The endocrine disruptor atrazine accounts for a dimorphic somatostatinergic neuronal expression pattern in mice.

It has now been established that a large number of man-made and natural chemicals are capable of interfering with the action of natural hormones. In this category "endocrine disruptors" such as the herbicide atrazine, when administered at ecological low doses (1 or 100 microg/kg per day) from gestational day 14 to postnatal day 21, provided a clear dimorphic neurodegenerative pattern in some brain areas of the domestic mouse (Mus musculus). Indeed, the high concentration (100 microg/kg per day) with respect to the low concentration (1 microg/kg per day) induced relevant neuronal damage in extrahypothalamic sites, such as the cortical and striatal areas in both sexes. Marked alterations in other areas, including the hippocampal and hypothalamic nuclei, were mostly typical of the female. At the neuronal level, the neuropeptide somatostatin, specific for the secretion of growth hormone, seemed to be a major target of atrazine effects, as demonstrated by evident subtype2,3,5 receptor mRNA differences of this neuropeptide, at least for the first two subtypes. In particular, a very strong (p < 0.001) upregulation of subtype2 expressing neurons was detected in female hypothalamic areas, specifically the suprachiasmatic nucleus, whereas a similar downregulatory trend was reported for some extrahypothalamic areas such as the striatum. Interestingly, very strong upregulatory and downregulatory actions were detected for neurons expressing subtype3 in male hypothalamic and amygdalar regions and in the cortical and hippocampal areas, respectively. Overall, it appears that these first neurotoxicological effects of atrazine are very likely linked to dimorphic expression patterns of specific somatostatin subtypes in discrete but key hypothalamic and extrahypothalamic areas of Mus musculus.

Dimorphic distribution of the two main GABA(A) binding sites in cortical and limbic areas of a rodent living in natural environmental conditions.

Labeling of the two more important gamma-aminobutyric acidA (GABA(A)) supramolecular sites with [3H] muscimol (GABA(A)) and [3H] flunitrazepam (benzodiazepine) provided saturable, stable, and dimorphic binding activities in cortical and limbic regions of the wood mouse Apodemus sylvaticus. Of the cortical layers, which contained the highest [3H] muscimol binding levels, only the female lamina V supplied a greater (51%; P <0.01) receptor density than in the male. Areas of the limbic system instead proved to be the more favorable targets for differential GABA(A) binding levels. The highest (P <0.001) and higher levels were found in the oriens-pyramidalis CA1 layer of the hippocampus (65%) and in the vertical limb diagonal band-medial septal nucleus (48%), basolateral amygdala nucleus (45%), and ventromedial hypothalamic nucleus (43%), respectively, of the female. A similar pattern was obtained for [3H]flunitrazepam binding activity, especially in the presence of GABA. The highest and higher binding activities were obtained in the female central amygdala nucleus (78%) and in the ventromedial hypothalamic nucleus (52%), basolateral amygdala nucleus (48%), and oriens-pyramidalis CA1 layer of the hippocampus (47%), respectively, whereas higher levels were observed only in the male vertical limb diagonal band-medial septal nucleus (56%). Even in the cortical regions, the female exhibited higher (42%; cortex lamina V) and moderately higher (38%; cortex lamina VI) levels, with binding differences in the latter site plus in the basolateral amygdala nucleus occurring in a GABA-nondependent manner. From the saturation binding analyses it was possible to reveal that both maximal number of binding sites (Bmax) and mean dissociation constant (K(D)) modifications were responsible for receptor differences of the two GABAergic sites. These findings tend to suggest that dimorphic variations of the GABA(A) supramolecular sites, in some cortical and limbic regions, are strongly involved in sex-specific aggressive and reproductive activities of rodents living in their natural habitats.

Gonadal-GABAergic interaction is an important factor involved in photoperiod-induced 2-[125I] iodomelatonin binding changes in the Japanese quail brain.

