Maria Mele

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.

ANTIHYPERTENSIVE AND NEUROPROTECTIVE EFFECTS OF CATESTATIN IN SPONTANEOUSLY HYPERTENSIVE RATS: INTERACTION WITH GABAERGIC TRANSMISSION IN AMYGDALA AND BRAINSTEM

The chromogranin A-derived peptide catestatin (CST) exerts sympathoexcitatory and hypertensive effects when microinjected into the rostral ventrolateral medulla (RVLM: excitatory output); it exhibits sympathoinhibitory and antihypertensive effects when microinjected into the caudal ventrolateral medulla (CVLM: inhibitory output) of vagotomized normotensive rats. Here, continuous infusion of CST into the central amygdalar nucleus (CeA) of spontaneously hypertensive rats (SHRs) for 15 days resulted in a marked decrease of blood pressure (BP) in 6-month- (by 37 mm Hg) and 9-month- (by 65 mm Hg)old rats. Whole-cell patch-clamp recordings on pyramidal CeA neurons revealed that CST increased both spontaneous inhibitory postsynaptic current (sIPSC) amplitude plus frequency, along with reductions of sIPSC rise time and decay time. Inhibition of GABAA receptors (GABAARs) by bicuculline completely abolished CST-induced sIPSC, corroborating that CST signals occur through this major neuroreceptor complex. Hypertension is a major risk factor for cerebrovascular diseases, leading to vascular dementia and neurodegeneration. We found a marked neurodegeneration in the amygdala and brainstem of 9-month-old SHRs, while CST and the GABAAR agonist Muscimol provided significant neuroprotection. Enhanced phosphorylation of Akt and ERK accounted for these neuroprotective effects through anti-inflammatory and anti-apoptotic activities. Overall our results point to CST exerting potent antihypertensive and neuroprotective effects plausibly via a GABAergic output, which constitute a novel therapeutic measure to correct defects in blood flow control in disorders such as stroke and Alzheimers disease.

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.

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

UNLABELLED 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.

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.

Catestatin and orexin-A neuronal signals alter feeding habits in relation to hibernating states.

Hibernation is a physiological state that by putting vital biological processes at rest enables mammals to protect all organs, especially the brain against ischemic insults and reperfusion injuries. Earlier studies have highlighted the role of hypothalamic (HTH) sites like the periventricular nucleus (Pe) toward sleep-wake and cardiovascular activities of hibernators. In the present work, infusions of Pe with the orexigenic neuropeptide orexin-A (ORX-A) or the novel anti-obesity sympathoinhibitory neuroactive peptide catestatin (CST) have been correlated to differing feeding and motor behaviors in the facultative hibernating hamster Mesocricetus auratus. Behavioral observations showed that treatment with CST provided an anti-obesity activity via the reduction of food intake and body weight for all hibernating states, while ORX-A promoted orexigenic events during mainly the entrance phase. Moreover, hamsters treated with this neuropeptide during the entrance and the arousal hypertensive phases also featured elevated ORX 2 receptor (ORX2R) levels in the third layer of the parietal cortex and lateral HTH (LH), areas involved with feeding, motor plus sleep-wake rhythms. Conversely, ORX-A down-regulated ORX2Rs in the ventromedial (VMH) and supraoptic (SO) HTH nuclei that are associated with anorexigenic effects. Even CST induced mixed ORX2R expression patterns in mostly HTH areas like the evident down-regulation in LH along with the up-regulation in VMH and SO. Overall treatments, especially ORX-A+CST led to reduced neurodegenerative phenomena in HTH supporting their importance together with ORX2Rs in preserving hemodynamic activities, feeding and sleep-wake rhythms of this diencephalic station, which may supply useful therapeutic indications for treating cardiovascular disturbances linked with brain dysfunctions.

Expression variations of chromogranin A and α1,2,4 GABAARs in discrete limbic and brainstem areas rescue cardiovascular alterations

Recent interferences of hemodynamic functions via modified brain neuronal mechanisms have proven to be major causes of dementia and sleeping disorders. In this work, cerebral expression differences of the neuroactive vesicular chromogranin A (CgA) and distinct α GABA(A)R subunits were detected in the facultative hibernating hamster. In particular, damaged neuronal fields of hypotensive torpor (TORP) state were correlated to elevated CgA and GABA(A)R α1, α4 mRNA levels in the paraventricular hypothalamic nucleus (PVN), central amygdalar nucleus (CeA) plus solitary tractus nucleus (NTS). Conversely, few neurodegeneration signals of hypertensive arousal (AROU) state, accounted for mostly lower CgA levels in the same areas. This state also provided increased α2-containing sites in amygdala, hippocampal and NTS neurons together with elevated α4-containing receptors in the periventricular hypothalamic nucleus (Pe). Interestingly in our hibernating model, CgA appeared to preferentially feature inhibitory neurosignals as indicated by preliminary perfusion of amygdalar sites with its highly specific antihypertensive derived peptide (catestatin) promoting GABA-dependent sIPSCs. Overall, evident neuronal damages plus altered expression capacities of CgA and α1-, α2-, α4-GABA(A)Rs in CeA, Pe, PVN as well as NTS during both hibernating states corroborate for the first time key molecular switching events guaranteeing useful cardiovascular rescuing abilities of neurodegenerative 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.

Distinct anxiogenic/anxiolytic effects exerted by the hamster lateral amygdalar nucleus injected with ORX-A or ORX-B in the presence of a GABAergic agonist.

Recently, there has been growing interest in the neurobiological role of some amygdalar neuromediators, such as GABA and orexins (ORX), that are responsible for stressful behaviors. Infusion of the major fear-related and panic-related basolateral amygdalar station, the lateral nucleus, with ORX-A and ORX-B, alone or in combination with the main &agr;1-containing GABAA receptor agonist (zolpidem), modified anxiety states of the Syrian hamster. Single daily doses of ORX-A led to evident anxiogenic features, as pointed out by more time spent in the dark compartment of the light–dark exploration test, effects that were suppressed by zolpidem. Conversely, doses of ORX-B induced anxiolytic effects, whereas the concomitant administration of this neuropeptide with zolpidem strongly favored anxiogenic responses. In addition, these behavioral responses resulted in a widely correlated upregulation of the ORX-2 receptor in some key feeding and motor limbic areas, such as the ventromedial hypothalamic nucleus, central amygdalar nucleus, and hippocampal CA1 layer. Overall, these first indications on the differing anxiety states induced by ORX-A and ORX-B injected into the lateral amygdalar nucleus alone or in combination with zolpidem may constitute useful future therapeutic alternatives for the treatment of panic disorders as well as stressful behaviors.