Stefano Gotti

Abstract: Trisomy 21, also referred to Down syndrome (DS), is the most common genetic cause of mental retardation, affecting 1 each 800-1000 newborn children all over the world. DS is a complex disease, determined by an extra copy of human chromosome 21 that causes an imbalanced gene dose effect. The syntenies that exist between mouse chromosomes 10, 16, and 17 and human chromosome 21 offer the opportunity for a genotype-phenotype correlation and several mouse models of DS have been developed to improve our knowledge about cognitive disabilities and brain alterations. We present here the different murine models available up to now and we discuss the neural alterations that have been described in these strains. The largest amount of studies involved the so called Ts65Dn mouse showing early alterations of nitrergic, noradrenergic and cholinergic systems at the level of the basal forebrain. Neurogenesis and spine formations are decreased in the hippocampus, as well as the whole size of the cerebellum and the number of granule cells.

Abstract: Endocrine-disrupting chemicals (EDC) are molecules that interfere with endocrine signaling pathways and produce adverse consequences on animal and human physiology, such as infertility or behavioral alterations. Some EDC act through binding to androgen or/and estrogen receptors primarily operating through a genomic mechanism regulating gene expression. This mechanism of action may induce profound developmental adverse effects, and the major targets of the EDC action are the gene products, i.e., mRNAs inducing the synthesis of various peptidic molecules, which include neuropeptides and enzymes related to neurotransmitters syntheses. Available immunohistochemical data on some of the systems that are affected by EDC in lower and higher vertebrates are detailed in this review.

Abstract: In the present study we used a transgenic mouse model, carrying the neuropeptide Y (NPY) Y1 receptor gene promoter linked to the LacZ reporter gene (Y1R/LacZ mice) to test the hypothesis of its up-regulation by gonadal hormones. Y1 receptor gene expression was detected by means of histochemical procedures and quantitative image analysis in the paraventricular nucleus, arcuate nucleus, medial preoptic nucleus, ventromedial nucleus and bed nucleus of stria terminalis of two-month-old female mice at different stages of estrous cycle. Qualitative and quantitative analyses showed that Y1R/LacZ transgene expression was higher in the paraventricular, arcuate, and ventromedial nuclei of proestrus mice as compared to mice in the other stages of the estrous cycle. In addition, we performed a comparison with a group of sexually active males. In this comparison a significant difference (less in males) was observed between males and proestrus females in the same nuclei. In conclusion, these data indicate that fluctuations in circulating levels of gonadal hormones, depending by estrous cycle, are paralleled by changes in the expression of NPY Y1 receptor in the hypothalamic nuclei involved in the control of both energy balance and reproduction.

Abstract: Nitric oxide (NO)-containing neurons are widely distributed within the central nervous system, including regions involved in the control of reproduction and sexual behavior. Nitrergic neurons may co-localize with gonadal hormone receptors and gonadal hormones may influence neuronal NO synthase expression in adulhood as well as during development. In rodents, the female, in physiological conditions, is exposed to short-term changes of gonadal hormones levels (estrous cycle). Our studies, performed in mouse hypothalamic and limbic systems, reveal that the expression of neuronal NO synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This is in accordance with the hypothesis that gonadal hormone activation of NO-cGMP pathway is important for mating behavior. NO-producing system appears particularly sensitive to alterations of endocrine balance during development, as demonstrated by our experiments utilizing perinatal exposure to bisphenol A, an endocrine disrupting chemical. In fact, significant effects were detected in adulthood in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis. Therefore, alteration of the neuronal NO synthase expression may be one of the causes of the important behavioral alterations observed in bisphenol-exposed animals.

Abstract: Bisphenol A (BPA) is a well-known plastic-derived pollutant that can bind to oestrogen receptors and is considered an endocrine-disrupting chemical. Its impact on different behaviours in rodents has been largely investigated, however, only a few data are available on its effects upon neural circuits. In the present study, we investigated the long-term effects of early exposure of mice of both sexes to BPA on the nitrinergic system, one of the neural systems involved in the control of sexual behaviour and under the control of gonadal hormones. Mice of both sexes were exposed for eight prenatal and eight postnatal days to BPA that was administered to the mothers. The maternally-exposed mice were sacrificed at the age of 2 months and their brains were sectioned and immunohistochemically treated for the detection of neuronal nitric oxide synthase (nNOS). Significant effects of BPA exposure were detected for the number of immunoreactive cells in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis, in a sex-oriented and dose-dependent way. These results indicate that BPA has a powerful effect on specific portions of the nNOS-immunoreactive system belonging to the accessory olfactory system that are particularly important for the control of sexual behaviour. In addition, they confirm that perinatal exposure to endocrine-disrupting chemicals, in particular to BPA, may have a high impact on the organisation of specific neural pathways that can later affect complex behaviours and functions.

