Giuseppina Giusi

Some environmental contaminants influence motor and feeding behaviors in the ornate wrasse (Thalassoma pavo) via distinct cerebral histamine receptor subtypes.

Common environmental contaminants such as heavy metals and pesticides pose serious risks to behavioral and neuroendocrine functions of many aquatic organisms. In the present study, we show that the heavy metal cadmium and the pesticide endosulfan produce such effects through an interaction of specific cerebral histamine receptor subtypes in the teleost ornate wrasse (Thalassoma pavo). Treatment of this teleost with toxic cadmium levels for 1 week was sufficient to induce abnormal swimming movements, whereas reduced feeding behaviors were provoked predominantly by elevated endosulfan concentrations. In the brain, these environmental contaminants caused neuronal degeneration in cerebral targets such as the mesencephalon and hypothalamus, damage that appeared to correlate with altered binding levels of the three major histamine receptors (subtypes 1, 2, and 3). Although cadmium accounted for reduced binding activity of all three subtypes in most brain regions, it was subtype 2 that seemed to be its main target, as shown by a very great (p < 0.001) down-regulation in mesencephalic areas such as the stratum griseum central layer. Conversely, endosulfan provided very great and great (p < 0.01) up-regulating effects of subtype 3 and 1 levels, respectively, in preoptic-hypothalamic areas such as the medial part of the lateral tuberal nucleus, and in the suprachiasmatic nucleus. These results suggest that the neurotoxicant-dependent abnormal motor and feeding behaviors may well be tightly linked to binding activities of distinct histamine subtypes in localized brain regions of the Thalassoma pavo.

A sexually dimorphic distribution pattern of the novel estrogen receptor G-protein-coupled receptor 30 in some brain areas of the hamster

The isolation of the G-protein-coupled receptor 30 (GPR30), an orphan membrane receptor unrelated to nuclear estrogen receptors (ERs), has become a key factor towards the unraveling of rapid estrogen action. This membrane receptor together with cellular signaling intermediaries, i.e., extracellular signal-dependent kinases 1 and 2, may promote neuronal proliferation and differentiation activities. In the present study, an evident gene expression pattern of GPR30 characterized postnatal 7 (young) and 60 (adult) days of age hamsters as shown by its heterogeneous mRNA distribution in hypothalamic, amygdalar and cerebellar areas of both sexes. In particular, most of the brain areas considered in the adult hamster plus only the amygdala and cerebellum of young animals behaved in a sexually dimorphic fashion. This similar pattern was also detected for the ERalpha and beta, as shown by the latter receptor prevailing in young and adult females, while the former predominated in young females. Even for the two kinases, a sexually dimorphic distribution was featured above all for young hamsters. Overall, the findings of the present study established a distinct expression pattern of the novel ER (GPR30) that may operate differently in some brain areas of the hamster and this may provide interesting insights regarding its probable neuroprotective role during the execution of some hibernating states, which are typical of our rodent model.

Feeding behaviors and ORXR–β-GABAAR subunit interactions in Carassius auratus

Orexins are one of the most potent orexigenic factors in fish that through their interaction with the GABA(A) receptor system assures the successful execution of feeding, motor and sleep-wake activities. In the present study, the effects of ORX-A (10ng/g BW) very greatly enhanced (p<0.001) the time spent in feeding behaviors while at the same time moderately increased (p<0.05) food intake of the goldfish. It is worthy to note that the great variations of time spent in feeding behaviors induced by β GABA(A)R agonist (muscimol, MUS) and antagonist (bicuculline, BIC) did not result to be correlated to any significant variations of food intake. It was, however, a T-maze study allowing us to establish that learning and mnemonic events very likely also operated in an ORX-A+GABA(A)R-dependent fashion in our fish model. Indeed, animals conditioned by red/blue lights greatly reduced latency time in the presence of ORX-A while neither MUS nor BIC alone modified such a parameter, with the exception of ORX-A+MUS being responsible for a moderate decrease of latency time with respect to conditioned fish treated with a saline solution. Conversely, ORX-A+MUS/BIC seemed to interfere with ORX-A actions as shown by their very great increase in latency time. Moreover, T-maze results appeared to be strengthened by evident ORXR transcriptional variations especially by the very great mRNA densities detected in some telencephalic regions of animals treated with ORX-A. Of all telencephalic regions Dl, considered homologous to the mammalian hippocampus, proved to be a major target for ORX-A effects. Overall, these data suggest that it is mainly the ORXergic system that promotes feeding behaviors via reward pathways in teleost fish as in mammals. Surprisingly, β GABA(A)R drugs did not modify such behaviors when given alone while the inhibitory effect on cognitive/reward processes was evoked when given together with ORX-A, suggesting that more than β subunits other GABA(A)R subunits could be promoting mnemonically guided motor behaviors.

