Ennio Avolio

Amygdalar orexinergic-GABAergic interactions regulate anxiety behaviors of the Syrian golden hamster.

At present neurobiological interests are directing more attention towards the major role of the amygdalar GABA(A) receptor on orexin-dependent behaviors. This telencephalic region has been widely studied especially in view of its control on various psychiatric disorders such as anxiety and depression. Recently, cross-talking relationships between these two specific neuroreceptor systems of the central-cortical amygdalar complex has been considered an important element for anxiety type of behaviors. In the present study, we investigated the effects of central amygdalar infusions with orexin-A, orexin-B±GABA(A) receptor α₂ subunit agonist (flunitrazepam) on elevated plus-maze and light-dark explorative behaviors of the facultative hibernating Syrian hamster. In a first case, it seemed that doses of orexin administered directly into the central nucleus were responsible for greater anxiogenic type of effects as shown by more time being spent both in the dark compartment and the closed arm of the elevated plus-maze, whereas, these effects were suppressed in the presence of flunitrazepam. At the cellular level, the effects of orexin accounted for evident argyrophilic reactions (neurodegeneration phenomena) including altered cell membrane and loss of cytoplasmic architecture in most amygdalar and hippocampal neuronal fields, while in the presence of flunitrazepam these reactions resulted to either be unappreciable or absent. Overall the actions of α₂-dependent inhibitory signals tend to corroborate, for the first time, a neuroprotective role against the over-excitatory orexinergic neurodegeneration reactions and thus its abnormal anxiety-like indications may prove to be therapeutically useful for orexin-dependent sleeping disorders.

Excitatory/inhibitory equilibrium of the central amygdala nucleus gates anti-depressive and anxiolytic states in the hamster

Several studies have pointed to the amygdala as a main limbic station capable of regulating different stressful states such as anxiety and depression. In this work it was our intention to determine the role of the central amygdala nucleus (CeA) on the execution of either anxiolytic and/or anti-depressant behaviors in the hibernating hamster (Mesocricetus auratus) via infusion of CeA with the antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) specific for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) plus the specific agonist for α4 GABAAR i.e. 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP). Treatment with CNQX appeared to mainly prompt anti-depressant effects as shown by the achievements of swimming feats during forced swim test while THIP prevalently accounted for evident bouts of climbing when exposed to the same test. Moreover, even in the presence of the concomitant administration of both of these compounds, hamsters continued to spend more time in swimming despite this significant behavioral effect resulted to be numerically reduced for hamsters treated with only the α4 GABAAR agonist. Conversely, when these animals were tested in elevated plus maze (EPM), THIP tended to mostly favor anxiolytic activities as exhibited by stressed animals spending more time entering and remaining in EPM open arms. It was interesting to note that behavioral changes induced by both drugs appeared to be also responsible for glutamate receptor (GluR) expression differences as indicated by CNQX favoring an evident up-regulation of GluR2-containing neurons whereas THIP induced an up-regulation, this time of GluR1-containing neurons. Overall, the anti-depressant role of CNQX seems to be mostly attributed to elevated GluR2 levels while an anxiolytic-like effect of THIP was correlated to high GluR1values thereby proposing distinct GluRs as useful therapeutic sites against degenerative diseases such as depression-like behaviors.

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

BackgroundExcitatory 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.ResultsFrom 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 < 0.001) drinking rhythms while moderately increased feeding (p < 0.05) responses during arousal with respect to moderately increased drinking levels in euthermics. Conversely, treatment with CNQX did not modify drinking rhythms and so animals spent more time executing exploratory behaviors. These same antagonists accounted for altered glutamatergic transcription activities as displayed by greatly reduced GluR1, NR1 and GluR2 levels in hippocampus, ventromedial hypothalamic nucleus (VMN) and amygdala, respectively, plus a great (p < 0.01) up-regulation of GluR2 in VMN of hibernators.ConclusionWe 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.

