Ketogenic diet ameliorates autism spectrum disorders-like behaviors via reduced inflammatory factors and microbiota remodeling in BTBR T+ Itpr3tf/J mice.

Autism Spectrum Disorder (ASD) is increasing, but its complete etiology is still lacking. Recently, application of ketogenic diet (KD) has shown to reduce abnormal behaviors while improving psychological/sociological status in neurodegenerative diseases. However, KD role on ASD and underlying mechanism remains unknown. In this work, KD administered to BTBR T+ Itpr3tf/J (BTBR) and C57BL/6J (C57) mice reduced social deficits (p = 0.002), repetitive behaviors (p < 0.001) and memory impairments (p = 0.001) in BTBR. Behavioral effects were related to reduced expression levels of tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 in the plasma (p = 0.007; p < 0.001 and p = 0.023, respectively), prefrontal cortex (p = 0.006; p = 0.04 and p = 0.03) and hippocampus (p = 0.02; p = 0.09 and p = 0.03). Moreover, KD accounted for reduced oxidative stress by changing lipid peroxidation levels and superoxide dismutase activity in BTBR brain areas. Interestingly, KD increased relative abundances of putatively beneficial microbiota (Akkermansia and Blautia) in BTBR and C57 mice while reversing the increase of Lactobacillus in BTBR feces. Overall, our findings suggest that KD has a multifunctional role since it improved inflammatory plus oxidative stress levels together with remodeling gut-brain axis. Hence, KD may turn out be a valuable therapeutic approach for ameliorating ASD-like conditions even though more evidence is required to evaluate its effectiveness especially on a long term.

Emotional and Spontaneous Locomotor Behaviors Related to cerebellar Daidzein-dependent TrkB Expression Changes in Obese Hamsters.

Current evidence supports the beneficial role of phytoestrogens in metabolic diseases, but their influences on spontaneous motor and anxiety behaviors plus neuroprotective effects have still not been completely elucidated. With the present study, neuro-behavioral activities were correlated to daidzein (DZ)-dependent expression changes of a high affinity catalytic receptor for several neurotrophins, and namely tropomyosin-related kinase B receptor (TrkB) in the cerebellar cortex of high-fat diet (HFD) hamsters (Mesocricetus auratus). Indeed, these changes appear to be tightly linked to altered plasma lipid profiles as shown by reduced low-density lipoproteins plus total cholesterol levels in DZ-treated obesity hamsters accounting for increased spontaneous locomotor together with diminished anxiety activities in novel cage (NCT) and light/dark box (LDT) tests. For this latter case, the anxiolytic-like hamsters spent more time in the light compartment, which was retained the aversive area of the LDT box. As for the evaluation of the neurotrophin receptor site, significantly elevated TrkB levels were also detected, for the first time, in the cerebellum of obese hamsters treated with DZ. In this condition, such a treatment widely led to an overall improvement of HFD-induced neurodegeneration damages, above all in the Purkinje and granular layers of the cerebellum. In this context, the notably active TrkB signaling events occurring in a DZ-dependent manner may turn out to be a key neuroprotective element capable of restoring normal emotional and spontaneously linked locomotor behaviors regulated by cerebellar cortical areas especially in obesity-related conditions.

Modifications of Behavior and Inflammation in Mice Following Transplant with Fecal Microbiota from Children with Autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder displaying the modification of complex human behaviors, characterized by social interaction impairments, stereotypical/repetitive activities and emotional dysregulation. In this study, fecal microbiota transplant (FMT) via gavage from autistic children donors to mice, led to the colonization of ASD-like microbiota and autistic behaviors compared to the offspring of pregnant females exposed to valproic acid (VPA). Such variations seemed to be tightly associated with increased populations of Tenericutes plus a notable reduction (p < 0.001) of Actinobacteria and Candidatus S. in the gastrointestinal region of FMT mice as compared to controls. Indeed altered behaviors of FMT mice was reported when evaluated in the different maze tests (light dark, novel object, three chamber tests, novel cage test). Contextually, FMT accounted for elevated expression levels of the pro-inflammatory factors IL-1ß, IL-6, COX-1 and TNF-α in both brain and small intestine. Villous atrophy and inflammatory infiltration (Caspase 3 and Ki67) were increased in the small intestine of FMT and VPA mice compared to controls. Moreover, the observed FMT-dependent alterations were linked to a decrease in the methylation status. Overall, findings of the present study corroborate a key role of gut microbiota in ASD. However, further investigations are required before any possible manipulation of gut bacteria with appropriate diets or probiotics can be conducted in ASD individuals.