Abstract: Connective tissue grafts are routinely procedures in the treatment of gingival defects. The clinical success of the gingival tissue graft procedures anyway should ensure not only the aesthetic integration between the tissues but also the physiological activity of the graft in terms of sensitivity and immunity because the skin and the mucosae constitute the first natural aspecific borders against pathogens. The aim of this paper was to investigate nervous net recovery after connective graft procedure, in relation with sensorial alteration in the injured area. Results showed that there is a close link among the number of Merkel cells and the alteration of sensations. Merkel cells can be found isolated standing in the basal layer, supposed to have neuroendocrine functions in the epithelia or in larger group not associated with nerves; when found in association with nerves they are named Merkel complexes, acting as slow adapter mechanical receptor. Our data can be explained in two ways: Merkel cells increase as a consequence of tissue injury, a sort of "SOS cells" that secrete neuroendocrine signals to guide tissue healing; as an alternative the presence of the Merkel cells could be read as a derailment of tissue regeneration with the stop of cellular differentiation in the direction of an abnormal proliferation, a sort of mad stem cell.

Abstract: Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei including the hippocampus, where some neurons express estrogen receptors. Nitric oxide plays a wide range of functions in the nervous system generally by acting as a neurotransmitter-like molecule. It has been demonstrated that long-term treatments with estradiol in ovariectomized females and with testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in rat hypothalamus, whereas changes in nNOS immunoreactivity or in associated NADPH-diaphorase activity were observed both in hypothalamus and in amygdala during different phases of estrous cycle. Estradiol could induce nNOS expression in several brain regions in rodents. Therefore, to clarify if the hippocampal NO producing system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity on two-month-old intact female mice. Immunoreactive cells were observed in all hippocampal subregions: the higher number was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate gyrus. The number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant in CA1, CA2 and CA3 regions. These results suggest that the nitrinergic system is a target for estrogen action in the hippocampus, and that this action may take place in physiological conditions according to the short-term variations of gonadal hormones during the estrous cycle.

Abstract: Several environmental chemicals have the capability of impacting endocrine function (endocrine disrupting chemicals [EDCs]), and therefore they may have long-term consequences, especially if exposure occurs during embryonic development. In this study we present data relative to two widely used animal models: the Japanese quail and the mouse. These two species have been used to understand neural, neuroendocrine, and behavioral components of reproduction and are optimal models to understand how these components are altered by precocious exposure to EDCs. In particular, we discuss the effects of embryonic exposure to diethylstilbestrol, genistein, or ethylene,1,1-dichloro-2,2-bis(p-chlorophenyl) on the sexually dimorphic parvocellular vasotocin system and male copulatory behavior in quail and the effects of bisphenol A on the nitrinergic and kisspeptin systems and their behavioral impact in the mouse. In both models the exposure to EDCs during the critical period (early embryonic period in birds, perinatal period in rodents) alters the differentiation of relevant sexually dimorphic pathways, often inducing the appearance of a sex-reversed neurochemical phenotype that is the most probable cause of the final alteration of sexually differentiated behaviors in the adult animal. In conclusion, the data presented here should stimulate a critical reanalysis of the way to determine the "safe" exposure levels to EDCs for wild species and humans, considering behavior and related neural circuits among the factors to be analyzed.

Abstract: Several environmental chemicals have the capability of impacting endocrine function (endocrine disrupting chemicals [EDCs]), and therefore they may have long-term consequences, especially if exposure occurs during embryonic development. In this study we present data relative to two widely used animal models: the Japanese quail and the mouse. These two species have been used to understand neural, neuroendocrine, and behavioral components of reproduction and are optimal models to understand how these components are altered by precocious exposure to EDCs. In particular, we discuss the effects of embryonic exposure to diethylstilbestrol, genistein, or ethylene,1,1-dichloro-2,2-bis(p-chlorophenyl) on the sexually dimorphic parvocellular vasotocin system and male copulatory behavior in quail and the effects of bisphenol A on the nitrinergic and kisspeptin systems and their behavioral impact in the mouse. In both models the exposure to EDCs during the critical period (early embryonic period in birds, perinatal period in rodents) alters the differentiation of relevant sexually dimorphic pathways, often inducing the appearance of a sex-reversed neurochemical phenotype that is the most probable cause of the final alteration of sexually differentiated behaviors in the adult animal. In conclusion, the data presented here should stimulate a critical reanalysis of the way to determine the "safe" exposure levels to EDCs for wild species and humans, considering behavior and related neural circuits among the factors to be analyzed.

