Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ) are linked to hibernating state in hamsters

<p>Abstract</p> <p>Background</p> <p>The structural arrangement of the γ-aminobutyric acid type A receptor (GABA_AR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, <it>in vitro </it>quantitative autoradiography and <it>in situ </it>hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABA_AR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators.</p> <p>Results</p> <p>Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α₁ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α₂subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α₄expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (β and γ), elevated β₃and γ₃mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β₃and γ₂during hibernating conditions.</p> <p>Conclusion</p> <p>We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABA_AR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.</p

HSP90 and pCREB alterations are linked to mancozeb-dependent behavioral and neurodegenerative effects in a marine teleost

The pesticide mancozeb (mz) is recognized as a potent inducer of oxidative stress due to its ability to catalyze the production of reactive oxygen species plus inhibiting mitochondrial respiration thus becoming an environmental risk for neurodegenerative diseases. Despite numerous toxicological studies on mz have been directed to mammals, attention on marine fish is still lacking. Thus, it was our intention to evaluate neurobehavioral activities of ornate wrasses (Thalassoma pavo) exposed to 0.2mg/l of mz after a preliminary screening test (0.07-0.3mg/l). Treated fish exhibited an evident (p1000%) while exploratory attitudes (total arm entries) diminished (-50%; p<0.05) versus controls during spontaneous exploration tests. Moreover, they showed evident enhancements (+111%) of immobility in the cylinder test. Contextually, strong (-88%; p<0.01) reductions of permanence in light zone of the Light/Dark apparatus along with diminished crossings (-65%) were also detected. Conversely, wrasses displayed evident enhancements (160%) of risk assessment consisting of fast entries in the dark side of this apparatus. From a molecular point of view, a notable activation (p<0.005) of the brain transcription factor pCREB occurred during mz-exposure. Similarly, in situ hybridization supplied increased HSP90 mRNAs in most brain areas such as the lateral part of the dorsal telencephalon (Dl; +68%) and valvula of the cerebellum (VCe; +35%) that also revealed evident argyrophilic signals. Overall, these first indications suggest a possible protective role of the early biomarkers pCREB and HSP90 against fish toxicit

Overstimulation of glutamate signaling in hamster hippocampal neurons: what’s new?

It is known that ischemic complications arise from neuronal and glial dysfunctions occurring in almost all brain areas. Within some neuronal networks, an early excitatory/inhibitory circuit imbalance tends to account for premature neuronal damages especially during the initial stages of perinatal development. Interestingly, cellular conditions reported in ischemia were also detected during the different phases of hibernation cycle and above all arousal state. Hibernating animals are able to survive under these conditions without neurological damage, so their neuronal circuits present an opportunity to investigate molecular strategies involved in mammalian cell survival under unfavorable conditions. We reported a contextual alterations of both ionotropic and metabotropic Glutamatergic systems in perinatal hippocampal neurons in response to ischemic-like condition, according to their early activation during neuronal development (Giusi et al., 2009; Di Vito et a.l, 2012). In addition, an altered expression was also reported for specific PSD scaffold proteins, which regulate Glutamate receptors targeting (Al-Hallaq et al., 2007). From our preliminary results, we can suggest that specific alterations of glutamatergic receptors, which differ significantly from those reported in other rodent, could play a major role toward the correction of neuronal development aberrations linked to clinical disorders

Ruolo neuroprotettivo del sistema istaminergico e delle HSPs nella risposta allo stress ambientale nell’encefalo del Teleosteo Thalassoma pavo

Dottorato di Ricerca in Biologia Animale, XIX CicloAt date, a plethora of evidence regarding adverse morpho-functional and neurobiological aspects provoked by environmental stressors has been considered. Following exposure to stress factors, the activation of both specific neurosignaling mechanisms and molecular pathways account for the modulation of complex adaptative processes in animal targets. In this context, the aim of the present work is to analyze the neuroprotective role of histaminergic system and heat shock proteins towards environmental neurotoxicants such as heavy metals and pesticides in the Teleost Thalassoma pavo. Such environmental stressors account for significative alterations on motor and feeding behaviors, which are tightly correlated to neurodegenerative processes in key brain regions. In this work, the molecular characterization of H2R and H3R permits to demonstrate a conservation of specific sequences, which appear to be determinant for the function of such subtypes in phylogenetically distant Vertebrates. Moreover, the inactivation of H2R and H3R, via the application of selective antagonists (Cimetidine and Thioperamide, respectively), induces in Thalassoma pavo abnormal behaviors and trascriptional alterations, suggesting a clear physiological role of this neuronal system in our model. The expression pattern of histaminergic system results to be highly modified following exposure to environmental stressors in a region-dependent manner. In particular, the heavy metals induce downregulations of H2R mRNA in some brain regions such as mesencephalon, which is involved in the regulation of motor activities. On the other hand, both heavy metal and pesticides account for an increasement of H3R trascriptional levels in hypothalamic and telencephalic areas. From the concomitant exposure to histaminergic antagonists and environmental stressors, it was possible to demonstrate that H2R blockade is responsible for enhanced stressors-dependent neurotoxic effects. On the contrary, the inhibition of H3R activities accounts for an amelioration of both abnormal motor behaviors and neuronal damage induced by such environmental stressors. Consistent with the effects on histaminergic system, heavy metals and pesticides also promote the activation of cellular defence processes through the stimulation of heat shock proteins trascription, i.e. HSP90 e HSP70. The histaminergic antagonists are able to influence heat shock proteins expression, inducing a heterogeneous pattern of HSP90 trascription levels, while in the case of HSP70 an enhanced expression is typical of all encephalic areas. The results of the present work demonstrate, for the first time in an aquatic Vertebrate, a possible interactions between histaminergic system-dependent neurosignaling activities and HSPs network, which could be represent an important neurophysiological mechanism operating during neuronal stress conditions.Università della Calabri