Cerebral pCREB-dependent social behavioral adversities following a short-term exposure to obesogenic diets in young hamsters.

Excessive fat and sugar intake represents a risk towards the development of different pathologies, such as obesity, diabetes, sociability and memory deficits. Although the adolescence stage is a susceptible period for these and other risks, effects of energy-dense nutrients in such an age period have not been fully investigated. In the present study, neurobehavioral alterations following a 4-week exposure to either normal diet (ND) or high-fat diet (HFD) plus normal water (NW) or liquid sugar (LS) were evaluated in young hamsters. HFD + LS and ND + LS significantly reduced food intake and water consumption, which was, in the latter group, almost completely substituted by LS. All obesogenic diets accounted for increased abdominal fat and liver weight with respect to body weight (p < 0.05-0.001). Additionally, glucose levels notably increased (p < 0.0001) together with insulin and triglycerides in HFD + LS (p < 0.001) and ND + LS (p < 0.01) while cholesterol displayed only a moderate increase (p < 0.05) in HFD + NW and HFD + LS. Animals fed with HFD and/or LS exhibited impaired social memory plus increased winning percentages (0.05 < p < 0.01) during the tube test. Interestingly, these same treatments led to a down-regulation of phosphorylated cAMP Response-Element Binding Protein (pCREB) in HFD + NW (p < 0.0001) for all areas, but rather was upregulated (p < 0.05) in ND + LS of the amygdala. Overall, in view of a brief exposure to palatable foods interfering with normal metabolic and social memory activities, the downregulation of pCREB constitutes a key indicator of neurobehavioral deficits during obesogenic diets. Compensatory mechanisms may be also occurring in the amygdala that strongly regulates emotional states via connections with other limbic areas.

Correlation of distinct behaviors to the modified expression of cerebral Shank1,3 and BDNF in two autistic animal models.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder featuring altered neuronal circuitry and consequently impaired social interactions, restrictive interests plus repetitive stereotypic activities. In the present study, differentiated behaviors of valproic (VPA) and propionic (PPA) acid-mediated autism rats were correlated to cerebral scaffolding proteins (Shank1,3) and BDNF expression variations. Sprague-Dawley offspring that received VPA during pregnancy displayed a notably diminished permanence (-78 %, p < 0.01) in the light chamber of light dark (LD) test, reduced exploratory tasks, i.e. grooming (-90 %) and rearing (-65 %). Moreover, they executed extremely greater climbing intervals (+300 %, p < 0.001) in novel cage (NC) test, plus exhibited an extremely reduced (-331 %) discrimination index in novel object recognition (NOR) test when compared to controls. PPA-treated postnatal days (PND) 12-16 rats also displayed anxiety-like behaviors, although in a less evident manner, as indicated by a moderate time (+55 %; p < 0.05) spent in dark chamber along with notable and moderate decreases in digging (-78 %) plus grooming (-52 %), respectively. Contextually, VPA- more than PPA supplied opposite Shank1,3 expression changes in cerebellum (CB; -62 %; +78 %), dorsomedial prefrontal cortex (DM-PFC; +95 % -76 %), respectively, while resulting extremely upregulated in hippocampus (HIP; +125 % - +155 %). Even BDNF resulted to be substantially and notably diminished in HIP (-85 %) and DM-PFC (-72 %), respectively, of VPA rats while it was only moderately reduced (-35 % to -45 %) in these same areas of PPA rats. The early altered brain-specific expression levels accounting for different behavioral performances may provide useful diagnostic indications and constitute valuable therapeutic strategies for autistic patients.

