Giuseppe Paino

The weaver mutant mouse: a model to study the ontogeny of dopamine transmission systems and their role in drug addiction

Dopaminergic neurons and their projection-systems are important in some fundamental human activities like locomotion, feeding and sex, essential for survival and procreation, and are relevant to pathologies like Parkinsons disease and drug abuse. Three main dopaminergic projection-systems, namely the nigrostriatal, mesocortical and mesolimbic pathways are the major targets of the neuropharmacological actions of psychomotor stimulants such as cocaine and amphetamine. Studies on knockout mice for dopamine or its receptors provide substantial information but fail to reveal the role of individual dopaminergic projection-systems. Mutant animals with defects specific to one or more projection-systems might be useful for studying the role of individual dopaminergic projection-systems. We propose the weaver mutant mouse, with a defective nigrostriatal dopaminergic projection-system and dopamine depletion in the dorsal striatum but with intact mesocorticolimbic projection-systems, as a suitable model to study the role of individual dopaminergic systems in diverse biological processes including Parkinsons disease and drug abuse.

Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity and mRNA expression in the duck gastrointestinal tract

Abstract. The presence and distribution of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity were studied in the duck gastrointestinal tract using immunohistochemistry and radioimmunoassays. Expression and distribution of PACAP mRNA were also studied using reverse transcriptase polymerase chain reaction (RT-PCR) and hybridization techniques. In addition, a partial coding sequence (cds) of the duck growth hormone-releasing hormone (GRF)/PACAP gene was identified. The presence of both PACAP-38 and PACAP-27 was demonstrated, the former being the predominant form. PACAP immunoreactivity was found in neurons and fibers of the enteric nervous system (ENS), in endocrine cells and in the gut associated lymphoid tissue (GALT). Double immunostaining showed that PACAP is almost completely colocalized with vasoactive intestinal peptide (VIP) in the ENS. Moreover, PACAP was also found in nitric oxide synthase (NOS)-containing neurons and nerve fibers. Radioimmunoassay (RIA) performed on denervated gut showed that more than one-half of the duodenal PACAP is extrinsic in origin. RT-PCR, Northern blot analysis and in situ hybridization confirmed the immunohistochemical data. The findings of the present study suggest that, in birds, PACAP may have multiple roles in regulating gastrointestinal functions.

Innervation of vas deferens and accessory male genital glands in the water buffalo (Bubalus bubalis). Neurochemical characteristics and relationships to the reproductive activity.

Autonomic nerves supplying mammalian male internal genital organs have an important role in the regulation of reproductive function. To find out the relationships between the neurochemical content of these nerves and the reproductive activity, we performed a histochemical and immunohistochemical study in a species, the water buffalo, exhibiting a seasonal sexual behaviour. The distribution of noradrenergic and nitric oxide synthase (NOS)- and peptide-containing nerves was evaluated during the mating and non-mating periods. Fresh segments of vas deferens and accessory genital glands were collected immediately after slaughter and immersed in 4% paraformaldehyde. Frozen sections were obtained and processed according to single and double labelling immunofluorescent procedures or NADPH-diaphorase histochemistry. During the mating period, a dense noradrenergic innervation was observed to supply the vas deferens as well as the accessory genital glands. NOS- and peptide-containing nerves were also observed but with a lower density. During the non-mating period noradrenergic nerves dramatically reduced. In addition, neuropeptide Y (NPY)- and vasoactive intestinal peptide (VIP)-containing nerves were also reduced. These findings suggest the presence of complex interactions between androgen hormones and the autonomic nerve supply in the regulation of male water buffalo reproductive functions.