Abstract: A satisfactory management to ensure a full restoration of peripheral nerve after trauma is not yet available. Using an experimental protocol, in which crush injury was applied 1 cm above the bifurcation of the rat sciatic nerve for 20 s, we here demonstrate that the levels of neuroactive steroids, such as pregnenolone and progesterone (P) metabolites (i.e. dihydroprogesterone, DHP, and tetrahydroprogesterone, THP) present in injured sciatic nerve were significantly decreased. On this basis, we have focused our attention on DHP and its direct precursor, P, analyzing whether these two neuroactive steroids may have neuroprotective effects on biochemical, functional and morphological alterations occurring during crush-induced degeneration-regeneration. We demonstrate that DHP and/or P counteract biochemical alterations (i.e. myelin proteins and Na(+),K(+)-ATPase pump) and stimulate reelin gene expression. These two neuroactive steroids also counteract nociception impairment, and DHP treatment significantly decreases the up-regulation of myelinated fibers' density occurring in crushed animals. Altogether, these observations suggest that DHP and P (i.e. two neuroactive steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury.

Abstract: Nitric oxide-containing neurons are widely distributed within the CNS, including regions involved in the control of reproduction and sexual behavior. The expression of neuronal nitric oxide synthase is influenced by testosterone in male rat, and by estrogens in female. Moreover, nitric oxide synthase may co-localize with gonadal hormones' receptors. Gonadal hormones may influence nitric oxide synthase expression in adulthood as well as during the development. In fact, in mice knockout for estrogen receptor alpha, the nitric oxide synthase-expressing population is deeply reduced in specific regions. In physiological conditions, the female in mammalian species is exposed to short-term changes of gonadal hormones levels (estrous cycle). Our recent studies, performed in the rat vomeronasal system and in mouse hypothalamic and limbic systems reveal that, in rodents, the expression of nitric oxide synthase-producing elements within regions relevant for the control of sexual behavior is under the control of gonadal hormones. The expression of nitric oxide synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This seems in accordance with the hypothesis that gonadal hormone activation of nitric oxide-cyclic guanosine-monophosphate pathway is important for lordosis behavior, as well as that this system is activated during mating behavior. Finally, comparative data available for other vertebrates suggest that class-specific and species-specific differences occur in the nitric oxide synthase system of hypothalamus and limbic structures. Therefore, particular caution is needed to generalize data obtained from studies in rodents.

Abstract: Nitric oxide (NO) is a gaseous intercellular messenger with a wide range of neural functions. NO is synthesized by activation of different isoforms of nitric oxide synthases (NOS). At present NOS immunoreactivity has been described in mouse brain in restricted and definite areas and no detailed mapping studies have yet been reported for NOS immunoreactivity. We have studied the distribution of neuronal NOS-containing neurons in the brain of three months male mice, using a specific commercial polyclonal antibody against the neuronal isoform of nitric oxide synthase (nNOS). Neuronal cell bodies exhibiting nNOS immunoreactivity were found in several distinct nuclei throughout the brain. The neurons that were positively stained exhibited different intensities of reaction. In some brain areas (i.e., cortex, striatum, tegmental nuclei) neurons were intensely stained in a Golgi-like fashion. In other regions, immunoreactive cells are moderately stained (i.e., magnocellular nucleus of the posterior commissure, amygdaloid nucleus, interpeduncular nucleus, lateral periaqueductal gray) or weakly stained (i.e., vascular organ of the lamina terminalis, hippocampus, inferior colliculus, reticular nucleus). In the mouse, the NO-producing system appears well developed and widely diffused. In particular, nNOS immunoreactive neurons seem chiefly present in several sensory pathways like all the nuclei of the olfactory system, as well as in many regions of the lymbic system. These data suggest a widespread role for the NO system in the mouse nervous system.

Abstract: Ts65Dn mice have been developed as a model for Down syndrome (DS). Because of its involvement in complex behaviors, including sexual and aggressive behaviors, we investigated the nitric oxide (NO) system in specific brain regions of these mutant mice (TS) after isolation-induced aggression. Male TS mice displayed significantly higher aggression than wild type (WT) mice and the comparison of the NO system, both with immunohistochemical and histochemical methods, resulted in robust differences between TS and WT mice in the hypothalamic paraventricular nucleus, in the nucleus of the diagonal band and in the medial septum, but not in the striatum of TS mice. In conclusion, we document alterations in the neuronal NO system of the TS mouse model of DS, suggesting a correlation of the behavioral aggressiveness with deficient NO production.