Daidzein Pro-cognitive Effects Coincided with Changes of Brain Neurotensin1 Receptor and Interleukin-10 Expression Levels in Obese Hamsters.

At present, concerns are pointing to "tasteful" high-fat diets as a cause of conditioning physical-social states that through alterations of some key emotional- and nutritional-related limbic circuits such as hypothalamic and amygdalar areas lead to obesity states. Feeding and energetic homeostatic molecular mechanisms are part of a complex neuronal circuit accounting for this metabolic disorder. In an attempt to exclude conventional drugs for treating obesity, daidzein, a natural glycosidic isoflavone, which mimics estrogenic neuroprotective properties against increased body weight, is beginning to be preferred. In this study, evident anxiolytic-like behaviors were detected following treatment of high-fat diet hamsters with daidzein as shown by extremely evident (p < 0.001) exploration tendencies in novel object recognition test and a notably greater amount of time spent (p < 0.01) in open arms of elevated plus maze. Moreover, the isoflavone promoted a protective role against neurodegeneration processes as shown by few, if any, amino cupric silver granules in amygdalar, hypothalamic and hippocampal neuronal fields when compared with obese hamsters. Interestingly, elevated expression levels of the anorexic neuropeptide receptor neurotensin1 in the above limbic areas of obese hamsters were extremely reduced by daidzein, especially during recovery of cognitive events. Contextually, such effects were strongly paralleled by increased levels of the anti-neuroinflammatory cytokine, interleukin-10. Our results corroborate a neuroprotective ability of this natural glycosidic isoflavone, which through its interaction with the receptor neurotensin1 and interleukin-10 pathways is correlated not only to improved feeding states, and subsequently obesity conditions, but above all to cognitive performances.

Probiotics modify body weight together with anxiety states via pro-inflammatory factors in HFD-treated Syrian golden hamster.

Emerging studies are beginning to suggest that emotional states together with healthful measures constitute pertinent features of our lifestyle in which bad eating habits but more importantly what our gut has to host are causing great concern. It is well known that humans have established mutual relationships with a wide array of colonized microbes (collectively called gut microbiota) consisting of bacteria, fungi, eukaryotic parasites and viruses. The gut microbiota has exhibited a notable ability of communicating with the brain via a two-way system that includes the vagus nerve, immune sites, and a number of neurotransmitters. Interestingly, stressful along with obesity, cognitive, and brain developmental states are strongly influenced by microbiota homeostatic conditions. It was our aim to investigate behavioral and obesity effects evoked by treatment with probiotics via neuroinflammatory factors and namely IL-1ß, NLRP3, Caspase-1 and NF-kB levels in the Syrian golden hamster. Following treatment with a high-fat diet (HFD), in the presence or absence of a multi-species probiotic formulation, hamsters were exposed to an unpredictable chronic mild stress (UCMS) test for 4 weeks. Independently of the diet, probiotics treatment markedly reduced stress-like behaviors in the different mazes. Moreover, probiotics decreased hypothalamic expression levels of the pro-neuroinflammatory factors like IL-1ß, NLRP3, Caspase-1 and NF-kB, whereas HFD increased them. Contextually, they decreased plasmatic levels of IL-1ß, NLRP3 and caspase-1 but not NF-kB. Our findings clearly support probiotics as a potentially valuable treatment strategy in obesity and anxiety, thereby proposing them for clinical treatments in patients with metabolic and mood disorders.

Genistein Modifies Hamster Behavior and Expression of Inflammatory Factors following Subchronic Unpredictable Mild Stress.

