Leptin uptake by serotonergic neurones of the dorsal raphe.

The effects of leptin on food intake, metabolism, sleep patterns and reproduction may be mediated, in part, by the midbrain serotonergic systems. Here, we report on the distribution of neurones that accumulate leptin in the raphe nuclei of male and female rats after intracerebroventricular administration of mouse recombinant leptin labelled with digoxigenin. Direct leptin-targeted cells were present in the periventricular grey, pontine and raphe nuclei. Confocal microscopy revealed that raphe neurones which accumulated leptin were predominantly serotonergic. The temporal pattern of leptin accumulation by raphe neurones showed a marked gender difference: 6 h after leptin administration, all male and female rats showed massive leptin binding in the dorsal raphe, while 30 min after leptin treatment, only 10% of male rats exhibited leptin-labelled cells in contrast to 50% of females. The present observations reveal that leptin can be selectively accumulated by serotonergic neurones in the raphe nuclei and that this mechanism is gender specific. These findings support the idea that the midbrain serotonergic system is an important mediator of the effects of leptin on brain function and may provide an explanation for gender differences in metabolism regulation and its coordination with higher functions of the brain.

Diurnal oscillation in glial fibrillary acidic protein in a perisuprachiasmatic area and its relationship to the luteinizing hormone surge in the female rat.

It is well known that the reproductive cycle in the female rat is closely associated with the circadian rhythms of motor activity and that this phenomenon requires the presence of estrogens. Estrogens induce plastic changes in neural connectivity and these changes could be the result of glial modifications. We have measured glial fibrillary acidic protein (GFAP) immunoreactivity in order to localize the area in which the coupling of the circadian rhythms to the generation of the luteinizing hormone (LH) surge may occur. As circadian rhythms are driven by the suprachiasmatic nucleus (SCN), GFAP immunoreactivity was measured in 5 areas of the SCN and surrounding regions. It was measured at two times during daylight (10.00 and 17.00 h) in ovariectomized (OVX) females implanted with Silastic capsules containing either estradiol benzoate (EB) or oil (control). Differences between morning and afternoon GFAP immunoreactivity were observed in a peri-SCN area, dorsal to the SCN and close to the 3rd ventricle, in estrogen-treated as well as in control OVX females. However, this difference increased in the subgroup of EB-treated females which displayed the strongest LH rhythmicity. These results suggest that the peri-SCN area could be an important locus for synaptic changes linking circadian rhythms with the estrogen-induced LH surge.

Phasic synaptic remodeling of the rat arcuate nucleus during the estrous cycle depends on insulin-like growth factor-I receptor activation.

Insulin-like growth factor-I (IGF-I) has trophic and plastic effects on neurons and glial cells and modulates neuroendocrine events by acting at the level of the hypothalamus. IGF-I and estrogen signaling interact to regulate in vitro hypothalamic neuronal survival and differentiation. In vivo, IGF-I levels fluctuate in the rat hypothalamic arcuate nucleus during the estrous cycle in parallel with a phasic remodeling of synaptic contacts and glial cell processes. Both the fluctuation of IGF-I levels and the synaptic and glial changes are induced by estrogen. The possible role of IGF-I in the regulation of arcuate nucleus synaptic plasticity has been assessed in the present study by intracerebroventricular administration to cycling female rats of a specific IGF-I receptor antagonist. In agreement with previous findings, the number of synaptic inputs to arcuate neuronal somas in control rats showed a significant decrease between the morning of proestrus and the morning of estrus. This decline in synaptic inputs and the accompanying increase in glial ensheathing of neuronal somas were blocked by the IGF-I receptor antagonist. In contrast, the IGF-I receptor antagonist did not affect the basal number of synapses or the morphology of synaptic terminals or length of the synaptic contacts. These findings indicate that IGF-I receptor activation may be involved in the phasic remodeling of arcuate nucleus synapses during the estrous cycle. Res.

Role of astroglia and insulin-like growth factor-I in gonadal hormone-dependent synaptic plasticity.