Postnatal development of intestinal endocrine cell populations in the water buffalo

The frequency and distribution of 11 endocrine cell populations were studied in the intestine of differently aged buffalo, grouped on the basis of diet: 2‐d‐olds (suckling), 5‐mo‐olds (weaning) and 5‐y‐olds (ruminant adult diet). The endocrine cell populations were identified immunocytochemically using antisera against 5‐hydroxytryptamine (5‐HT), somatostatin, gastrin, cholecystokinin (CCK), COOH‐terminal octapeptide of gastrin/CCK, neurotensin, motilin, gastric inhibitory polypeptide (GIP), secretin, glucagon/glicentin (GLU/GLI) and polypeptide YY (PYY). In adult buffalos the regional distribution of endocrine cells is similar to that of other adult ruminants. During postnatal development, these cell types showed the following changes in their frequency and distribution: (1) 5‐HT, neurotensin and gastrin/CCK immunoreactive cells (i.c.) showed a decrease in frequency with age; (2) somatostatin i.c. frequency remained stable with age; (3) motilin, GIP, secretin and CCK i.c. showed a slight increase in frequency with age; (4) GLU/GLI and PYY i.c. decreased in frequency with age in the small intestine, caecum and proximal colon and an increase in frequency in the rectum. It was hypothesised that the endocrine cell types, whose presence and localisation is substantially stable in all examined ages, probably contain substances that are strictly necessary for intestinal function. In contrast the hormones contained in the cell populations that decreased with age, are probably involved in physiological needs during the milk and weaning diet or play a role in intestinal growth.

Expression of agouti-related protein (AgRP) in the hypothalamus and adrenal gland of the duck ( Anas platyrhynchos )

The presence and distribution of agouti-related protein (AgRP) immunoreactivity were investigated in the hypothalamus and adrenal gland of the duck using immunohistochemistry and Western blot analysis. Expression of AgRP mRNA was also studied using reverse transcriptase polymerase chain reaction (RT-PCR). A partial coding sequence (cds) of the duck AgRP gene was identified. Western blot analysis showed the presence of an AgRP-like peptide having a molecular weight consistent with the number of predicted amino acids of the avian AgRP. In the hypothalamus, AgRP immunoreactivity was found in neurons of the nucleus infundibularis and in fibers projecting to the median eminence. In the adrenals, AgRP immunoreactivity was observed in medullary cells. These findings suggest that in the duck, AgRP may play a role in regulating energy homeostasis and adrenal endocrine functions.

Effects of castration on the expression of brain-derived neurotrophic factor (BDNF) in the vas deferens and male accessory genital glands of the rat

Brain-derived neurotrophic factor (BDNF) is a growth factor belonging to the family of neurotrophins. Although neurotrophins in the male genital organs have been well documented, their role in the biology of these organs is far from clear. In particular, little is known about the influence of sex hormones on neurotrophin expression. In the present study, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), we investigated the distribution and tissue concentration of BDNF in the vas deferens and accessory male genital glands in normal and castrated rats. The expression of BDNF mRNA was also investigated. In normal rats, BDNF immunoreactivity was localized in the musculature of the vas deferens and vesicular gland and in the fibromuscular stromal cells of the prostate. In the ventral prostatic lobes, BDNF immunoreactivity was localized in basal, secretory and neuroendocrine epithelial cells. Innervating ganglia and nerves were immunoreactive in all the examined tracts. After castration, BDNF immunoreactivity increased in the musculature of the vesicular gland and in the fibromuscular stromal cells of both dorsal and ventral prostatic lobes. BDNF immunoreactivity also increased in the nerves. ELISA and reverse transcription/real-time polymerase chain reaction confirmed the findings of the immunohistochemical study. In the accessory glands, castration induced an increase of both BDNF tissue concentration and mRNA expression. These results suggest that BDNF is expressed in the internal male genital organs of the rat and that its expression is downregulated by androgen hormones. We hypothesize that the observed BDNF increases are related to the castration-induced regression of the sympathetic nerves.