BACKGROUND: Previous studies have pointed to the protective role of genistein against stress adaptations although neuromolecular mechanisms are not yet fully known. With this work, we evaluated the influence of such a phytoestrogen on hamster behavioral and molecular activities following exposure to subchronic unpredictable mild stress. METHODS: The motor behaviors of hamsters (n = 28) were analyzed using elevated plus maze (EPM) test, hole board (HB) test, and forced swim test (FST). In addition, neurodegeneration events were assessed with amino cupric silver stain, while expression variations of tropomyosin receptor kinase B (TrkB), nuclear factor kappa-B1 (NF-κB1), and heat shock protein 70 (Hsp70) mRNAs were highlighted in limbic neuronal fields via in situ hybridization. RESULTS: Genistein accounted for increased motor performances in EPM and HB tests but reduced immobility during FST, which were correlated with diminished argyrophilic signals in some limbic neuronal fields. Contextually, upregulated Hsp70 and TrkB mRNAs occurred in hippocampal (HIP) and hypothalamic neuronal fields. Conversely, diminished NF-κB1 levels were mainly obtained in HIP. CONCLUSION: Hormonal neuroprotective properties of genistein corroborating anxiolytic and antidepressant role(s) through elevated expression levels of stress proteins and trophic factors may constitute novel therapeutic measures against emotional and stress-related motor performances.

Orexin receptor expression is increased during mancozeb-induced feeding impairments and neurodegenerative events in a marine fish.

Food intake ensures energy resources sufficient for basic metabolism, immune system and reproductive investment. It is already known that food-seeking performances, which are crucially controlled by orexins (ORXs), may be under the influence of environmental factors including pollutants. Among these, mancozeb (mz) is becoming an environmental risk for neurodegenerative diseases. Due to few studies on marine fish exposed to mz, it was our intention to correlate feeding latency, food intake and feeding duration to potential neurodegenerative processes in key diencephalic sites and expression changes of the ORX neuroreceptor (ORXR) in the ornate wrasses (Thalassoma pavo). Hence, fish exposed for 4 days (d) to mz 0.2 mg/l (deriving from a 0.07, 0.14, 0.2, 0.3 mg/l screening test) displayed a significant reduction (p < 0.05) of food intake compared to controls as early as 1d that became more evident (p < 0.01) after 3d. Moreover, significant enhancements of feeding latency were reported after 1d up to 3d (p < 0.001) and even feeding duration was enhanced up to 3d (p < 0.001), which instead moderately increased after 4d (p < 0.05). A reduction (-120%; p < 0.001) of mean body weight was also detected at the end of exposure. Likewise, a notable (p < 0.001) activation of ORXR protein occurred together with mRNA up-regulations in diencephalic areas such as the diffuse nucleus of the inferior lobe (+48%) that also exhibited evident degenerative neuronal fields. Overall, these results highlight an ORX role as a vital component of the neuroprotective program under environmental conditions that interfere with feeding behaviors.

Reduced learning and memory performances in high-fat treated hamsters related to brain neurotensin receptor1 expression variations.

Recent indications are suggesting that high fat and sugar-enriched foods do not only evoke harmful physiological conditions, but they also endure evident structural alterations in cerebral regions controlling cognitive and feeding behaviors. Food consumption plus neuronal energy regulatory mechanisms seem to constitute a complex system assuring that food calories do not exceed body requirements. At the same time obesogenic-related properties of limbic feeding stations like the hypothalamus (HTH), hippocampus (HIP) and amygdala (AMY) tend to control eating habits through the interaction of distinct neuropeptides. For this purpose, it was our intention to correlate expression differences of a key anti-obesogenic neuropeptide receptor i.e. neurotensin1 (NTR1) on mnemonic performances in the hibernating hamster (Mesocricetus auratus) exposed to a high fat diet (HFD). Interestingly, these hamsters exhibited a notable enhanced (p < 0.01) body weight from the fifth on to the twelfth week of treatment, which was accompanied by elevated blood lipid cholesterolo and triglycerides and glucose levels. At the same time these hamsters provided diminished locomotor activities such as exploratory bouts, rearing and grooming behaviors. Of greater relevance was their very extreme (p < 0.001) inability of identifying new objects during novel object recognition (NOR) tests along with not having correctly chosen the chamber of the conditioned place preference (CPP) apparatus, which contained the gratifying reward. Surprisingly the altered behavioral plus mnemonic tasks of HFD hamsters were tightly related to elevated NTR1 expression changes in the above limbic sites thus proposing this neuronal system as a highly probable alternative for treating obesity-dependent mnemonic dysfunctions.