Gonadal hormones exert a critical influence over the architecture of specific brain areas affecting the formation of neuronal contacts. Cellular mechanisms mediating gonadal hormone actions on synapses have been studied extensively in the rat arcuate nucleus, a hypothalamic center involved in the feed-back regulation of gonadotropins. Gonadal steroids exert organizational and activational effects on arcuate nucleus synaptic connectivity. Perinatal testosterone induces a sexual dimorphic pattern of synaptic contacts. Furthermore, during the preovulatory and ovulatory phases of the estrous cycle there is a transient disconnection of inhibitory synaptic inputs to the somas of arcuate neurons. This synaptic remodeling is induced by estradiol, blocked by progesterone, and begins with the onset of puberty in females. Astroglia appear to play a significant role in the organizational and the activational hormone effects on neuronal connectivity by regulating the amount of neuronal membrane available for the formation of synaptic contacts and by releasing soluble factors, such as insulin-like growth factor I (IGF-I), which promote the differentiation of neural processes. Recent evidence indicates that gonadal steroids and IGF-I may interact in their trophic effects on the neuroendocrine hypothalamus. Estradiol and IGF-I promote the survival and morphological differentiation of rat hypothalamic neurons in primary cultures. The effect of estradiol depends on IGF-I, while the effects of both estradiol and IGF-I depend on estrogen receptors. Furthermore, estrogen activation of astroglia in hypothalamic tissue fragments depends on IGF-I receptors. These findings indicate that IGF-I may mediate some of the developmental and activational effects of gonadal steroids on the brain and suggest that IGF-I may activate the estrogen receptor to induce its neurotrophic effects on hypothalamic cells. In addition, IGF-I levels in the neuroendocrine hypothalamus are regulated by gonadal steroids. IGF-I levels in tanycytes, a specific astroglia cell type present in the arcuate nucleus and median eminence, increase at puberty, are affected by neonatal androgen levels, show sex differences, and fluctuate in accordance to the natural variations in plasma levels of ovarian steroids that are associated with the estrous cycle. These changes appear to be mediated by hormonal regulation of IGF-I uptake from blood or cerebrospinal fluid by tanycytes. These results suggest that tanycytes may be involved in the regulation of neuroendocrine events in adult rats by regulating the availability of IGF-I to hypothalamic neurons. In summary, IGF-I and different forms of neuron-astroglia communication are involved in the effects of estradiol on synaptic plasticity in the hypothalamic arcuate nucleus.

Endocrine-dependent accumulation of IGF-I by hypothalamic glia.

Tanycytes are specialized glial cells of the hypothalamus and median eminence. Immunoreactive insulin-like growth factor I (IGF-I) levels fluctuate in tanycytes with the natural variations in sex steroids associated with the ovarian cycle. To determine whether these changes are as a result of differences in IGF-I accumulation, the peptide was labelled with digoxigenin and injected into the lateral cerebral ventricle. Tanycyte-like cells specifically accumulated digoxigenin-labelled IGF-I. This accumulation was mediated by IGF-I receptors and showed marked differences during the oestrous cycle, being low in the afternoon of pro-oestrus and high in the afternoon of oestrus. These results indicate that the accumulation by tanycytes of IGF-I or IGF-I fragments capable of receptor-mediated internalization is under endocrine control, suggesting that hypothalamic glia may be involved in neuroendocrine regulation.

Interaction of the signalling pathways of insulin-like growth factor-I and sex steroids in the neuroendocrine hypothalamus.