Distinct alpha subunits of the GABAA receptor are responsible for early hippocampal silent neuron-related activities.

The modulatory actions of GABAA receptor subunits are crucial for morphological and transcriptional neuronal activities. In this study, growth of hamster hippocampal neurons on biohybrid membrane substrates allowed us to show for the first time that the two major GABAA α receptor subunits (α2,5) are capable of early neuronal shaping plus expression differences of some of the main neuronal cytoskeletal factors (GAP‐43, the neurotrophin––BDNF) and of Gluergic subtypes. In a first case the inverse α5 agonist (RY‐080) seemed to account for the reduction of dendritic length at DIV7, very likely via lower BDNF levels. Conversely, the effects of the preferentially specific agonist for hippocampal α2 subunit (flunitrazepam) were, instead, directed at the formation of growth cones at DIV3 in the presence of greatly (P < 0.01) diminished GAP‐43 levels as displayed by strongly reduced axonal sprouting. It is interesting to note that concomitantly to these morphological variations, the transcription of some Gluergic receptor subtypes resulted to be altered. In particular, flunitrazepam was responsible for a distinctly rising expression of axonal NR1 mRNA levels from DIV3 (P < 0.01) until DIV7 (P < 0.001), whereas RY‐080 evoked a very great (P < 0.001) downregulation of dendritic GluR2 at only DIV7. Together, our results demonstrate that GABAA α2,5 receptor‐containing subunits by regulating the precise synchronization of cytoskeletal factors are considered key modulating neuronal elements of hippocampal morphological growth features. Moreover, the notable NR1 and GluR2 transcription differences promoted by these GABAA α subunits tend to favorably corroborate the early role of α2 + α5 on hippocampal neuronal networks in hibernating rodents through the recruitment and activation of silent neurons, and this may provide useful insights regarding molecular neurodegenerative events.

α GABAA subunit-orexin receptor interactions activate learning/motivational pathways in the goldfish

Orexins (ORXs) cross-talking with γ-aminobutyric acid(A) receptor (GABA(A)R) is beginning to constitute a key neuronal signaling feature responsible for the successful promotion of sleep-wake cycle, feeding and motor behaviors plus reward/motivational activities. In this work, ORX-A and the two α GABA(A)R agonists (zolpidem, ZOL; diazepam, DZP) accounted for very great (p<0.001) increases of feeding while only DZP elicited great (p<0.01) levels of food intake in the goldfish (Carassius auratus). It was, however, T-maze and conditioned place preference (CPP) methods that allowed us to specifically establish learning/reward-related events operating in an ORX-A+GABA(A)R-dependent fashion in our experimental model. T-maze data showed that conditioned ORX-A treated-fish were capable of reaching the red/blue chamber and ingesting their food reward in a very greatly reduced latency time with respect to untreated conditioned fish while DZP and ZOL greatly and moderately (p<0.05) reduced their latency time, respectively. Regarding CPP study, conditioned ORX-A- and DZP-treated animals showed comparably greater preferences for the conditioned compartment that became even greater in ORX-A+DZP-treated fish. Surprisingly, ORX receptor expression of the telencephalon was preferentially activated by ORX-A treatments while diencephalic/mesencephalic structures and namely the tuberculum posterioris (TPp) were more sensitive to DZP especially following treatment with ORX-A+DZP. Overall, behavioral performances along with ORX receptor transcriptional properties tend to point to α GABA(A)R agonists as enhancers of palatability while the ORXergic system constitutes a crucial link between satiety-related and cognitive centers through the activation of TPp thus proposing this ascending dopaminergic system as a key target of learning/reward processes in fish.