Role of Brain Neuroinflammatory Factors on Hypertension in the Spontaneously Hypertensive Rat.

It is already widely known that the different brain areas involved in blood pressure control, are highly vulnerable to the deleterious effects of this condition. Of particular concern are hypertensive and neuroinflammatory-dependent injuries that by modifying blood flow account for artery structural and functional alterations. It was thus our intention to establish if expression changes of some key brain neuroinflammatory factors like caspase-1,3, NF-kB, IL-1ß and NLRP3, which are known to control blood pressure, are actively involved with inflammation regulatory events in a highly valuable spontaneously hypertensive rat (SHR) model. Indeed, notably increased (p < 0.001) caspase-1, NLRP3 and IL-1ß mRNA levels were detected in amygdalar plus hypothalamic areas of SHR. Contextually, similar up-regulated levels of these factors were also reported in brainstem nuclei with respect to the few hippocampal areas. This trend was supported by moderate increases (p < 0.05) of NLRP3 in amygdalar and brainstem sites, while notably greater expression differences of NF-kB protein were observed in hippocampal and hypothalamic areas of SHR. At the same time, moderately increased levels of iNOS were typical of all of the above brain areas with the exception of the consistently (p < 0.01) increased levels featured in the brainstem. Moreover, even immunohistochemical evaluations supplied notably and moderately increased cleaved caspase-3 cell levels in hippocampus and hypothalamus areas, respectively. Overall, evident hypertensive bouts correlated to neuroinflammatory events, especially in brain areas controlling blood pressure, tend to underlie the value of novel therapeutic approaches designed to improve brain blood flow and subsequently reduce hypertensive-dependent cerebral complications.

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 (p<0.001) latency to reach T-maze arms (>1000%) 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 toxicity.

Orexin-A Rescues Chronic Copper-Dependent Behavioral and HSP90 Transcriptional Alterations in the Ornate Wrasse Brain.

Recently, orexin (ORX)ergic system has gained great attention for its major neuroregulatory role on fish motor, circadian, feeding activities and above all during water toxic conditions. Fish are particularly sensitive to acute sublethal copper (Cu) concentrations while little is known about neurobehavioral data after chronic Cu exposure. For this work, the marine teleost ornate wrasse (Thalassoma pavo) was monitored for 21 days during exposure to a relatively low CuCl2 concentration (0.25 mg/l). In particular, at 1 day fish displayed moderate reductions (-32%) of feeding behaviors versus controls that decreased up to -94% at 21 days. Swimming activities were also moderately (-44%) reduced at 1 day, which diminished even further (~ -75%) from 7 to 21 days. The lethargic attitudes seemed to be frequently interrupted by aquatic surface respiration and abnormal motor behaviors such as rapid and abrupt changes of direction. Contextually, an upregulatory trend of HSP90 mRNAs occurred especially in the lateral part of the dorsal telencephalon (Dl; +72%), medial preglomerular nucleus (NPGm; +87%), and torus longitudinalis (TLo; +108%). Moreover, some of these nuclei displayed extensive argyrophilic signals that are typical of notable neurodegenerative events. Interestingly, a single intraperitoneal administration of ORX-A (10 ng/g of body weight) at 21 days attenuated not only some behavioral impairments but also HSP90 mRNA levels and neurodegenerative events. Behavioral indications of the present results tend to underlie the valuable role of HSP90 transcript together with the ORXergic system as probable rescuing factors operating in marine fish during persistent adverse environmental states with eventual bearings on human health issues.

Role of Leptin and Orexin-A Within the Suprachiasmatic Nucleus on Anxiety-Like Behaviors in Hamsters.