The type of mechanism(s) by which melatonin alone and/or through the intervention of other putative neurotransmitters is able to control circadian rhythms remains unresolved. Comparison of 2-[125I]iodomelatonin binding pattern in the brain of castrated and gonadally intact Japanese quail (Coturnix japonica), using quantitative receptor autoradiography, displayed that the combination of the intact gonadal condition and a long-day (16L:8D) photostimulatory schedule is responsible for major binding changes. In fact, high and low binding levels were obtained in the suprachiasmatic area and nucleus ectomamillaris (p < 0.01) and in the nucleus preopticus anterior and paleostriatum primitivum (p < 0.001), respectively. A gonadal modulatory role was not always evident in all brain areas as revealed by long-day photic cycles producing diminished (p < 0.01) binding levels in the anterior neostriatum and the nucleus rotundus of both castrated and gonadally intact animals, although elevated values were also found in the substantia grisea centralis (p < 0.05) of the same animals. Saturation binding studies revealed that gonadal and/or photic effects induce alterations in the number of binding sites, whereas the affinity constant varied only in some hypothalamic sites. Testing of GABAergic activity on 2-[125I] iodomelatonin binding levels showed that this inhibitory neurotransmitter was responsible for increasing low receptor values. Moreover, GABA-dependent influences were shown to be mediated via a GABAA receptor subtype since bicuculline (specific antagonist of this site) inhibited the elevated GABA-induced melatonin binding levels in the above brain sites of the gonadally intact quail exposed to both photoperiod cycles. Even in this case, melatonin binding changes were due to the variations in the number of binding sites. The apparent GABAergic-gonadal influence resulting in changes of the 2-[125I] iodomelatonin binding values, under the different photic conditions, provides evidences of other probable neural mechanisms that entrain circadian rhythmicity in neuroendocrine activities and in sociosexual behaviors.

Sexual dimorphism of GABAA receptor levels in subcortical brain regions of a woodland rodent (Apodemus sylvaticus).

This is the first report of quantitative autoradiography results showing sex differences of GABAA receptor levels in brain regions of a wild rodent (wood mouse, Apodemus sylvaticus) living in its natural habitat. The labeling of this GABAergic site with its specific high affinity radioligand [3H] muscimol provided a heterogeneous and dimorphic binding pattern in some of the neural centers. In the female, higher (> or = 50 < or = 65%) to moderately higher (< 50%) binding levels than in the male, even after correction of the specific binding values using the calculated quenching coefficients, were observed in the substantia nigra pars reticulata and ventral lateral thalamic nucleus, brain centers that are relays of motor circuits. In the male, on the other hand, a higher level was only obtained in the caudateputamen. Relays of the stria terminalis-hypothalamic-central gray pathway such as the bed nucleus of the stria terminalis, the pontine central gray and the ventromedial hypothalamic nucleus, were among the other female brain areas with an extremely higher (> 65%) to higher and moderately higher binding activity than in the male. From the saturation analyses, it appeared that the binding differences were mainly due to Bmax variations, although closer examinations revealed that changes in the KD might have also accounted for [3H] muscimol binding differences, as shown by the high KD and Bmax values in the bed nucleus of the stria terminalis, the substantia nigra pars reticulata and the pontine central gray of the female wood mouse. These findings suggest that the dimorphic binding activity of GABAA receptors in the above brain regions might be involved in neuronal circuitry mechanisms related to sex-specific social behaviors in rodents living in their natural environmental conditions.

Effects of N omega-nitro-L-arginine methyl ester on benzodiazepine binding in some limbic areas of hyperlipidaemic rats.