GABAergic influences on ORX receptor-dependent abnormal motor behaviors and neurodegenerative events in fish.

At date the major neuroreceptors i.e. gamma-aminobutyric acid(A) (GABA(A)R) and orexin (ORXR) systems are beginning to be linked to homeostasis, neuroendocrine and emotional states. In this study, intraperitoneal treatment of the marine teleost Thalassoma pavo with the highly selective GABA(A)R agonist (muscimol, MUS; 0.1 microg/g body weight) and/or its antagonist bicuculline (BIC; 1 microg/g body weight) have corroborated a GABA(A)ergic role on motor behaviors. In particular, MUS induced moderate (p<0.05) and great (p<0.01) increases of swimming towards food sources and resting states after 24 (1 dose) and 96 (4 doses) h treatment sessions, respectively, when compared to controls. Conversely, BIC caused a very strong (p<0.001) reduction of the former behavior and in some cases convulsive swimming. From the correlation of BIC-dependent behavioral changes to neuronal morphological and ORXR transcriptional variations, it appeared that the disinhibitory action of GABA(A)R was very likely responsible for very strong and strong ORXR mRNA reductions in cerebellum valvula and torus longitudinalis, respectively. Moreover these effects were linked to evident ultra-structural changes such as shrunken cell membranes and loss of cytoplasmic architecture. In contrast, MUS supplied a very low, if any, argyrophilic reaction in hypothalamic and mesencephalic regions plus a scarce level of ultra-structural damages. Interestingly, combined administrations of MUS+BIC were not related to consistent damages, aside mild neuronal alterations in motor-related areas such as optic tectum. Overall it is tempting to suggest, for the first time, a neuroprotective role of GABA(A)R inhibitory actions against the overexcitatory ORXR-dependent neurodegeneration and consequently abnormal swimming events in fish.

Aestivation and hypoxia-related events share common silent neuron trafficking processes

BackgroundThe availability of oxygen is a limiting factor for neuronal survival since low levels account not only for the impairment of physiological activities such as sleep-wake cycle, but above all for ischemic-like neurodegenerative disorders. In an attempt to improve our knowledge concerning the type of molecular mechanisms operating during stressful states like those of hypoxic conditions, attention was focused on eventual transcriptional alterations of some key AMPAergic silent neuronal receptor subtypes (GluR1 and GluR2) along with HSPs and HIF-1α during either a normoxic or a hypoxic aestivation of a typical aquatic aestivator, i.e. the lungfish (Protopterus annectens).ResultsThe identification of partial nucleotide fragments codifying for both AMPA receptor subtypes in Protopterus annectens displayed a putative high degree of similarity to that of not only fish but also to those of amphibians, birds and mammals. qPCR and in situ hybridization supplied a very high (p < 0.001) reduction of GluR1 mRNA expression in diencephalic areas after 6 months of aerial normoxic aestivation (6mAE). Concomitantly, high (p < 0.01) levels of HSP70 mRNAs in hypothalamic, mesencephalic and cerebellar areas of both 6mAE and after 6 months of mud hypoxic aestivation (6mMUD) were detected together with evident apoptotic signals. Surprisingly, very high levels of GluR2 mRNAs were instead detected in thalamic along with mesencephalic areas after 6 days of normoxic (6dAE) and hypoxic (6dMUD) aestivation. Moreover, even short- and long-term hypoxic states featured high levels of HIF-1α and HSP27 transcripts in the different brain regions of the lungfish.ConclusionsThe distinct transcriptional variations of silent neurons expressing GluR1/2 and HSPs tend to corroborate these factors as determining elements for the physiological success of normoxic and hypoxic aestivation. A distinct switching among these AMPA receptor subtypes during aestivation highlights new potential adaptive strategies operating in key brain regions of the lungfish in relation to oxygen availability. This functional relationship might have therapeutic bearings for hypoxia-related dysfunctions, above all in view of recently identified silent neuron-dependent motor activity ameliorations in mammals.