It is well established that the maintenance of energy expenditure is linked to active hypothalamic neural mechanisms controlling adaptive stimuli such as food intake. Variations of glucose levels and hormonal (leptin plus orexin-A) parameters, which are involved with energy homeostasis during different behavioral states, have not yet been fully defined. In this first study, behavioral analyses of an unpredictable stress model dealing with the actions of a sub-chronic administration of orexin-A (ORX-A) and the anti-hunger neuropeptide, i.e., leptin (LEP) within the hypothalamic suprachiasmatic (SCH) nucleus, were conducted on the valuable hibernating rodent (hamster; Mesocricetus auratus) model noted for its distinct depression and anxiety states. Treatment with LEP accounted for a notable reduction (p < 0.01) of body weight in stressed hamsters that not only executed very evident (p < 0.001) movements to and from elevated plus maze (EPM) but also spent less time in the dark area of the light-dark box test (LDT). Conversely, ORX-A predominantly evoked anxiogenic effects that were inverted by LEP. Interestingly, the anti-hunger neuropeptide accounted for both down-regulated NPY1 transcripts in mostly lateral-posterior hypothalamic areas while up-regulated levels were detected in the parietal cerebral cortex, hippocampus, and amygdala, which largely behaved in an opposite manner to ORX-A-dependent effects. Overall, the present findings corroborate a predominating LEPergic effect of the SCH toward the reduction of hamster anxiety-like behaviors with respect to that of ORX-A signaling, which may constitute useful therapeutic targets for stress-related obesity states.

Unpredictable Chronic Mild Stress Paradigm Established Effects of Pro- and Anti-inflammatory Cytokine on Neurodegeneration-Linked Depressive States in Hamsters with Brain Endothelial Damages.

The mechanisms by which inflammation affects the different emotional moods are only partially known. Previous works have pointed to stress hormones like glucocorticoids plus the vascular factor endothelin-1 as key factors evoking stressful states especially in relation to endothelial dysfunctions. With this work, it was our intention to establish the role of pro- and anti-inflammatory cytokine expression variations towards depression-like behaviors and consequently the development of neurodegeneration events caused by endothelial damages in the hamster (Mesocricetus auratus). Such a rodent, which is considered a valuable animal model to test depression and anxiety states, exhibited a variety of depression-like behaviors including reduction in sucrose consumption, locomotion, and exploration (p < 0.01) following exposure to unpredictable chronic mild stress. Contextually, a tight correlation between unpredictable chronic mild stress-induced depressive states and expression of the pro-inflammatory cytokines was detected as shown by marked expression levels (p < 0.01) of IL-1ß and NF-kB in the hippocampus, amygdala, and prefrontal cortex. Even the anti-inflammatory cytokine IL-10 supplied notably significant (p < 0.001) expression levels in the same areas of resilient hamsters. Application of hemodynamic and endothelial functional studies pointed to altered arterial endothelial activities in depressed with respect to resilient animals. Moreover, evident damaged neuronal fields in the above areas of depressed hamsters allowed us to correlate such a behavioral phenomenon to the upregulation of IL-1ß and NF-κB. Overall, the differing roles of pro- and anti-inflammatory cytokines on depressive states, especially in view of brain endothelial damages, may provide novel therapeutic measures against mood disorders linked to neurodegenerative diseases.

Application of the Co-culture Membrane System Pointed to a Protective Role of Catestatin on Hippocampal Plus Hypothalamic Neurons Exposed to Oxygen and Glucose Deprivation.

Depletion of oxygen and glucose even for brief periods is sufficient to cause cerebral ischemia, which is a predominant worldwide cause of motor deficits with the reduction of life quality and subsequently death. Hence, more insights regarding protective measures against ischemic events are becoming a major research goal. Among the many neuronal factors, N-methyl-D-aspartate receptors (NMDAR), orexinergic neuroreceptors (ORXR), and sympatho-inhibitory neuropeptide catestatin (CST) are widely involved with ischemic episodes. In this study, it was possible to induce in vitro ischemic conditions of the hamster (Mesocricetus auratus) hippocampal and hypothalamic neuronal cultures, grown on a newly compartmentalized membrane system, via oxygen and glucose deprivation (OGD). These cultures displayed notably differentiated NMDARergic and ORXergic receptor expression activities along with evident brain-derived neurotrophic factor (BDNF) plus orexin A (ORX-A) secretion, especially under co-cultured conditions. Interestingly, addition of CST in OGD-insulted hippocampal cells accounted for upregulated GluN1 and ORX1R transcripts that in the case of the latter neuroreceptor was very strongly (p < 0.001) increased when co-cultured with hypothalamic cells. Similarly, hypothalamic neurons supplied very evident upregulations of GluN1, ORX1R, and above all of GluN2A transcripts along with increased BDNF and ORX-A secretion in the presence of hippocampal cells. Overall, the preferential CST effects on BDNF plus ORX-A production together with altered NMDAR and ORXR levels, especially in co-cultured hypothalamic cells pointed to ORX-containing neurons as major protective constituents against ischemic damages thus opening new scenarios on the cross-talking roles of CST during ischemic disorders.