Quantitative autoradiography techniques were used to evaluate the chronic effects of the potent nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester, on the binding pattern of [3H]flunitrazepam (benzodiazepine agonist) in some behaviorally key limbic areas of the genetic hyperlipidaemic Pittsburg Yoshida rat. Administration of this potent synthase inhibitor was capable of supplying higher and moderately higher binding levels in the basolateral amygdala nucleus (+52%) and in the oriens-pyramidalis CA1 hippocampus layer (+38%), respectively. When we tested for the binding changes in the presence of GABA (principal benzodiazepine modulator) we noticed that a physiological concentration (20 microM) of this inhibitory neurotransmitter was sufficient to induce notable changes in other limbic areas. In fact, lower binding values (-65%) were reported for the bed nucleus of stria terminalis whereas moderately higher values (+38%) were obtained for the radiatum-lacunosum molecular CA1 hippocampus layer. From the saturation studies, it was possible to observe that the major receptor variations provoked by the potent synthase inhibitor were not only due to changes in the total number of binding sites because there were variations, as in the case of the basolateral amygdala nucleus, that were instead due to differences in the affinity binding state. These results provide evidences of a GABAergic-nitric oxide synthase inhibitor interaction that might also be involved in the regulation of convulsive, anxiolytic, and aggressive behaviors that are modulated at the benzodiazepine site.

Antihypertensive and neuroprotective effects of catestatin in spontaneously hypertensive rats: interaction with GABAergic transmission in amygdala and brainstem.

The chromogranin A-derived peptide catestatin (CST) exerts sympathoexcitatory and hypertensive effects when microinjected into the rostral ventrolateral medulla (RVLM: excitatory output); it exhibits sympathoinhibitory and antihypertensive effects when microinjected into the caudal ventrolateral medulla (CVLM: inhibitory output) of vagotomized normotensive rats. Here, continuous infusion of CST into the central amygdalar nucleus (CeA) of spontaneously hypertensive rats (SHRs) for 15 days resulted in a marked decrease of blood pressure (BP) in 6-month- (by 37 mm Hg) and 9-month- (by 65 mm Hg)old rats. Whole-cell patch-clamp recordings on pyramidal CeA neurons revealed that CST increased both spontaneous inhibitory postsynaptic current (sIPSC) amplitude plus frequency, along with reductions of sIPSC rise time and decay time. Inhibition of GABAA receptors (GABAARs) by bicuculline completely abolished CST-induced sIPSC, corroborating that CST signals occur through this major neuroreceptor complex. Hypertension is a major risk factor for cerebrovascular diseases, leading to vascular dementia and neurodegeneration. We found a marked neurodegeneration in the amygdala and brainstem of 9-month-old SHRs, while CST and the GABAAR agonist Muscimol provided significant neuroprotection. Enhanced phosphorylation of Akt and ERK accounted for these neuroprotective effects through anti-inflammatory and anti-apoptotic activities. Overall our results point to CST exerting potent antihypertensive and neuroprotective effects plausibly via a GABAergic output, which constitute a novel therapeutic measure to correct defects in blood flow control in disorders such as stroke and Alzheimer's disease.

Brain excitatory/inhibitory circuits cross-talking with chromogranin A during hypertensive and hibernating states.

To date, many scientific attempts have been directed towards the development of experimental models for the identification of neuronal mechanisms evoking cardiovascular and hemodynamic dysfunctions. The spontaneously hypertensive rat (SHR), a genetic model of essential hypertension, has become a valuable rodent for the characterization of molecular markers in hypertensive-related diseases. Recently, growing interests have also been directed to a new experimental paradigm i.e. hibernation, a physiological state consenting the hamster (Mesocricetus auratus) to activate protective mechanisms against ischemic-like complications during the arousal phase. With this intention, the present review will focus attention on specific neurosignaling systems involved with the preservation of hemodynamic conditions in those brain areas that play a pivotal role on such a feature. It is widely known that healthy neurons conserve their structural and responsiveness properties in presence of a constant blood supply, which is assured by their coupling to microvessels and perivascular astrocytes as well as by secretory proteins such as chromogranin A (CgA). So, it will be interesting to establish if this protein alone or with the participation of excitatory/inhibitory neurosignals is capable of influencing some brain areas controlling cardiovascular conditions in both SHRs and hibernating hamsters. In this context, the present work will deliver the most important findings regarding neuronal CgA and its cross-talking ability with major inhibitory (GABA/adenosine) and/or excitatory (glutamate) neuroreceptor systems in relation to hypertensive/hypotensive states of both animal models. Indications deriving from such approaches may provide clinically useful insights regarding their role as protective factors of hemodynamic and neurological disorders.

