Recent studies have demonstrated that an acute, low dose of ketamine, a non-competitive NMDA receptor antagonist, provides rapid and sustained antidepressant effects in patients with major depressive disorder. Studies in rodents have shown that the antidepressant properties of ketamine are due to an increase in dendritic spine density in the cortex. Our goal was to determine whether these effects are specific to ketamine and whether they are dependent on dose, drug regimen and brain region. We observed that the effects of ketamine on spine density were dependent on dose and drug regimen and were also brain region specific. In addition, MK-801, another NMDA receptor antagonist, did not demonstrate the same effects on spine density as ketamine. Furthermore, genetic NMDA receptor hypofunction significantly reduced spine density. Our studies demonstrate that while acute ketamine treatment leads to an increase in cortical spine density, chronic administration has opposite and potentially detrimental effects.

Calcium (Ca2+) influx through N-methyl-D-asparate receptors (NMDARs) is widely held to be the requisite step initiating delayed neuronal death following ischemic stroke. However, blocking NMDARs fails to prevent the accumulation of intracellular Ca2+ ([Ca2+]i) and subsequent neurotoxicity. This suggests that alternate, as yet uncharacterized Ca2+-influx pathways exist in neurons. Transient receptor melastatin 2 (TRPM2) is a Ca2+-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the central nervous system (CNS), the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. Here, I employ a combination of molecular, biochemical and electrophysiological approaches to demonstrate that functional TRPM2 channels are expressed in pyramidal neurons of the hippocampus. Unlike in heterologous expression systems, the ADPR-dependent activation of TRPM2 in neurons required a concomitant rise in [Ca2+]i via either voltage-dependent Ca2+ channels or NMDARs. While short, repeated NMDA applications activated a TRPM2-like current in the absence of exogenous ADPR, sustained NMDA application to hippocampal neurons resulted in the activation of a pannexin1 (Px1) hemichannel. Px1 hemichannels are large conductance, nonjunctional gap junction channels that can be activated following periods of oxygen-glucose deprivation (OGD) in neurons. Activation of Px1 required the influx of Ca2+ through NMDARs. Supplementing the intracellular milieu with adenosine triphosphate (ATP) prevented Px1 activation, suggesting that hemichannels may be activated during periods of mitochondrial dysfunction and metabolic failure. Our findings have potential implications for the treatment of diseases such as cerebral ischemia and Alzheimer’s disease (AD) as they implicate two novel ion channels in the excitotoxic signaling cascade activated downstream of NMDARs.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Recent studies have demonstrated that an acute, low dose of ketamine, a non-competitive NMDA receptor antagonist, provides rapid and sustained antidepressant effects in patients with major depressive disorder. Studies in rodents have shown that the antidepressant properties of ketamine are due to an increase in dendritic spine density in the cortex. Our goal was to determine whether these effects are specific to ketamine and whether they are dependent on dose, drug regimen and brain region. We observed that the effects of ketamine on spine density were dependent on dose and drug regimen and were also brain region specific. In addition, MK-801, another NMDA receptor antagonist, did not demonstrate the same effects on spine density as ketamine. Furthermore, genetic NMDA receptor hypofunction significantly reduced spine density. Our studies demonstrate that while acute ketamine treatment leads to an increase in cortical spine density, chronic administration has opposite and potentially detrimental effects.