Catestatin and GABA(A)R related feeding habits rely on dopamine, ghrelin plus leptin neuroreceptor expression variations.

Catestatin (CST), an endogenously small sympathoinhibitory peptide is capable of interfering with the major cerebral neuroreceptor-blocking site, i.e. γ-aminobutyric acidA receptor (GABAAR) system especially in limbic brain areas that are involved with feeding behaviors. The GABAARergic-related effects seem to derive from its interaction with other molecular neuroreceptors such as dopaminergic, ghrelin and leptinergic. In this context, the present study aimed to investigate probable feeding responses (eating and drinking) induced by treatment with CST and the GABAAR antagonist bicucullin (BIC) alone or simultaneously (CST+BIC) in the Syrian hibernating hamster (Mesocricetus auratus) model. Hamsters that received these compounds via intracerebroventricular infusions displayed notable variations of feeding and drinking bouts. In particular, an anorexigenic response was evident following treatment with CST while BIC evoked a significant increase of eating and drinking behaviors. Surprisingly when both agents were given simultaneously, a predominating anorexigenic response was detected as shown by evident CST-dependent reduction of feeding bouts. Contextually such behaviors, especially those following the combined treatment were tightly correlated with the significantly increased cerebral dopamine receptor 1 (D1) plus reduced ghrelin receptor (GhsR) and leptin receptor (LepR) transcript levels. Overall, the anorexigenic effect of CST deriving from its tight interaction with GABAARs activity plus D1 and GhsR transcripts tends to propose these neuronal elements as pivotal factors responsible for feeding disorders.

Neurobehavioral alterations plus transcriptional changes of the heat shock protein 90 and hypoxia inducible factor-1α in the crucian carp exposed to copper.

The various physiological alterations caused by copper (Cu) exposure in fish have attracted great interests toward neuronal strategies against Cu toxicity. Here, neurobehavioral activities (including anxiety-like behaviors) and transcriptional levels of heat shock protein (Hsp)90 and hypoxia inducible factor-1alpha (HIF-1α) were evaluated in the crucian carp (Carassius carassius) treated with nominal sub-lethal higher (1.45mg/L) and lower (0.30mg/L) concentrations of CuCl2. Both concentrations accounted for diminished swimming speed and food intake plus a strong preference for the dark side of the light/dark apparatus together with a reduction of crossings between the two compartments with respect to controls. Contextually, Hsp90 and HIF-1α transcripts were largely down- and up-regulated, respectively, in some brain areas such as in the medial part of the dorsal telencephalon (Dm, -52%) and in the ventral part of the ventral telencephalon (Vv, +68%). When carps were transferred to CuCl2-free water, some behaviors were rescued especially for fish previously exposed to 0.30mg/L concentration. In this same condition, Hsp90 mRNA levels were recuperated (-94%) in the medial preglomerular nucleus (NPGm) with respect to exposed fish while HIF-1α mRNA was mostly down-regulated in telencephalic stations. Moreover, recovery capacities of this extraordinary resistant fish was exhibited by evident reductions (-80%) of the dark argyrophilic granules such as in the ventral telencephalon (VTel). Overall, our results point to interesting responses against Cu toxicity involving Hsp90 and HIF-1α transcripts that may constitute early indicators of environmental stressors leading to the repair of metal-induced damages in fish with notable brain plasticity properties.