Amygdalar glutamatergic neuronal systems play a key role on the hibernating state of hamsters

Abstract Background Excitatory transmitting mechanisms are proving to play a critical role on neuronal homeostasis conditions of facultative hibernators such as the Syrian golden hamster. Indeed works have shown that the glutamatergic system of the main olfactory brain station (amygdala) is capable of controlling thermoregulatory responses, which are considered vital for the different hibernating states. In the present study the role of amygdalar glutamatergic circuits on non-hibernating (NHIB) and hibernating (HIB) hamsters were assessed on drinking stimuli and subsequently compared to expression variations of some glutamatergic subtype mRNA levels in limbic areas. For this study the two major glutamatergic antagonists and namely that of N-methyl-D-aspartate receptor (NMDAR), 3-(+)-2-carboxypiperazin-4-yl-propyl-1-phosphonate (CPP) plus that of the acid α-amine-3-hydroxy-5-metil-4-isoxazol-propionic receptor (AMPAR) site, cyano-7-nitro-quinoxaline-2,3-dione (CNQX) were infused into the basolateral amygdala nucleus. Attempts were made to establish the type of effects evoked by amygdalar glutamatergic cross-talking processes during drinking stimuli, a response that may corroborate their major role at least during some stages of this physiological activity in hibernators. Results From the behavioral results it appears that the two glutamatergic compounds exerted distinct effects. In the first case local infusion of basolateral complexes (BLA) with NMDAR antagonist caused very great (p Conclusion We conclude that predominant drinking events evoked by glutamatergic mechanisms, in the presence of prevalently down regulated levels of NR1/2A of some telencephalic and hypothalamic areas appear to constitute an important neuronal switch at least during arousal stage of hibernation. The establishment of the type of glutamatergic subtypes that are linked to successful hibernating states, via drinking stimuli, may have useful bearings toward sleeping disorders.</p

Effetti neuro-comportamentali del rame nei teleostei: ruolo dell'HSP90 e del sistema ORXergico

