Localization of cholecystokinin in the zebrafish retina from larval to adult stage.

The peptide hormone cholecistokinin (CCK) plays a key role in the central and peripheral nervous system. It is known to be involved in the digestive physiology and in the regulation of food intake. Moreover, the CCK expression has also been detected in the retina of different vertebrates, including fish, although its biological activity in this tissue remains to be elucidated. In literature no data are yet available about the CCK-immunoreactivity in the zebrafish retina during development. Therefore, the aim of the study was to investigate the distribution of sulfated cholecystokinin octapeptide (CCK8-S) as a well preserved form during evolution in the zebrafish retina from 3days post hatching (dph) until adult stage, using immunohistochemistry in order to elucidate the potential role of this protein in the development and maintenance of normal retinal homeostasis. The cellular distribution of CCK in the retina was similar from 3 dph to 40days post fertilization (dpf) when immunoreactivity was found in the photoreceptors layer, in the outer plexiform layer, in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer (GCL). Immunohistochemical localization at 50 dpf as well as in the adult stage was observed in a subpopulation of amacrine cells in the proximal inner nuclear layer, in the inner plexiform layer, in displaced amacrine cells and in retinal ganglion cells in the GCL. Our results demonstrate for the first time the occurrence of CCK in the zebrafish retina from larval to adult stage with a different pattern of distribution, suggesting different roles of CCK during retinal cells maturation.

Lack of interaction of endocannabinoids and 5-HT(3) neurotransmission in associative fear circuits of the amygdala: evidence from electrophysiological and behavioural experiments.

Both the serotonergic and the endocannabinoid system play a major role in mediating fear and anxiety. In the basolateral amygdala (BLA) it has been shown that the cannabinoid receptor 1 (CB1) is highly co-expressed with 5-HT3 receptors on GABAergic interneurons suggesting that 5-HT3 receptor activity modulates CB1-mediated effects on inhibitory synaptic transmission. In the present study, we investigated the possible interactions of CB1 and 5-HT3-mediated neuronal processes in the BLA using electrophysiological and behavioural approaches. Whole-cell patch-clamp recordings were performed in coronal brain slices of mice. Electric stimuli were delivered to the lateral amygdala to evoke GABAA receptor-mediated inhibitory postsynaptic currents (GABAA-eIPSCs) in the BLA. The induction of LTDi, a CB1-mediated depression of inhibitory synaptic transmission, was neither affected by the 5-HT3 antagonists ondansetron (OND; 20 µM) and tropisetron (Trop; 50 nM) nor by the 5-HT3 agonists SR57227A (10 µM). In auditory fear conditioning tests, mice treated with SR57227A (3.0mg/kg i.p.) showed sustained freezing, whereas treatment with Trop (1.0 mg/kg i.p.) decreased the expression of conditioned fear. These effects were overruled by the CB1 antagonist rimonabant (RIM; 3.0 mg/kg), which caused increased freezing with or without co-treatment with Trop. In summary, these experiments do not support a functional interaction between CB1 and 5-HT3 receptors at the level of GABA neurotransmission in the BLA nor in terms of fear regulation.

Prolonged fear incubation leads to generalized avoidance behavior in mice.

Long-lasting presence of avoidance and emotional numbing are reliable behavioral markers for PTSD, but little is known about its psychological and biological underpinnings. We employed our recently established mouse model of PTSD (i) to study the emergence of avoidance behavior in the aftermath of a trauma, (ii) to disentangle the impact of context generalization vs. lack of motivation vs. novelty fear and (iii) to assess the therapeutic value of benzodiazepines and selective serotonin reuptake inhibitors (SSRIs). Specific conditioned avoidance to shock-paired odor turned into generalized avoidance after 28 days of fear incubation. Combination of habituation to the novel environment and extinction of contextual fear abolished both generalized and specific avoidance behavior. Chronic fluoxetine treatment partially reversed the phenotype, whereas acute treatment with diazepam did not. Our animal model may help understanding the mechanisms underlying psychological and biological mechanisms of PTSD for the benefit of developing pharmacotherapeutic strategies, which specifically address generalized avoidance.

