derived hypothalamic alpha-MSH through an alpha 1-adrenoreceptor. The data suggest that the control mechanism in the two alpha-MSH hypothalamic pools are different.

A central action of alpha-melanocyte-stimulating hormone on serum levels of LH and prolactin in rats.

We have investigated the effect of administration of alpha-MSH into the median eminence (ME) of rats on the release of LH and prolactin. Continuous infusion of alpha-MSH (0.5 micrograms/h) into the ME from the afternoon of the second day of dioestrus and over the 24 h of pro-oestrus inhibited the preovulatory LH and prolactin surge and the occurrence of ovulation. This inhibitory effect on LH and prolactin release was also observed in chronically ovariectomized rats given a single injection of alpha-MSH (1 micrograms/ml per rat) into the ME (blood samples were collected 0, 20, 60, 90, 105 and 120 min after injection). The intraperitoneal injection of the dopamine receptor blocker, haloperidol (2 mg/kg), 30 min before the injection of alpha-MSH into the ME prevented the inhibitory effect of alpha-MSH on the release of LH and prolactin. These results suggest that hypothalamic alpha-MSH might be involved in the regulation of LH and prolactin release via the tuberoinfundibular dopaminergic system and that this system also modifies the serum concentrations of alpha-MSH.

Photoaffinity labelling of melanoma cell MSH receptors.

UV-irradiation at 365 nm of cultured Cloudman S91 mouse melanoma cells in the presence of photoreactive alpha-MSH analogues induced longlasting receptor stimulation as revealed by the ensuring activation of tyrosinase. Receptor labelling was more efficient with 4-diazirinophenyl and 2-nitro-4-azidophenyl photolabels than with 4-azidophenyl, and was further increased when superpotent [Nle4,D-Phe7]-alpha-MSH was used as ligand. Incubation of B16 melanoma cell membranes with mono-iodinated [Nle4,D-Phe7,Trp-(Naps)9]-alpha-MSH followed by UV-irradiation at 310-550 nm labelled a single band on SDS-PAGE with a molecular mass approximately or equal to 45 kDa. The displacement curve obtained in a competitive photolabelling experiment paralleled that of the binding assay, demonstrating that the labelling was specific.

Effect of gonadectomy and reposition of gonadal steroids on alpha-melanocyte-stimulating hormone concentration in discrete hypothalamic areas during a 24-hour period.

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) was measured by radioimmunoassay in males after orchidectomy and after orchidectomy plus testosterone replacement, and in females after ovariectomy and after ovariectomy plus estradiol or progesterone, or estradiol and progesterone (EP) replacement. Gonadectomy inverted the diurnal rhythm of the alpha-MSH content observed in intact males in the medial basal hypothalamus (MBH) and preoptic hypothalamic area (POA), and produced a notable decrease of alpha-MSH total content in the three regions studied (MBH, POA and dorsolateral hypothalamus, DLH). The addition of testosterone restored the rhythm of the intact males and increased alpha-MSH content in MBH and POA. No diurnal variations in alpha-MSH content were observed in ovariectomized females. A circadian rhythm similar to that of proestrus was observed in MBH after estradiol or EP replacement, and in POA after the addition of any steroid. In DLH the injection of estradiol produced variations through the day, but they are somehow different from those described for proestrus. Treatment with progesterone significantly decreased alpha-MSH content in MBH and DLH, but not in POA. In this region an increase in alpha-MSH content was noticed after EP replacement. We conclude that gonadal steroids can alter the content of hypothalamic alpha-MSH and influence the diurnal variations of the peptide. This may be important in the modulation of several types of behavior or in the neuroendocrine control of gonadotropin release in females, where alpha-MSH seems to modulate the release of luteinizing hormone and prolactin.

Changes in alpha-melanocyte-stimulating hormone content in discrete hypothalamic areas of the male rat during a twenty-four-hour period.

Circadian variations in alpha-melanocyte-stimulating hormone (alpha-MSH) content of discrete hypothalamic areas of the male rat were observed either with radioimmunoassay or bioassay. In the medial basal hypothalamus and preoptic area the alpha-MSH content increased sharply between 02.00 and 06.00 h, showing the highest concentration at 06.00 h. In contrast, no significant changes in alpha-MSH content were detected in the lateral hypothalamus during a 24-hour period. Pituitary alpha-MSH also showed a diurnal variation which was different from that in the two hypothalamic areas. The finding that alpha-MSH values in the brain are maximal during the activity period of the rat is in agreement with results demonstrating a role of alpha-MSH in behaviour and locomotor activity.

Modifications in alpha-MSH concentrations in different hypothalamic areas during the estrous cycle in the rat.

The present experiments were undertaken to examine the pattern of alpha-MSH during the estrous cycle in different hypothalamic areas and assess if during the whole estrous cycle the MSH which arises from proopiomelanocortin (POMC) has the same concentrations pattern as MSH from the dorso-lateral hypothalamus. MSH concentration of the mediobasal hypothalamus (MBH), preoptic area (POA) and dorso-lateral hypothalamus (DLH) was measured using a specific radioimmunoassay. The rats were sacrificed every four hours by perfusion. It is possible to observe a circadian rhythm in all the areas studied except in MBH during estrus. The MSH concentration in MBH was high during the light period, inversely, in POA the concentration remained low. In general, when MSH is high in MBH, it is low in POA and vice versa. The pattern of MSH in DLH is very similar to that observed for the peptide in POA.

Inhibition by L-prolyl-L-leucyl-glycinamide (PLG) of alpha-melanocyte stimulating hormone release from hypothalamic slices.

Release of alpha-MSH from male rat hypothalamic slices was studied using a sensitive bioassay (1-2 pg). Addition of 60 mM KCl to superfusion medium resulted in a twofold increase in alpha-MSH release compared to spontaneous release. Both spontaneous and potassium-induced release were inhibited in a dose-response manner by the tripeptide Pro-Leu-Gly-NH2 (PLG, or MIF-1); 0.04 microgram to 1 microgram PLG inhibited the alpha-MSH release but the lowest dose demonstrated a greater inhibitory effect; high concentrations of PLG, on the other hand, did not modify either spontaneous or potassium-evoked alpha-MSH release from the slices. Contrarily, DA did not modify either spontaneous or potassium- induced alpha-MSH release at any of the doses tested. These findings demonstrate that the inhibitory behavior of PLG and DA in the central nervous system (CNS) differs from their behavior towards alpha-MSH release in the pituitary. This suggests differences in the regulation of alpha-MSH release from the pituitary and the CNS.