The experiments reported in this thesis studied the contributions of opioid and NMDA receptors to predictive fear learning, as measured by freezing in the rat. The first series of experiments (Chapter 2) used a within-subject one-trial blocking design to study whether opioid receptors mediate a direct action of predictive error on Pavlovian association formation. Systemic administrations of the opioid receptor antagonist naloxone or intra-vlPAG administrations of the selective μ-opioid receptor antagonist CTAP prior to Stage II training prevented one-trial blocking. These results show for the first time that opioid receptors mediate the direct actions of predictive error on Pavlovian association formation. The second series of experiments (Chapter 3) then studied temporal-difference prediction errors during Pavlovian fear conditioning. In Stage I rats received CSA ?? shock pairings. In Stage II they received CSA/CSB ?? shock pairings that blocked learning to CSB. In Stage III, a serial overlapping compound, CSB → CSA, was followed by shock. The change in intra-trial durations supported fear learning to CSB but reduced fear of CSA, revealing the selective operation of temporal-difference prediction errors. This bi-directional change in responding was prevented by systemic NMDA receptor antagonism prior to Stage III training. In contrast opioid receptor antagonism differentially affected the learning taking place during Stage III, enhancing learning to CSB while impairing the loss of fear to CSA. The final series of experiments (Chapter 4) then examined potential neuroanatomical loci for the systemic effects reported in Chapter 3. It was observed that intra-BLA infusion of ifenprodil, an antagonist of NMDA receptors containing the NR2B subunit, prevented all learning during Stage III, whereas intra-vlPAG infusion of the μ-opioid receptor antagonist CTAP facilitated learning to CSB but impaired learning to CSA. These results are consistent with the suggestion that opioid receptors in the vlPAG provide an important contribution to learning. Importantly, this contribution of the vlPAG is over and above its role in producing the freezing conditioned response. Furthermore, the findings of this thesis identify complementary but dissociable roles for amygdala NMDA receptors and vlPAG μ-opioid receptors in predictive fear learning.

Universiẗat, Diss., 2006--Frankfurt (Main).

Zugl.: Leipzig, Univ., Diss., 2009

Το γλουταμινικό οξύ αποτελεί τον κύριο διεγερτικό νευροδιαβιβαστή στο Κεντρικό Νευρικό Σύστημα των θηλαστικών, παίζοντας σημαντικό ρόλο σε διαδικασίες μάθησης και μνήμης, αλλά και σε διεργασίες διεγερσιμοτοξικότητας. Το γλουταμινικό οξύ ασκεί τις δράσεις του μέσω δύο ομάδων υποδοχέων, των ιοντοτρόπων και των μεταβοτροπικών υποδοχέων. Οι ιοντοτρόποι υποδοχείς είναι δίαυλοι ιόντων, των οποίων η ενεργοποίηση προκαλεί ταχεία διεγερτική νευροδιαβίβαση και διακρίνονται σε δύο μεγάλες υποομάδες ,τους NMDA και τους μη- NMDA υποδοχείς. Οι μεταβοτροπικοί υποδοχείς, συνδέονται με G-πρωτεΐνες και προκαλούν αργή συναπτική νευροδιαβίβαση μέσω ενεργοποίησης δεύτερων αγγελιοφόρων. Διακρίνονται σε τρεις μεγάλες ομάδες: το Group I που περιλαμβάνει τους mGluR1 και mGluR5, οι οποίοι ενεργοποιούν την Φωσφολιπάση C (PLC), το Group II που περιλαμβάνει τους mGluR 2 και 3 και το Group III που περιλαμβάνει τους mGluRs 4, 6, 7και 8, οι οποίοι ρυθμίζουν αρνητικά την αδενυλική κυκλάση. Οι μεταβοτροπικοί