Yaacov Egozi

Inhibition of dopamine release by melatonin: regional distribution in the rat brain

Dopamine release evoked by electrical field stimulation of slices from various regions of rat brain was assessed in the presence of 10-10-10-5 M melatonin. Inhibition of dopamine release by melatonin was observed in the ventral hippocampus, medulla pons, preoptic area and median and posterior hypothalamus. No inhibitory effect of melatonin on dopamine release was observed in the cerebral cortex, cerebellum, dorsal hippocampus and striatum. Equal concentrations of melatonin were needed to produce half-maximal inhibition in all the regions affected. The results indicate that the brain sites for inhibitory effect of melatonin on dopamine neurosecretion overlap the sites reportedly involved in its modulation of neuroendocrine functions.

Agonist and antagonist binding to rat brain muscarinic receptors: Influence of aging

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [3H]-antagonist as well as of the labeled natural neurotransmitter, [3H]-acetylcholine [( 3H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [3H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [3H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [3H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [3H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.

Studies on Muscarinic Receptors in Mouse and Rat Hypothalamus: A Comparison of Sex and Cyclical Differences

The ligand binding properties of muscarinic receptors present in whole hypothalamus as well as in its three regions (preoptic area, median and posterior regions) were studied in male rats and mice, as well as in female rats during various stages of the estrous cycle, using the tritiated antagonist N-methyl-4-piperidyl benzilate. Kinetic and equilibrium analysis of antagonist binding as well as competition experiments with agonists, were used to probe the nature of the binding sites and possible differences between specific muscarinic sites in the various preparations. We could detect differences in agonist binding parameters between male and female rats in the preoptic area. In female rats binding of agonist to high affinity state is characterized by a lower affinity (27 nM) than in males (3.7 nM). Secondly, the population of agonist high affinity sites at the proestrous stage is much higher than that at other stages of cycle (66 vs. 38%). In addition, the binding properties of muscarinic receptors following intracisternal 6-hydroxydopamine lesion were investigated. This treatment resulted in a decrease of about one sixth of the total muscarinic receptors in the hypothalamus. The data are best interpreted as a degeneration of existing presynaptic muscarinic receptors located on catecholamine terminals in the hypothalamus. The pre- and postsynaptic nature of the muscarinic receptors, their localization in view of their binding properties, and their possible physiological role are discussed.

Inhibition by Melatonin of Dopamine Release from Rat Hypothalamus in vitro: Variations with Sex and the Estrous Cycle

The inhibitory effect of melatonin on evoked dopamine release from whole hypothalamus as well as from its three regions (preoptic area, median and posterior hypothalamus) was studied in male rats and in female rats during various stages of the estrous cycle. Inhibition of dopamine release by 1 microM melatonin was significantly higher at the estrous and lower at the diestrous than at the metestrous and proestrous stages. The variations in inhibition of dopamine release during the estrous cycle occurred predominantly in the preoptic area, and were nonsignificant in the other hypothalamic regions. In male rats, a constant level of inhibition was observed. In addition, the concentration of melatonin needed to produce half maximal effect in the female shifted from approximately 0.6 nM at the estrous and metestrous stages to more than 1 microM at the proestrous and diestrous stages. The corresponding concentration in male rats was of the order of 1 microM, and did not change.

Biochemical Characterization and Sex Dimorphism of Muscarinic Receptors in Rat Adenohypophysis

The ligand binding properties of muscarinic receptors present in rat adenohypophysis were studied using the tritiated antagonists N-methyl-4-piperidyl benzilate, 3-quinuclidinyl benzilate, and methylscopolamine. Equilibrium analysis of antagonists binding as well as competition experiments with several agonists were used to probe the nature of binding sites. The nature of antagonist binding in the adenohypophysis points to heterogeneity of binding sites, in contrast to other brain regions, in which homogeneous populations of muscarinic receptors were detected. Some of the binding properties, e.g. affinity constants, population of high affinity agonist binding sites, etc., clearly indicated differences between male and female rats as well as differences in female rats at various stages of estrous cycle. The nature of these receptors and their possible physiological role are discussed.