Chronic morphine alters calbindin D-28k immunostaining patterns in mouse forebrain

The influence of chronic morphine treatment on the brain of adult mouse has been studied. Female Swiss mice were daily administered saline or morphine (30 or 60 mg/kg body weight) for a period comprising 7 days before mating, during gestation and until 21 days post-partum. Their brains were then perfusion-fixed and examined for histology and calbindin D-28k protein-immunoreactivity. Histological observations revealed no significant changes in the various brain regions; whereas a reduced number of calbindin-positive cells was encountered in the cingulate and parietal cortices and the lateral septal regions of morphine-treated brains compared with those of controls. The alteration in the expression-patterns of this neuroprotective calcium-binding protein in specific regions of the adult brain might be one of the mechanisms by which the addictive drugs modify the functional aspects of the CNS.

Maternal morphine alters parvalbumin immunoreactivity patterns in neonatal mouse brain

The influence of chronic maternal morphine on the parvalbumin immunoreactive patterns in developing mouse brain was studied. Female Swiss mice were administered daily saline or morphine (30 or 60 mg/kg) for a period of 7 days before mating, gestation, and 21 days postpartum. Their pups were sacrificed on postnatal day 18 and the brains were examined histologically and immunohistochemically for parvalbumin‐positive neurons. Histological observations revealed no significant changes in the cell number of the morphine‐exposed neonatal forebrain, whereas the number of parvalbumin‐positive neurons increased in layers II–IV of the parietal cortex I. Moreover, the number of parvalbumin‐positive dendrites increased remarkably in the cingulate and parietal I cortices of the morphine‐exposed neonates, indicating the region‐specific increase in the PV immunoreactive profiles. These results are consistent with the key roles played by the above brain regions in the altered behavioral patterns of the maternally addicted neonates, such as impaired somatosensory and cognitive performances. The mechanism of morphine action on parvalbumin expression in neonatal mouse brain is not evident, but alterations in the expression patterns of parvalbumin in specific regions of the developing brain might be one of the cellular mechanisms by which addictive drugs modify the functional aspects of the developing CNS. Synapse 35:265–271, 2000.

The distribution and colocalisation of nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-/nitric oxide synthase (NOS)-containing neurons in the innervation of the duck ureter.

The distribution and colocalisation of nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-/nitric oxide synthase (NOS)-containing (nitrergic) neurons in the innervation of the duck ureter have been studied using histochemistry and immunohistochemistry. Quantitative analysis showed that nitrergic neurons made up 60% and 70% of the total intramural and adventitial neuronal populations, respectively. About 40% of intramural nitrergic neurons expressed VIP-immunoreactivity, and about 75% of nitrergic adventitial neurons expressed TH-immunoreactivity. The density of nitrergic adventitial neurons was significantly greater in the lower tract than in the upper and intermediate tracts. Nerve lesioning experiments showed that the majority of ureteral nitrergic innervation was extrinsic in origin; nitrergic adventitial neurons primarily projected caudocranially, whereas NOS-immunoreactive and NOS-/VIP-immunoreactive intramural neurons primarily projected craniocaudally. These findings suggest that, in birds, the nitrergic innervation plays a role in ureteral functions such as epithelial mucosecretion, muscular motility, and the closing and/or opening of the ureteral papilla.

The vascularization of the Bursa cloacalis (of Fabricius) in the duck

The morphological and structural features of the vascular component of the bursa of Fabricius in the duck are described. By means of an intravasal perfusion of suitably colored neoprene Latex or Microscopaque, or a perfusion of Indian ink, it was possible to follow visually the routes of arteries, veins and the microcirculation; this last represents a major characteristic of the bursal follicle in the duck. The microcirculation has its origins in several roots arising from the follicular arterioles, which in the region of the cortex constitute an open vascular layer. From these pre‐capillary arterioles, adjacent to the limiting layer between cortex and medulla, a dense capillary network is formed. With the aid of an electron microscope the presence of a cortico‐medullary barrier of an epithelio‐reticular nature was reveled; close to it there are the terminal branches of the intrafollicular network. The barrier appears to be structurally more compact than that of the cortical capillaries of the mammalian thymus.