υποδοχείς διαδραματίζουν σημαντικό νευροτροποποιητικό ρόλο σε ολόκληρο τον εγκέφαλο και αποτελούν στόχο για θεραπευτική αγωγή διαφόρων ψυχιατρικών και νευρολογικών διαταραχών όπως το άγχος, η κατάθλιψη, το σύνδρομο του εύθραυστου Χ και η σχιζοφρένεια. Στον ιππόκαμπο, μια δομή όπου διενεργούνται διεργασίες μάθησης και μνήμης, οι mGluR5 υποδοχείς είναι οι πλέον άφθονοι από την οικογένεια των Group I Μεταβοτροπικών υποδοχέων και παίζουν ρόλο στη ρύθμιση της συναπτικής πλαστικότητας και χωρικής μάθησης. Όπως έχει δειχθεί από ηλεκτροφυσιολογικές μελέτες, μια από τις πιο αξιοσημείωτες επιδράσεις των mGluR5 υποδοχέων είναι η ενίσχυση των NMDA-εξαρτώμενων ρευμάτων. Η δράση τους αυτή φαίνεται να παίζει ρόλο στη δημιουργία του LTP ( μακροχρόνια συναπτική ενίσχυση), καθώς η χορήγηση του ειδικού ανταγωνιστή των mGluR5 υποδοχέων (ΜΡΕΡ) σε ιππόκαμπειους νευρώνες επίμυος, αναστέλλει την επαγωγή του LTP. Στο ραβδωτό σώμα, οι mGluR5 υποδοχείς βρίσκονται υπό τον έλεγχο των Α2Α υποδοχέων αδενοσίνης, με τους οποίους σχηματίζουν ετεροδιμερή συμπλέγματα Α2Α-mGluR5 υποδοχέων. Στον ιππόκαμπο, οι Α2Α υποδοχείς αδενοσίνης εκφράζονται σε πολύ χαμηλά επίπεδα, ενώ είναι ελάχιστα τα βιβλιογραφικά δεδομένα για την πιθανή αλληλεπίδρασή τους με τους mGluR5 υποδοχείς. Προκειμένου να διερευνήσουμε το μοριακό μηχανισμό μέσω του οποίου οι mGluR5 υποδοχείς του γλουταμινικού οξέος ενισχύουν τα ρεύματα των NMDA υποδοχέων στον ιππόκαμπο επίμυος, εξετάσαμε την in vitro επίδραση της ενεργοποίησης των mGluR5, στο επίπεδο φωσφορυλίωσης της NR2B υπομονάδας του NMDA υποδοχέα, στα κατάλοιπα τυροσίνη-1472 και σερίνη-1303. Εν συνεχεία, διερευνήσαμε in vitro, την ενδεχόμενη αλληλεπίδραση των mGluR5 και Α2Α υποδοχέων στον ιππόκαμπο, μελετώντας την επίδραση της διέγερσης ή της αναστολής τους στην mGluR5-επαγόμενη φωσφορυλίωση των NMDA υποδοχέων. Τέλος, προκειμένου να ανιχνεύσουμε το ενδοκυττάριο μονοπάτι μεταγωγής σήματος των mGluR5 υποδοχέων στον ιππόκαμπο, εξετάσαμε την επίδραση της ενεργοποίησης των mGluR5 υποδοχέων στο επίπεδο φωσφορυλίωσης της ERK ½ κινάσης καθώς και την επίδραση της διέγερσης των Α2Α υποδοχέων αδενοσίνης στην ενεργοποίηση αυτή. Τα αποτελέσματά μας δείχνουν ότι η διέγερση των mGluR5 υποδοχέων του γλουταμινικού οξέος με τον ειδικό αγωνιστή τους, CHPG, επάγει μια ισχυρή φωσφορυλίωση του καταλοίπου τυροσίνη- 1472 της NR2B υπομονάδας, η οποία είναι εξειδικευμένη στη θέση αυτή δεδομένου ότι δεν εμφανίζονται στο κατάλοιπο σερίνη-1303. Σύμφωνα με τα υπάρχοντα βιβλιογραφικά δεδομένα, η φωσφορυλίωση του καταλοίπου τυροσίνη -1472 της NR2B υπομονάδας, παίζει ρόλο στη μετακίνηση του υποδοχέα στη μεμβράνη και στην ορθή τοποθέτηση του στη σύναψη (trafficking). Συγκεκριμένα, η φωσφορυλίωση στη θέση αυτή καταστέλλει την κλαθρίνη-εξαρτώμενη ενδοκυττάρωση των NMDA υποδοχέων, με αποτέλεσμα τη συσσώρευσή τους στη συναπτική μεμβράνη. Η συσσώρευση των NR2B-NMDA υποδοχέων και η σωστή τοποθέτηση τους στη συναπτική μεμβράνη προάγει τις NMDA εξαρτώμενες αποκρίσεις. Το γεγονός αυτό, συμφωνεί με τα ηλεκτροφυσιολογικά δεδομένα που δείχνουν ότι η διέγερση των mGluR5, ενισχύει τα ρεύματα των NMDA υποδοχέων και προάγει την επαγωγή του LTP στον ιππόκαμπο. Σύμφωνα με τα αποτελέσματά μας, θεωρούμε ότι η φωσφορυλίωση στη τυροσίνη-1472, αποτελεί το μοριακό μηχανισμό ( ή μέρος αυτού), μέσω του οποίου η ενεργοποίηση των mGluR5 υποδοχέων προάγει τα ρεύματα των NMDA διαύλων. Είναι ενδιαφέρον ότι, οι mGluR5 υποδοχείς του γλουταμινικού οξέος