Amygdalar excitatory/inhibitory circuits interacting with orexinergic neurons influence differentially feeding behaviors in hamsters

Recently, environmental stimuli on different neurobiological events, via participation of distinct amygdalar (AMY) ORXergic fibers have aroused wide interests in view of their ability to modify neuronal linked stressful and physiological homeostatic conditions. Results of the present study indicate that ORXergic (ORX-A/B) circuits of the facultative hibernating golden hamster (Mesocricetus auratus) central AMY (CeA) and basolateral AMY (BlA) nuclei constitute major sites of feeding behaviors. Indeed, hamsters after treatment of BlA with ORX-A frequently ingested greater quantities of food as compared to controls, while ORX-B in CeA induced a very (p<0.001) great consumption of water. The same nuclei treated separately with either ORX-A or ORX-B ± the selective α(1) GABA(A) benzodiazepine receptor agonist (zolpidem) dedicated less time to eating and drinking sessions. Conversely, hamsters that received the same neuropeptides but this time with the glutamatergic agonist NMDA displayed greater hyperphagic effects above all for ORX-A. When behavioral changes were compared to the expression of the specific ORXergic receptor (ORX-2R), an up-/down-regulating pattern was detected in some limbic areas (AMY, hippocampus and hypothalamus) following treatment with ORX-A or ORX-B plus NMDA. Overall, indications deriving from this study strongly point to hamster BlA-enriched ORX-A fibers in combination with either inhibitory or excitatory signals as main targets of hyperphagic responses while CeA ORX-B activities in presence of these same neuronal signals predominantly induced drinking motivational behaviors. The distinct behavioral activities of these two neuropeptides may have useful clinical bearings toward psychiatric and sleeping disorders such as bulimia and narcolepsy.

Distinct Amygdalar AMPAergic/GABAergic Mechanisms Promote Anxiolitic-Like Effects in an Unpredictable Stress Model of the Hamster

Studies have pointed to both α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) antagonists and GABAA receptor (GABAAR) agonists as potent antistress agents. In this work, separate subchronic injections of the AMPAR antagonist, 6-ciano-7-nitroquinoxaline-2,3-dione (CNQX), and α1 GABAAR subunit agonist (Zol) within the central amygdala nucleus modified the elevated plus maze performances of hamsters exposed randomly to one of the following stressful conditions: food/water deprivation, forced swimming test, and permanence in cold room. Indeed, stressed hamsters treated with CNQX or Zol displayed a very great (p < 0.001) increase of entrance plus a moderate (p < 0.05) time spent into open arms, respectively. At the cellular level, Zol-treated animals supplied a moderately evident argyrophilic reaction (indicative of neurodegeneration) in the hippocampus while it was absent in the hypothalamus. Interestingly, this reaction was significantly reduced by CNQX supporting its preferential protective role. Furthermore, both agents were responsible for a mixed expression pattern of GluR1 and GluR2 mRNA levels in which Zol overall upregulated GluR1 mRNAs, while they were downregulated by CNQX in the hippocampal oriens-pyramidalis layer and in layer III of the cerebral cortex. These findings support the amygdalar AMPAergic protective response against anxiety states in chronically stressed hamsters, which may constitute useful therapeutic strategies for panic-related mood disorders.

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

BackgroundThe structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) 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, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR 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.ResultsBoth autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (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 α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression 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 β3 and γ3 mRNAs 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 β3 and γ2 during hibernating conditions.ConclusionWe 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 GABAAR 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.

Central amygdalar nucleus treated with orexin neuropeptides evoke differing feeding and grooming responses in the hamster.

Interaction of the orexinergic (ORXergic) neuronal system with the excitatory (glutamate, l-Glu) or the inhibitory (GABA) neurosignaling complexes evokes major homeostatic physiological events. In this study, effects of the two ORXergic neuropeptides (ORX-A/B) on their receptor (ORX-2R) expression changes were correlated to feeding and grooming actions of the hibernating hamster (Mesocricetus auratus). Infusion of the central amygdala nucleus (CeA) with ORX-A caused hamsters to consume notable quantities of food, while ORX-B accounted for a moderate increase. Interestingly the latter neuropeptide was responsible for greater frequencies of grooming with respect to both controls and the hamsters treated with ORX-A. These distinct behavioral changes turned out to be even greater in the presence of l-Glu agonist (NMDA) while the α1 GABAA receptor agonist (zolpidem, Zol) greatly reduced ORX-A-dependent feeding bouts. Moreover, ORX-A+NMDA mainly promoted greater ORX-2R expression levels with respect to ORX-A-treated hamsters while ORX-B+Zol was instead largely responsible for a down-regulatory trend. Overall, these features point to CeA ORX-2R sites as key sensory limbic elements capable of regulating eating and grooming responses, which may provide useful insights regarding the type of molecular mechanism(s) operating during feeding bouts.

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.

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-1β 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-1β 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.

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.