Fine structure of spermatozoa in the blackspot sea bream Pagellus bogaraveo (Brünnich, 1768) with some considerations about the centriolar complex.

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Pagellus bogaraveo. The spermatozoon of P. bogaraveo belongs, like that of the other sparid fish, to the teleostean "type I" spermatozoon with the flagellar axis insert perpendicular to the nuclear fossa. It has an ovoidal head, a short, cylindrically shaped midpiece and a long tail region. The nucleus reveals a deep invagination (nuclear fossa), in which the centriolar complex is located, and a satellite nuclear notch shaped like a golf club. The two centrioles are perpendicular to each other and show a conventional "9+0" pattern. The distal centriole is attached to the nuclear envelope by means of basal feet and radial fibers made of electron-dense material. Below the basal plate, plasma membrane pinches in, and the necklace, a specialized connection joining axonemal doublets to the plasma membrane, is visible. The short midpiece houses one mitochondrion. The flagellum is perpendicularly and eccentrically with respect to the nucleus and contains the conventional "9+2" axoneme.

Physiological responses to starvation in the European eel (Anguilla anguilla): effects on haematological, biochemical, non-specific immune parameters and skin structures.

The physiological effects of short-term starvation on some haematological, biochemical and non-specific immune response parameters together with the histological structure of the skin, were investigated in the European eel, Anguilla anguilla. Blood haemoglobin and haematocrit, serum glucose and cortisol, hemolysins, haemagglutinins, and lysozyme in the plasma, kidney and epidermal extract, were measured in fish after 31, 42 and 58 days of starvation, and compared to those of fed fish. Starvation did not affect haemoglobin and haematocrit values, while an increase in glucose and cortisol levels was found in starved eels by day 42. Haemolytic and haemagglutinating activities decreased in starved eels. On the other hand, starvation caused an increase in the lysozyme content in the epidermal extracts, while no significant variations were observed in kidney and plasma. On the whole, no major changes in metabolic, haematological and non-specific immune parameters were observed when short-term (less than 2 months) starvation was applied to the European eel, suggesting an adaptive response to starvation, rather than a typical alarm-stress response, allowing this species to withstand food deprivation.

Rice protein-concentrate meal as a potential dietary ingredient in practical diets for blackspot seabream Pagellus bogaraveo: a histological and enzymatic investigation.

Field and laboratory studies were conducted to evaluate the intestinal responses to partial replacement of fish meal with rice protein concentrate (RPC) in practical diets for blackspot seabream Pagellus bogaraveo. Two experimental diets were formulated to be isoproteic and isoenergetic with an increasing level of RPC (20 and 35%, respectively) and were tested against a fish meal-based control diet (RPC0). The diets showed similar features for growth performances and both intestinal histology and digestive enzymes. This study confirmed that RPC does not induce intestinal mucosa alterations in this fish. The dietary RPC supplement caused a significant increase in trypsin activity, whereas lipase activity was reduced.

Ontogeny of the digestive tract in sharpsnout sea bream Diplodus puntazzo (Cetti, 1777).

The ontogeny of the digestive tract was studied histologically and histochemically in sharpsnout sea bream Diplodus puntazzo from hatching (0 DAH, Days After Hatching) until day 57 (57 DAH). At hatching, the digestive tract appeared as a histologically undifferentiated straight tube lying dorsally to the yolk sac. When the mouth opened at 3 DAH, the digestive tract was differentiated into buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage and both the bile and pancreatic duct had opened into the anterior intestine. Active feeding began in 50% of larvae at 4 DAH, although permanence of yolk reserves until 7 DAH suggests a period of both endogenous and exogenous feeding. Nutrient absorption was first visible from 5 DAH, as colourless supra- and infranuclear vacuoles in the anterior intestinal mucosa, suggesting a lipid content, as well as supranuclear, eosinophilic vacuoles, containing protein, in the posterior intestinal mucosa. Early caecal development could be detected from 10 DAH, whereas gastric glands appeared at 30 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion. Goblet cells appeared in the digestive tract of sharpsnout sea bream larvae shortly after first feeding. The mucus content of goblet cells varied with the digestive region and, in the buccal cavity and oesophagus, also with the developmental phase. This study provides knowledge for better husbandry practices in the aquaculture industry, as well as for the implementation of future nutritional studies.