Combined gonadal and photic influences on 2-[125I] iodomelatonin-binding level changes in some brain areas of the quail.

Quantitative receptor autoradiography was used to study the binding of 2-[125I] iodomelatonin in the brain of the castrated and gonadally intact male Japanese quail Coturnix japonica exposed to both long- and short-day photoperiod cycles. The distribution study displayed that these conditions were responsible for a heterogeneous binding pattern as shown by elevated receptor levels being located in visual brain centers, such as the stratum opticum, nucleus pretectalis, and nucleus geniculatus lateralis, pars ventralis, while lower values were found in the nucleus lateralis hypothalami and nucleus isthmi pars magnocellularis. Closer examination of the 2-[125I] iodomelatonin-binding pattern following the different gonadal and photic influences showed that combination of the gonadally intact condition and a 16L:8D (long-day) photoperiod cycle was required for the greater binding changes. These differences occurred in brain sites such as the area preoptica, paleostriatum primitivum, and nucleus ectomamillaris. Saturation binding studies, which were carried out only in some of the above areas, revealed that the combined gonadal- and photic-induced changes are basically due to the modifications of total number of binding sites. The importance of a gonadal steroid modulatory role in the photic-dependent melatonin binding activity suggests that other types of neuronal mechanisms might be involved in the regulation of neuroendocrine and socio-sexual behaviors in nonmammalian vertebrates.

Dimorphic features of the different alpha-containing GABA-A receptor subtypes in the cortico-basal ganglia system of two distantly related mammals (hedgehog and rat)

This investigation represents a first study dealing with the dimorphic differences of the main alpha-containing gamma-aminobutyric acid (GABA(A)) receptors in the brain of two distantly related mammals (hedgehog and rat). The labeling of these receptors in the presence of zolpidem (highly selective benzodiazepine agonist) and under the different degree of GABA(A)/benzodiazepine allosteric coupling activities accounted for a heterogeneous colocalization of alpha1-enriched and of alpha2/3-enriched and alpha5-enriched GABA(A) receptors in some areas of the cortico-basal ganglia system (including the important ventrolateral thalamic station) of both mammalian sexes. In the hedgehog, the greatest (P<0.001) GABA-dependent reduction of zolpidem inhibition constants was mostly registered in alpha1- and/or alpha5-enriched areas, such as the frontoparietal cortex lamina III (235%), ventrolateral thalamic nucleus (128%), and substantia nigra pars reticulata (110%) of the male. However, the greatest reductions in the rat were instead detected in the male substantia nigra pars reticulata (192%) and female striatum (120%), areas which are enriched either by the colocalization of alpha1- with alpha2/3-subunits or by all three alpha-subunits. These results support the contention that a sex-related alpha-containing GABA(A) receptor sensitivity constitutes an important element in the execution of skilled motor activities during the different socio-sexual behaviors of the two mammals.

Morphological and functional variations of Leydig cells in testis of the domestic pig during the different biological stages of development.

The relationship of morphometrical and androgen receptor evaluations of the main testicular interstitium cellular element (Leydig cells) in the domestic pig provided interesting numerical and morphological features during the different aging stages. As early as 25 days (a period in which the pig is sexually immature) there was a low number of Leydig cells (1.46 x 10(8)) with respect to a 78% and 35% increase in the adult (2.48 x 108) and aged (1.78 x 10(8)) animal, respectively. Interestingly, when the volume density of Leydig cells was considered, the average volume of these cells seemed to be high (75%) in the aged pig with respect to the young immature animal whereas a lower increase (27%) was observed for the adult animal. Moreover, the evaluation of testosterone receptor binding sites in the testis at the various stages of development also displayed a differentiated pattern since elevated testosterone receptor binding levels of the high dissociation affinity type were obtained for the adult pig. Thus, from the combined morphological variations of Leydig cells and testosterone receptor binding activity, it appears that this androgenic receptor component exerts distinct autocrine effects on the different functional features of some testicular tissue constituents at the different aging stages of the domestic pig.