φαίνεται να βρίσκονται υπό τον έλεγχο των Α2Α υποδοχέων αδενοσίνης και στον ιππόκαμπο, όπως και στο ραβδωτό σώμα, καθώς τα αποτελέσματά μας δείχνουν ότι φαρμακευτική αναστολή των Α2Α υποδοχέων εξασθενεί σημαντικά την 10 ικανότητα των ιπποκάμπειων mGluR5 να ελέγχουν τις NMDA-επαγόμενες απαντήσεις. Άρα στον ιππόκαμπο φαίνεται ότι οι Α2Α υποδοχείς δρουν αδειοδοτικά για τη λειτουργία των mGluR5 υποδοχέων ως προς τη ρύθμιση των NMDA-επαγόμενων απαντήσεων. Τέλος, τα αποτελέσματά μας καταδεικνύουν ότι η ενεργοποίηση των mGluR5 υποδοχέων, με το ειδικό αγωνιστή τους CHPG, στον ιππόκαμπο, επάγει τη φωσφορυλίωση της ERK 1/2 κινάσης με τρόπο δοσοεξαρτώμενο. Η επαγωγή αυτή δεν υπόκειται στον έλεγχο των Α2Α υποδοχέων αδενοσίνης, καθώς η φαρμακευτική αναστολή τους δεν είχε επίδραση στα επίπεδα φωσφορυλίωσης της ERK 1/2 κινάσης. Η λειτουργική σημασία της ενεργοποίησης αυτής δεν είναι ακόμα γνωστή και αποτελεί στόχο μελλοντικών πειραμάτων μας. / --

Ca2+ is an important second messenger which modulates a variety of signalling pathways in both excitable and non-excitable cells. In the CNS, Ca2+ plays an important role in neurons both physiologically and pathologically. Ca2+ influx following synaptic activity, is important in development, plasticity, redox balance, as well as in neuroprotection, largely through activation of pro-survival pathways downstream of synaptic NMDAR activation, including upregulation of antioxidant defences. However, excessive Ca2+ influx in neurons leads to neuronal damage and excitotoxicity, in which mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (Mcu) resulting in mitochondrial dysfunction is a key player. Excitotoxicity occurs due to glutamate efflux from astrocytes following stroke, traumatic brain injury and in chronic neurodegenerative diseases, leading to excessive neuronal NMDAR activation and triggering of its downstream pro-death pathways. This thesis focuses on understanding the pro-survival and pro-death effects of signalling pathways activated by Ca2+ in neurons, as well as the potential effect of neuronal synaptic activity on influencing neuroprotective gene transcription in astrocytes. I investigated the role of AMPK, a master regulator of metabolism, in NMDA excitotoxicity in cortical neurons as a potential downstream effector of Mcu-dependent excitotoxic death; and found the deletion of AMPKα1/2 to be neuroprotective against NMDA-mediated excitotoxicity, however I found AMPK activation to be independent of Mcu. I also investigated the expression pattern of Mcu and other mitochondrial calcium regulatory genes (MCRGs), and found MCRGs to be differentially expressed in different neural cells (primary neurons vs astrocytes), and neuronal subtypes (CA1 vs CA3 region of the hippocampus), suggesting differing dependence on the various MCRGs in mitochondrial Ca2+ handling in these cell types. A better functional understanding of these genes will allow for investigation of their importance in mitochondrial Ca2+ handling, including their potential role in excitotoxicity. I next investigated the neuroprotective effects of synaptic activity induced Ca2+ influx, focusing on antioxidant target genes of Nrf2, a transcription factor and major regulator of antioxidant genes. I found that unlike astrocytes, neurons express very low levels of Nrf2. However, synaptic activity increased the expression of several Nrf2 target genes in neurons, independently of astrocytes