Rice protein concentrate meal as potential dietary ingredient in practical diets for blackspot seabream (Pagellus bogaraveo).

The aim of this study was to evaluate the performances and the flesh quality of Pagellus bogaraveo fed with diets containing rice protein concentrate [RPC, 70% crude protein (CP) and 10% ether extract]. Three isoproteic and isoenergetic (CP 47%, 22 MJ/kg DM) diets were formulated with an increasing level of RPC: 0%, 20% and 35%. The fish (mean weight 75 g) from the Messina Straits were randomly distributed in 12 tanks (3 diets x 4 replications, 10 fish/tank). The daily ratio (1.5% of the fish biomass) was updated every 15 days. Biomass gain showed an opposite trend to the RPC diet inclusion. No differences appeared in the somatic indexes. Differences appeared between fatty acid profiles of the dorsal muscle. Fatty acid of series n-6 increased and fatty acid of series n-3 decreased in fillets of fish fed with increasing levels of RPC. The inclusion of RPC in the diets, as a partial replacement of fish meal (20%), is possible without affecting the growth performance and fillet quality.

Fine structure of spermatozoa in the gilthead sea bream (Sparus aurata Linnaeus, 1758) (Perciformes, Sparidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Sparus aurata L. The mature spermatozoon of gilthead sea bream belongs, like that of the other sparid fish, to a "type I" as defined by Mattei (1970). It has a spherical head which lacks an acrosome, a short, irregularly-shaped midpiece and a long cylindrical tail. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The two centrioles are approximately perpendicular to each other and show a conventional "9+0" pattern. The proximal centriole is associated with a cross-striated cylindrical body lying inside a peculiar satellite nuclear notch which appears as a narrow invagination of the nuclear fossa. The distal centriole is attached to the nuclear envelope by means of a lateral plate and radial fibres made of an electron-dense material. The short midpiece houses one mitochondrion. The flagellum is inserted perpendicularly into the base of the nucleus and contains the conventional 9+2 axoneme.

Endocannabinoids and beta-amyloid-induced neurotoxicity in vivo: effect of pharmacological elevation of endocannabinoid levels.

We investigated the involvement of endocannabinoids in the control of neuronal damage and memory retention loss in rodents treated with the beta-amyloid peptide (1-42) (BAP). Twelve days after stereotaxic injection of BAP into the rat cortex, and concomitant with the appearance in the hippocampus of markers of neuronal damage, 2-arachidonoyl glycerol, but not anandamide, levels were enhanced in the hippocampus. VDM-11 (5 mg/kg, i.p.), an inhibitor of endocannabinoid cellular reuptake, significantly enhanced rat hippocampal and mouse brain endocannabinoid levels when administered sub-chronically starting either 3 or 7 days after BAP injection and until the 12-14th day. VDM-11 concomitantly reversed hippocampal damage in rats, and loss of memory retention in the passive avoidance test in mice, but only when administered from the 3rd day after BAP injection. We suggest that early, as opposed to late, pharmacological enhancement of brain endocannabinoid levels might protect against beta-amyloid neurotoxicity and its consequences.

Dopaminergic drugs may counteract behavioral and biochemical changes induced by models of brain injury.