Regional heterogeneity of muscarinic receptors of mouse brain

Labelled and non-labelled compounds are the same as in [1-3] . Direct binding studies, competition experiments and the analysis of binding kinetics were carried out as detailed in [1-3] . Choline acetyltransferase and cholinesterase activities were determined according to [4,5]. Male 1CR mice (20-25 g) were decapitated, brains were rapidly removed and the brain areas were dissected in a cold room after identification with the aid of [6]. After dissection, brain regions (from 15-25 mice for the thalamus and 10-15 mice for other regions) were homogenized in ice-cold 0.32 M sucrose using a motor-driven Teflon pestle (950 rev./min) in a glass homogenizer to yield a 10% homogenate (w/v). Specific binding is defined as the total minus the non-specific, i.e., binding in the presence of 5 X 10-s M of unlabelled ligand.

Muscarinic Mechanisms and Sex Hormone Secretion in Rat Adenohypophysis and Preoptic Area

The highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate [(3H)-4NMPB] was used in direct binding and in competition experiments with the muscarinic agonist oxotremorine to investigate the influence of in vivo endocrine manipulations involving estrogen on muscarinic receptors in the adenohypophysis and hypothalamus. In the adenohypophysis the characteristics of antagonist binding in ovariectomized female rats, like those in androgenized females, resembled those in normal male rats; this trend was reversed in ovariectomized females after implantation of 17 beta-estradiol capsules, with the characteristics of antagonist binding now resembling those in normal female rats at estrus. Agonist binding characteristics also showed some distinct differences between treated and normal female rats: the proportion of high affinity binding sites decreased both in ovariectomized and androgenized rats, while the affinity of these sites became greater as compared to control animals. The results clearly show that changes in the levels of sex steroids, as a result of the above-mentioned endocrine manipulations, influence the behavior of the muscarinic receptors in both areas, and suggest muscarinic participation in the regulation of gonadotropin release in the hypothalamus-pituitary-ovary axis.

Muscarinic involvement in the regulation of gonadotropin-releasing hormone in the cyclic rat

The release of gonadotropin-releasing hormone (GnRH) from the median eminence (ME) in cyclic rats was stimulated to a significant extent by the selective muscarinic antagonists 11[(2)(diethylamino)methyl][-1-piperidinyl]-acetyl-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine, and to a lesser extent also by other ligands selectively antagonistic to m1 and m3 receptors. Such stimulation was estrous-cycle-dependent and was not achieved by muscarinic agonists. We suggest that the effect is induced via the m4 receptor subtype. Attempts to block the muscarinic-antagonist-induced stimulation of GnRH release with a variety of drugs were successful only in the presence of prazosin, an antagonist to alpha 1-adrenergic receptors. One possible explanation for this muscarinically mediated stimulation of GnRH release is that it results from cross-talk between the muscarinic and the alpha 1-adrenergic receptors, i.e., muscarinic agonists might inhibit the release induced by alpha 1-agonists, and muscarinic antagonists, by cancelling this inhibitory effect, might thus allow the endogenous alpha 1-agent, norepinephrine, to induce the release of GnRH.

Effect of Ca2+ on the Binding Characteristics of Muscarinic Receptors in Rat Adenohypophysis – Variation During the Estrous Cycle

The effect of Ca2+ on the biochemical characteristics of muscarinic receptors in the adenohypophysis of male and female rats at the various stages of the estrous cycle was investigated in binding experiments using the specific muscarinic antagonist N-methyl-4-piperidyl benzylate ( [3H]-4NMPB) and the muscarinic agonist oxotremorine. By using Ca2+ chelators such as EGTA, and Ca2+ channel blockers such as D-600, we showed that Ca2+ profoundly alters the binding characteristics of both antagonists and agonists to the muscarinic receptors. In female rats the effect of Ca2+ on antagonist binding is mainly on the maximal binding capacity of the receptors, while changes in the dissociation constants are much more moderate. The effect is expressed in the ability of Ca2+ to expose or to eliminate binding sites as a function of the estrous cycle. In agonist binding, the presence of Ca2+ has a pronounced effect on the proportion of high-affinity binding sites, which parallels the changes induced in antagonist binding throughout the estrous cycle. Interestingly, the natural progression of the cycle from diestrus 2 to the estrous stage undergoes a change identical to that occurring in vitro upon Ca2+ removal. D-600 can completely block the effect of Ca2+ on the binding of both [3H]-4NMPB and oxotremorine. The concentration of D-600 required in order to induce such blocking is also dependent on the estrous cycle. It appears that the progression of the estrous cycle is accompanied by changes in the muscarinic receptors which may in turn be coupled to Ca2+ channels.