Exposure to sub-chronic unpredictable stress accounts for antidepressant-like effects in hamsters treated with BDNF and CNQX.

Recent evidences indicate that cerebral neurotrophic factors like vascular endothelial growth factor plus signaling pathways of the glutamatergic neuroreceptor system (L-Glu) are determinant modulators of depression-like states. In the present study, the type of interaction(s) exerted by the AMPAergic antagonist, 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) and the brain derived neurotrophic factor (BDNF) on depression-like behaviors in hamsters (Mesocricetus auratus) were investigated. Sub-chronic administration of BDNF in the hippocampal dentate gyrus (DG) of stressed hamsters was responsible for very evident (p<0.001) sucrose consumption along with notably elevated swimming bouts and reduced immobility states in the forced swim test (FST). Meanwhile, CNQX displayed evident anxiolytic actions in the elevated plus maze (EPM) as shown by marked (p<0.01) increases of movements to and from both arms. Interestingly cerebral neurodegeneration events, which are viewed during depression states, were reduced following treatment with both compounds. Contextually, marked mRNA expression levels of the BDNF receptor (tropomyosin-related kinase B; TrkB) were detected in DG and the oriens-pyramidalis of HIP (Or-Py) while a moderate (p<0.05) up-regulation was registered in the amygdalar central nucleus (CeA) and the hypothalamic ventromedial nucleus (VMH) of hamsters treated with BDNF. Similarly, this treatment caused moderate increases of the major stress protein (Hsp70) in DG and Or-Py. Conversely, while CNQX induced similar TrkB expression levels, it instead accounted for a moderate reduction of Hsp70 mRNAs in the same brain areas. Overall these results support crucial roles played by BDNF and AMPAergic neurosignaling mechanisms during distinct adaptive responses of depression- and anxiety-like states in hamsters.

Neuroprotective effect of human mesenchymal stem cells in a compartmentalized neuronal membrane system.

In this work, we describe the development of a compartmentalized membrane system using neonatal rodent hippocampal cells and human mesenchymal stem cells (hMSCs) to investigate the neuroprotective effects of hMSCs. To elucidate this interaction an in vitro oxygen-glucose deprivation (OGD) model was used that mimics central nervous system insults in vivo. Cells were cultured in a membrane system with a sandwich configuration in which the hippocampal cells were seeded on a fluorocarbon (FC) membrane, and were separated by hMSCs through a semipermeable polyethersulfone (PES) membrane that ensures the transport of molecules and paracrine factors, but prevents cell-to-cell contact. This system was used to simulate a cerebral ischemic damage by inducing OGD for 120min. The core contribution of the work highlights the neuroprotective effects of hMSCs on hippocampal cells in a membrane system for the first time. The novel results show that hMSC secretome factors protect hippocampal cells against OGD insults as indicated by the conservation of specific structural and functional cell features together with the development of a highly branched neural network after the damage. Moreover, neuronal cells co-cultured with hMSCs before OGD insult were able to maintain BDNF production and O2 consumption and did not express the apoptotic markers that were expressed in similarly insulted neuronal cells that had not been co-cultured with hMSCs. This compartmentalized membrane system appears to be a very useful and reliable system for studying the neuroprotective effects of hMSCs and identifying secreted factors that may be involved. STATEMENT OF SIGNIFICANCE: This paper is based on a combined synergism of biomaterials technology and stem cell approach, focusing on the development of a compartmentalized membrane system that serves as an innovative tool for highlighting the role of hMSCs on hippocampal neurons upon damage. The membrane system consists of two different flat sheet membranes, giving rise to double and separated cell membrane compartments that prevent cell-to-cell contact but allow the transport of paracrine factors. This system strongly corroborates the paracrine mediated neuroprotection of hMSCs on ischemic damaged neurons. The challenging and pioneeristic approach by using biomaterials allowed to perform a stepwise analysis of the phenomena, providing new insights into the field of MSC therapy.