Jorge Belmar

Effect of castration and testosterone on norepinephrine storage and on the release of [3H]norepinephrine from rat vas deferens

Abstract Norepinephrine and dopamine-β-hydroxylase, used as noradrenergic vesicle markers, were found to be decreased in the rat vas deferens 10 days after castration. Five days of testosterone administration to castrated animals increased the enzyme activity over that of controls but did not modify norepinephrine content. In tissue fractions obtained by differential centrifugation, the highest activities of the noradrenergic markers appeared in the vesicular fraction of controls and in the soluble fraction of castrated animals. Testosterone reversed the effect of castration: it increased dopamine-β-hydroxylase activity in the vesicular and soluble fractions, while norepinephrine increased only in the vesicular fraction. Results obtained after continuous sucrose gradient centrifugation of vesicular fractions suggested that these changes principally affected the number of light noradrenergic vesicles while testosterone increased the number of vesicles reduced by castration. Hormonal manipulations also modified some functional properties of nerve endings: norepinephrine depletion after transmural stimulation in the presence of tetraethylammonium, as well as the release of the neurotransmitter, were decreased after castration. These effects were reversed by testosterone. The results suggest a modulatory effect of testosterone on the norepinephrine storage system and on the functional properties of the adrenergic innervation of vas deferens.

In vitro progesterone effects on 3H-dopamine release from rat corpus striatum slices obtained under different endocrine conditions

The release of dopamine (DA) from corpus striatum is affected by the endocrine state of the animal being progesterone suggested as a potential hormonal modulatory signal. Most of its actions have been described on endogenous DA release induced by amphetamine. However, the release of DA and the mechanism of the drug effect have been shown to be highly complexes. Considering that DA recently incorporated and/or synthetized is preferentially used we have characterized the effect of progesterone in vitro on the K(+)-induced release of 3H-dopamine (3H-DA) from rat corpus striatum slices. These were obtained during the estrous cycle or under conditions of high or low levels of endogenous progesterone (pregnant and ovariectomized rats). The release of 3H-DA was independent of the cycle. However, progesterone in vitro modified the induced release in a cycle-dependent way. Low concentrations of the hormone (100-200 mM) reduced the K+ (30 mM) effect while higher doses (300-500 mM) were facilitatories. After 7 days of ovariectomy, the induced release of 3H-DA was unchanged while in pregnant rats it was found decreased. In both cases the inhibitory effect of the hormone disappeared. Both progesterone (200 nM) and omission of Ca++ from the superfusion medium did not modified tyramine (20 microM) or K+ induced release, respectively. Data suggest that the pool of DA, related to exocytotic mechanism of release, could be specifically affected by progesterone, in a bimodal way, probably through independent genomic and non-genomic influences.

Early Malnutrition and Changes in the Induced Release of Noradrenaline in the Prefrontal Cortex of Adult Rats

The influence of early protein-energy malnutrition on the induced release of noradrenaline in the rat prefrontal cortex was studied: (i) by evaluating in vivo the release of the neurotransmitter as revealed by changes in the ability of pyramidal cells to integrate transient transmembrane currents generated by discrete packets of noradrenaline released by repetitive electrical stimulation of the locus coeruleus; and (ii) by measuring in vitro the potassium-induced release of 3H-noradrenaline in slices obtained from the brain frontal pole. Both electrophysiological and neurochemical data show that, in malnourished rats, weak stimulation produces an increased release, whereas strong stimulation results in a decreased release of the neurotransmitter. The results provide direct evidence that malnutrition alters the release of noradrenaline at the cortical level. Since the prefrontal cortex is involved in cognitive processing, the present results could provide functional evidence linking nutritional and behavioral deficits.

Clonidine treatment during gestation prevents functional deficits induced by prenatal malnutrition in the rat visual cortex.

It has been shown that prenatal malnutrition results at birth in increased concentration of noradrenaline (NA) in the brain. Besides, it is known that NA is an important regulator of normal regressive processes occurring during synaptogenesis such as cell death, axonal pruning and synaptic elimination. The present study was designed to investigate (i) whether prenatal malnutrition enhances the NA release in the visual cortex and (ii) whether or not chronic administration of clonidine during gestation may prevent long-term deleterious effects of fetal malnutrition on functional properties of interhemispheric connections of the visual cortex and on the interhemispheric asymmetry of visual evoked responses. Prenatal malnutrition was induced by restricting food consumption to pregnant rats from Day 8 postconception to parturition. Results show that at birth, prenatally malnourished rats had higher NA release than normals. At 45-50 days of age, the malnourished group exhibited (a) reduced peak-to-peak amplitude and diminished extent of the projecting field of transcallosal evoked responses, and (b) abolished interhemispheric asymmetry of visual evoked responses. Clonidine administration to malnourished mothers from Day 14 postconception to parturition (10 g/kg/day s.c.), prevented in the offspring disorders induced by prenatal malnutrition on cortical NA release, on interhemispheric connectivity of visual areas and on interhemispheric bioelectrical asymmetry, probably by restoring the normal trophic role of NA during synaptogenesis. Results are discussed in relationship to normal regressive events occurring during early brain development.