The dopaminergic drugs, bromocriptine, cabergoline, dihydroergocryptine, pergolide and ropinirole were injected subcutaneously (s.c.) at the dose of 0.1, 0.5 and 1 mg/kg/day for 7 days into male rats of the Sprague-Dawley strain. The drug pre-treatment reverted amnesia induced in rats by hypobaric hypopxia and tested in active and passive avoidance tasks. A restoration of memory retention, as assessed in a step-through passive avoidance task, was found in animals with a 2-month brain occlusive ischemia and exposed to dopaminergic drugs for 7 days. For behavioral effects in both active and passive avoidance tests in both experimental models, the rank of relative potency was ropirinole>bromocriptine=cabergoline>pergolide>dihydroergocryptine. Spontaneous ambulation of animals with brain occlusive ischemia was increased by the higher doses of drugs. All dopaminergic drugs reduced kainate mortality rate. The rank of relative potency for this effect was ropirinole=bromocriptine=cabergoline>pergolide=dihydroergocryptine. However, no change was found in other seizure parameters (latency to first convulsion and total number of convulsions) after drug treatment. A biochemical analysis of glutathione redox index (glutathione reduced/glutathione oxidized ratio) in discrete brain areas revealed that exposure to dopaminergic drugs increased this parameter in frontal cortex, striatum and hippocampus of animals subject to hypobaric hypoxia and brain occlusive ischemia. For this effect, the relative potency rank was ropirinole>bromocriptine=cabergoline>>pergolide=dihydroergocryptine. These behavioral and biochemical findings suggest that dopaminergic drugs may counteract either behavioral or biochemical changes induced by experimental models of brain injury. This activity was found after protective activity (as found in animals pre-treated with these drugs and exposed to hypobaric hypoxia) or reversal of brain injury (as found in animals treated after 2-month occlusive brain ischemia). Their neuroprotective activity probably involves the reduction/oxidation balance of the glutathione system in the brain.

Fine structure of spermatozoa in the common pandora (Pagellus erythrinus Linnaeus, 1758) (Perciformes, Sparidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the Sparid fish Pagellus erythrinus L. The spermatozoon of pandora has a spherical head lacking an acrosome, a cone-shaped midpiece and a long tail. The midpiece houses a single mitochondrion. The centriolar complex lies inside the nuclear fossa and is composed of a proximal and a distal centriole which are arranged at right angles to each other. The flagellum is inserted medio-laterally into the head, contains the conventional 9+2 axoneme and possesses one pair of lateral fins. On the basis of its ultrastructural organization, the pandora sperm can be regarded as an evolved form of the primitive spermatozoon found in Teleosts. According to the morphological classification proposed by Mattei (1970), the sperm of pandora belongs to a "type I" designation, like that of the other Sparid fish.

Placebo affects the performance of rats in models of depression: is it a good control for behavioral experiments?

Experimental design of behavioral studies in animals generally includes placebo-treated controls. However, when placebo is administered by injection in experimental models of psychiatric diseases such as depression, where stress may affect the execution of the behavioral test, it is possible that injection per se may influence the behavioral response. Rats injected with clomipramine hydrochloride (1, 10 or 50 mg/kg), as compared to animals injected with physiological saline as placebo, showed a dose-dependent decrease of the immobility time in the despair test and of the number of floor units explored in the open field in the reserpine test. However, when animals injected with placebo or clomipramine 50 mg/kg were compared with untreated intact controls, it was found that the immobility time in the despair test was higher in the placebo-treated animals than in untreated intact controls. A difference was found between clomipramine-injected animals and untreated intact controls. In contrast, rats tested in the reserpine test, which is based on repeated drug injections, no difference was found between placebo-treated animals and untreated intact controls. These results indicate that stressful procedure of the experimental design may change the response of animals in behavioral tests. Studies with experimental models of depression, where stressful procedures are used, should include a control group of untreated intact animals.

Dual effects of melatonin on barbiturate-induced narcosis in rats.

Melatonin affects the circadian sleep/wake cycle, but it is not clear whether it may influence drug-induced narcosis. Sodium thiopenthal was administered intraperitoneally into male rats pre-treated with melatonin (0.05, 0.5, 5 and 50 mg/kg). Melatonin pre-treatment affected in a dual manner barbiturate narcosis, however, no dose-effect correlation was found. In particular, low doses reduced the latency to and prolonged the duration of barbiturate narcosis. In contrast, the highest dose of melatonin (50 mg/kg) caused a paradoxical increase in the latency and produced a sustained reduction of the duration of narcosis, and a reduction in mortality rate. Melatonin 0.5 and 5 mg/kg influenced the duration but not the latency of ketamine- or diazepam-induced narcosis. Thus, the dual action of melatonin on pharmacological narcosis seems to be specific for the barbiturate mechanism of action.