Effects of the xenoestrogen bisphenol A in diencephalic regions of the teleost fish Coris julis occur preferentially via distinct somatostatin receptor subtypes

The xenoestrogen bisphenol A, a contaminant used in the manufacturing of polymers for many consumer products, has been shown to mimic estrogenic actions. This xenoestrogen regulates secretion and expression of pituitary lactotrophs plus morphological and structural features of estrogen target tissues in rodents. Recently, ecological hazards produced by bisphenol A have drawn interests towards the effects of this environmental chemical on neurobiological functions of aquatic vertebrates of which little is known. In this study, the effects of bisphenol A on the distribution of the biologically more active somatostatin receptor subtypes in diencephalic regions of the teleost fish Coris julis were assessed using nonpeptide agonists (L-779, 976 and L-817, 818) that are highly selective for subtype(2) and subtype(5), respectively. Bisphenol A proved to be responsible for highly significant increased binding levels of subtype(2) in hypothalamic areas, while markedly decreased levels of subtype(5) were found in these diencephalic areas, as well as in the medial preglomerular nucleus. The extensive distribution of somatostatin receptor subtype(2) and subtype(5) in the teleost diencephalic areas suggests that, like in mammals, this receptor system may not only be involved in enhanced hypophysiotropic neurohormonal functions but might also promote neuroplasticity events.

PAN hollow fiber membranes elicit functional hippocampal neuronal network.

This study focuses on the development of an advanced in vitro biohybrid culture model system based on the use of hollow fibre membranes (HFMs) and hippocampal neurons in order to promote the formation of a high density neuronal network. Polyacrylonitrile (PAN) and modified polyetheretherketone (PEEK-WC) membranes were prepared in hollow fibre configuration. The morphological and metabolic behaviour of hippocampal neurons cultured on PAN HF membranes were compared with those cultured on PEEK-WC HF. The differences of cell behaviour between HFMs were evidenced by the morphometric analysis in terms of axon length and also by the investigation of metabolic activity in terms of neurotrophin secretion. These findings suggested that PAN HFMs induced the in vitro reconstruction of very highly functional and complex neuronal networks. Thus, these biomaterials could potentially be used for the in vitro realization of a functional hippocampal tissue analogue for the study of neurobiological functions and/or neurodegenerative diseases.

Flat and tubular membrane systems for the reconstruction of hippocampal neuronal network.

The selection of appropriate biomaterials that promote cellular adhesion and growth is particularly important for the in vitro reconstruction of neuronal network. This study focused on the development of new polymeric membranes in flat and tubular (hollow‐fibre) configurations as novel biomaterials for neuronal outgrowth. Two membrane systems constituted by modified polyetheretherketone (PEEK‐WC) and polyacrylonitrile (PAN) membranes were developed and used for the culture of hamster hippocampal neurons. We demonstrated that all investigated membranes supported the adhesion and growth of hippocampal neurons enhancing neuronal differentiation and neurite alignment. The differences in cell behaviours between cells cultured on flat and hollow‐fibre (HF) membranes were highlighted by the quantitative analysis of neuronal marker fluorescence intensity, morphometric analysis, RT–PCR analysis and also by metabolic activity measurements. In particular, the PAN HF membranes showed ideal growth culture conditions, guaranteeing adequate levels of metabolic features. Primary hippocampal cells cultured on PAN HF membranes were able to recreate in vitro a 3D neural tissue‐like structure that, mimicking the hippocampal tissue, could be used as a tool for the study of natural and pathological neurobiological events. Copyright