Allopregnanolone-Induced Modification of Presynaptic Basal and K+-Induced [3H]-Norepinephrine Efflux from Rat Cortical Slices during the Estrous Cycle

Superfused frontal slices of cerebral cortex were preloaded with [³H]-norepinephrine ([³H]NE). Basal [³H]NE efflux and K⁺-induced [³H]NE release were studied during the estrous cycle and in the presence of neurosteroids. Basal [³H]NE efflux showed estrous cycle-related variations, with lowest values found during estrus and diestrus II. Allopregnanolone (10^–9 M) potentiated basal [³H]NE efflux from the 1st minute of its application; the effect of the steroid was still present after 20 min. This effect was also dependent upon the estrous cycle, since basal [³H]NE efflux was mainly increased during estrus diestrus I, and to a lesser degree only during proestrus. During diestrus II and after ovariectomy, basal [³H]NE efflux was no longer affected by the neurosteroid. In the presence of yohimbine (10^–6 M), the effect of allopregnanolone on basal efflux was potentiated only during the first 3 min but vanished thereafter. Allopregnanolone (10^–9 M) potentiated the K⁺-induced [³H]NE release during estrus, but pregnenolone (10^–9 M) was ineffective, suggesting specificity of the neurosteroid. Yohimbine (10^–6 M) also potentiated K⁺-induced [³H]NE release. When applied simultaneously with allopregnanolone (10^–9M), a potentiating effect on [³H]NE release was observed. The present results suggest that allopregnanolone is a neurosteroid able to modulate norepinephrine release in the cerebral cortex in an estrous cycle-dependent manner, and that the effect could involve noradrenergic alpha-2 receptors.

Changes of norepinephrine levels and release in rat cerebral cortex during the estrous cycle.

We studied the influence of the estrous cycle and ovariectomy on the noradrenergic innervation of the rat cerebral cortex. The lowest norepinephrine (NE) concentration was found during estrus in frontal and occipital cortex. At that stage and at diestrus-2, 20 mM K+ induced the lowest release of [3H]NE from occipital region slices, and the highest release was found at 60 mM K+. Ovariectomy (7 days) decreased the 20 mM K+ effect. Yohimbine (10 x 10(-6) M) increased the induced NE release through the cycle but its effects disappeared after ovariectomy. In the frontal cortex the drug effect was only found at diestrus-1 and no changes were observed in the K+ effect through the cycle. Results suggest that normal endocrine influences can modify noradrenergic neurotransmission in the rat cerebral cortex.

Effect of clonidine early in life on brain morphofunctional deficits induced by neonatal malnutrition in the rat

A great body of evidence indicates that malnutrition early in life induces central noradrenergic hyperactivity (CNH). On the other hand, it is known that noradrenaline (NA) is an important regulator of the regressive processes occurring during synaptogenesis such as cell death, axonal pruning and synaptic elimination. This leads to the hypothesis that some of the morphofunctional modifications induced by malnutrition on the brain could be due, at least in part, to an increase of NA activity during the period of accelerated brain growth. This study evaluates whether early reduction of CNH by the alpha-2 presynaptic adrenoreceptor agonist clonidine, prevents long-term morphofunctional deficits induced by protein-energy malnutrition in the rat. Results of experiments performed on 45 day-old malnourished animals that received clonidine during the suckling period, show that the pharmacological treatment prevented: (i) deficits in both NA levels and NA release in the visual cortex; (ii) deficit in brain weight but not in body weight; and (iii) reduction of the normal brain interhemispheric asymmetry of visual cortical evoked potentials. It is suggested that administration of clonidine early in life prevents brain morphofunctional deficits by malnutrition, by restoring the normal tropic role of NA during synaptogenesis.

Neuroactive steroids modulate GABA inhibition of hypothalamic somatostatin release

The reduced steroids 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) and 3α,21-di-hydroxy-5α-pregnan-20-one (allotetrahydroDOC) are potent ligands of GABAA receptors. This study examined the possible modulatory effect of these metabolites on GABA inhibition of somatostatin release in cultured hypothalamic neurones. Allopregnanolone potentiates GABA inhibition, and reversed picrotoxin and bicuculline-induced augmentation of somatostatin release in a dose-dependent manner. AllotetrahydroDOC also inhibits the stimulated release induced by the antagonists, but did not modify that induced by depolarizing concentrations of K+. Pregnenolone sulphate had no effect on picrotoxin-induced somatostatin release. These findings clearly establish that 3α-hydroxysteroids modulate GABA inhibition of hypothalamic somatostatin release.

Enhancement of Central Noradrenaline Release during Development Alters the Packing Density of Neurons in the Rat Occipital Cortex

The effect of chronic yohimbine treatment early in life on packing density of neurons was evaluated in the occipital cortex of young rats. Yohimbine administration to pups between days 5 and 16 of postnatal life (2.5 mg/kg/day i.p.) resulted at 45 days of age in significantly higher neuronal density in layers II-V of the occipital cortex, the effect being more marked in the dorsal region than in the dorsolateral and lateral ones. Results suggest a relationship between enhanced central noradrenaline activity and altered development of the neuropil in the occipital cortex.