Dottorato di Ricerca in Biologia animle, Scuola di Dottorato “Life Sciences”, Ciclo XXV, a.a. 2011-2012Tra i metalli, il rame (Cu2+), sebbene essenziale per il corretto metabolismo corporeo, può risultare un potente agente tossico in grado di promuovere pericolosi eventi nocivi, soprattutto nei pesci. Ad oggi, sono poche le informazioni sulla neurotossicità ramedipendente e le risposte attivate per la difesa e riparazione da tale tossicità. Su questi presupposti, nel presente lavoro sono stati investigati gli effetti del cloruro di rame (CuCl2), a diverse concentrazioni e tempi di esposizione, sia a livello comportamentale che neuronale nel teleosteo d’acqua dolce, Carassius carassius, e marino, Thalassoma pavo. Da un punto di vista molecolare, l’attenzione è stata indirizzata alle variazioni trascrizionali dell’Heat Shock Protein 90 (HSP90), chaperon con ruolo critico sia in condizioni fisiologiche che di stress. Alla luce di recenti evidenze che propongono il sistema orexinergico come cruciale nella coordinazione delle reazioni fisiologiche allo stress, è stata valutata anche la capacità dell’orexina-A (ORX-A), somministrata ogni giorno intraperitonealmente (10 ng/g peso corporeo) durante l’esposizione al Cu2+, di modulare gli effetti neuro-comportamentali di tale metallo nei suddetti teleostei. Il trattamento a breve termine (48h) con due concentrazioni di metallo, selezionate mediante screening preliminari in Carassius carassius (1.45 mg/L e 0.30 mg/L CuCl2) e Thalassoma pavo (1.07 mg/L e 0.25 mg/L CuCl2), ha causato evidenti alterazioni comportamentali. In particolare, il feeding è risultato notevolmente (p<0.001; p<0.01) ridotto, vs i controlli, in Carassius carassius e in Thalassoma pavo, rispettivamente. Nel primo pesce, la concentrazione di 1.45 mg/L ha causato anche una moderata (p<0.05) riduzione dello swimming, accompagnata da un simultaneo incremento del resting state. In modo analogo, in Thalassoma pavo, la riduzione dello swimming durante l’esposizione alla più bassa concentrazione, si è tradotta in un concomitante incremento del resting state. Nei teleostei marini, esposti a 1.07 mg/L di metallo, l’assenza di effetti significativi sul rest era dovuta alla comparsa di abnormal motor behaviors, caratterizzati da perdita di equilibrio e movimenti repentini ed improvvisi. In Carassius carassius tali atteggiamenti insorgevano solo moderatamente dopo 48h di esposizione alla alta concentrazione. Inoltre, durante l’esposizione, entrambi i teleostei mostravano una considerevole tendenza a nuotare verso la superficie (swimming towards surface). Accanto agli effetti sul comportamento, l’Amino Cupric Silver Stain ha anche mostrato segni di neurodegenerazione a carico dei neuroni, soprattutto nel nucleo laterale del telencefalo dorsale (Dl) degli esemplari esposti alle alte concentrazioni di contaminante. Parallelamente, l’ibridazione in situ ha fatto registrare nell’encefalo di Thalassoma pavo, trattato con la concentrazione più alta, un’up-regulation dell’HSP90 in differenti nuclei, come Dl (+87%). In Carassius carassius la risposta trascrizionale è stata invece meno evidente, con moderati incrementi dell’mRNA rinvenuti, ad esempio, nella valvola del cervelletto (VCe; +31%), in seguito a trattamento con la più alta concentrazione che causava anche moderate down-regulations a livello del telencefalo. E’ stato interessante notare che molte delle alterazioni comportamentali venivano ridotte dalla somministrazione di ORX-A, soprattutto a livello del feeding di entrambi i teleostei. In Carassius carassius si è potuto osservare, inoltre, una riduzione degli abnormal motor behaviors e dello swimming towards surface. Anche a livello neurodegenerativo, l’ORX-A è stata in grado di prevenire i danni indotti dal Cu2+, come nel Dl di Carassius carassius esposto alla più alta concentrazione. Dal punto di vista molecolare, l’ORX-A ha prevenuto la down-regulation indotta dal metallo in Dl, favorendo addirittura una moderata up-regulation del trascritto in TLo (+32%) di Carassius carassius esposto a 1.45 mg/L di CuCl2, rispetto ai controlli. Anche in Thalassoma pavo, il neuropeptide induceva una significativa up-regulation dell’espressione di HSP90, come nel nucleo laterale del telencefalo ventrale (Vl; +92%) durante esposizione alla concentrazione più bassa di CuCl2. Quando i pesci esposti al Cu2+ per 48h sono stati trasferiti in acqua priva di metallo, molti deficit neuro-comportamentali erano, almeno parzialmente, ripristinati. Inoltre, quasi tutti i nuclei encefalici di entrambi i teleostei, esposti alla più alta concentrazione, hanno mostrato incrementi significativi dell’mRNA per l’HSP90, sia rispetto al precedente trattamento che ai controlli. Alla luce dei dati ottenuti e in virtù del fatto che la bassa concentrazione aveva fatto rinvenire alterazioni neuro-comportamentali più lievi, in alcuni casi recuperabili, è stato interessante verificare gli effetti di questa stessa concentrazione in un trattamento cronico (21 giorni). Nel corso di tale trattamento, il feeding di Carassius carassius era drasticamente ridotto, rispetto ai controlli, a partire dal giorno 7 di esposizione mentre in Thalassoma pavo la riduzione diveniva elevata al giorno 14. A livello encefalico, si riscontrava una più estesa degenerazione che colpiva molti più campi neuronali, compreso il nucleo diffuso del lobo inferiore (NDLI). Al contempo, il trattamento cronico era responsabile di una generalizzata up-regulation dell’HSP90 in Thalassoma pavo e di un effetto trascrizionale sito-specifico in Carassius carassius. In quest’ultimo teleosteo, infatti, si riportavano down-regulations nel telencefalo (~-30%) e in NDLI (-55%), ed upregulations in TLo (+30%), VCe (+43%) e nel corpo del cervelletto (CCe; +52%). La somministrazione di ORX-A al 21 giorno, sebbene causasse un’attenuazione degli effetti del Cu2+ sul comportamento, non era però capace di ridurre i danni neuronali e, a livello trascrizionale, non influenzava in modo efficiente l’espressione dell’HSP90 Nel complesso, i risultati di questo lavoro di Dottorato forniscono importanti evidenze degli effetti neuro-comportamentali del Cu2+ in Carassius carassius e Thalassoma pavo, sottolineando il coinvolgimento dell’HSP90 nei meccanismi di protezione e riparazione innescati a livello encefalico. In aggiunta a ciò, per la prima volta, si suggerisce l’implicazione del sistema ORXergico nell’attivazione di risposte adattative allo stress indotto dal Cu2+, sia nei teleostei marini che d’acqua dolceUniversità della Calabri