Among the numerous endocrine signals that affect the central nervous system, sex steroids play an important role. It has been recently postulated that part of the effects of these hormones on the brain may be mediated by trophic factors, such as insulin-like growth factor I (IGF-I). Both estradiol and IGF-I increase the survival and differentiation of developing fetal rat hypothalamic neurons in culture. The effect of estradiol is blocked by the pure estrogen receptor antagonist ICI 182,780, by an antisense oligonucleotide to the estrogen receptor, and by an antisense oligonucleotide to IGF-I. In turn, the effect of IGF-I is blocked by ICI 182,780 and by the antisense oligonucleotide to the estrogen receptor. These findings indicate that estrogen-induced activation of the estrogen receptor in developing hypothalamic neurons requires the presence of IGF-I and that both estradiol and IGF-I use the estrogen receptor to mediate their trophic effects on hypothalamic cells. In vivo, sex steroids affect IGF-I levels in the endocrine hypothalamus. IGF-I levels in tanycytes, a specific subtype of glial cells present in the arcuate nucleus and median eminence, are sexually dimorphic in the rat, increase with the onset of puberty, and are regulated by perinatal and adult levels of sex steroids. These changes may be due to hormonal modifications of IGF-I uptake by tanycytes from blood or cerebrospinal fluid. Therefore, this type of glial cell appears to play a central role in the interaction of sex steroids and IGF-I in the hypothalamus.

Alteration of prolactin control in adult rats treated neonatally with sex steroids.

Androgenized, oestrogenized and control female and male rats were used to establish possible differences in the alteration of the prolactin control system. Neonatal treatment involved administration of oestradiol benzoate or testosterone propionate (TP) on days 1 and 5 to the males and on day 5 to the females. Oil-treated animals were used as controls. Plasma prolactin levels were measured in these animals during adulthood (a) before gonadectomy, performed on day 80, and 27 days after gonadectomy and (b) on the 2 days (at 10.00 and 17.00 h) after administration of a single dose of TP to gonadectomized animals. Oestrogenized rats had the highest plasma prolactin concentrations just before and after gonadectomy. Testosterone propionate increased plasma prolactin levels in all groups. This response was more notable in the female than in the male groups, and was highest in the oestrogenized animals. Temporal rhythms of the prolactin response to TP were daily, perhaps circadian, with increased levels in the afternoon compared with those in the morning. This pattern was not present in oestrogenized females and androgenized males. Results suggest (a) that oestrogens and androgens given neonatally differ in their ability to alter the prolactin control system, and (b) that females seem to be more sensitive than males to changes in hypothalamic differentiation induced by neonatal steroid treatment.

[Effect of administering prostaglandine f2alpha and e2 on lh secretion in rat (author's transl)]. / Efecto de la administración de prostaglandina F2alpha y E2 sobre la secreción de LH en la rata.

Intraperitoneal administration of 100 ng of synthetic LH-RH to castrated male rats, produces a significant elevation of plasma LH values, as determined by RIA. A greater increase is obtained, by administrating 400 ng of LH-RH. Intrayugular injection of physiologic saline reduces basal values of plasma LH. This pattern is not modified by administration of 10 microgram or 100 microgram of PGF2alpha. On the other hand 10 microgram of PGE2 compensates the fall and 100 microgram significantly increases plasma LH over the basal values. Intracarotidly a similar pattern is observed. Plasma LH values decreases in the control group and no modification is observed by the administration of 10 microgram of PGF2alpha. By injecting the same amount of PGE2, plasma LH values remains constant, compensating the fall observed in the controls. Our results show a stimulatory effect of PGE2 and no effect of PGF2alpha on LH secretion in the castrated male rat.

[Radioimmunoanalysis of plasma testosterone]. / Determinación por radioinmunoanálisis de la testosterona plasm

A simple radioimmunoassay procedure for the determination of testosterone concentration in plasma is described. Small amounts of plasma, 0,5 ml for males and 1 ml for females, are extracted with diethyl ether. Recoveries for the extraction are obtained by the addition of labelled tesosterone. The radioimmunoassay is performed directly on the extracts without further purification. Separation of the free from the antibodie bound testosterone is achieved with dextran coated charocoal. Under the used conditions, the calibration curve covers a range between 0,5 and 1,5 ng per sample. Reproducibility is acceptable with a coefficient of variation of 7%. The normal values are slightly higher than those obtained with methods using some purification step of the extract before the assay but due to its simplicity the described method is a suitable one for clinical purposes.