and Nrf2. Additionally, I found no effect of synaptic activity on increasing Nrf2 protein levels, despite previous reports in literature of Nrf2 pathway activation following synaptic activity. Finally, using RNA-seq I identified a list of genes strongly upregulated by a known Nrf2 activator in astrocytes, and found no evidence that neuronal activity triggers expression of these genes independently of neurons, providing further evidence that neuronal activity does not activate the Nrf2 pathway in astrocytes. This suggests that synaptic activity via pathways activated by Ca2+ signalling provides neurons with cell-autonomous antioxidant defences, independently of Nrf2; thus providing a distinct pathway for antioxidant defences in neurons from the Nrf2 pathway, which is activated in astrocytes providing neurons with non-cell autonomous antioxidant support. These results give us further insight into the mechanisms that underlie synaptic and non-synaptic Ca2+ signalling pathways mediating neuronal survival and death, which could help in identifying therapeutic targets to combat excitotoxicity and oxidative stress in various neurological diseases.

ARAUJO, João Ronielly Campelo. Efeito antidepressivo-símile da frutalina, lectina α-d-galactose ligante, isolada de sementes de Artocarpus incisa L., em camundongos. 2016. 79 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Anderson Silva Pereira (anderson.pereiraaa@gmail.com) on 2017-04-03T18:22:06Z No. of bitstreams: 1 2016_dis_jrcaraújo.pdf: 2086872 bytes, checksum: 3373789680d8de097182e323441225d7 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-04-04T22:36:31Z (GMT) No. of bitstreams: 1 2016_dis_jrcaraújo.pdf: 2086872 bytes, checksum: 3373789680d8de097182e323441225d7 (MD5) / Made available in DSpace on 2017-04-04T22:36:31Z (GMT). No. of bitstreams: 1 2016_dis_jrcaraújo.pdf: 2086872 bytes, checksum: 3373789680d8de097182e323441225d7 (MD5) Previous issue date: 2016 / Frutalin (FTL), an α-D-galactose-binding lectin isolated from breadfruit seeds (Artocarpus incisa L.), has a range biological activities, but has not been conclusively shown to act on CNS disorders. In this study we evaluated the effect of FTL on mouse behavior. Mice (n=6/group) were treated with FTL (0.25; 0.5 or 1 mg/kg; i.p.) or vehicle (NaCl 0.9 %;10 mL/kg; i.p.) and submitted to hole-board (HBT), elevated plus maze (PMT), open field (OFT), tail suspension (TST) and forced swimming (FST) tests. In a second set of experiments, yohimbine (1 mg/kg), ketamine (0.1 mg/kg), L-NAME (10 mg/kg) or methylene blue (10 mg/kg) were administered (i.p.) 30 min before FTL (0.5 mg/kg). In order to evaluate the subchronic effect of FTL, animals were injected with FTL (0.5 mg/kg) or vehicle for 7 days and submitted to FST on the first and last day of treatment. A molecular docking was conducted on the NOS enzyme and NMDA receptor. No changes were observed in HBT and OFT results. The smallest dose of FTL (0.25 mg/kg) was associated with an increase in the number of entries into closed arms in PMT (p<0.05). FTL reduced immobility in TST (0.25 and 0.5 mg/kg; p<0.05) and FST (0.25 mg/kg; p<0.05 and 0.5 mg/kg; p<0.01). In FST, the effect of FTL was dependent on carbohydrate interaction and protein structure integrity and it was reduced by ketamine (NMDA antagonist), L-NAME (non-selective NOS inhibitor) and methylene blue (soluble guanylyl cyclase inhibitor). The antidepressant-like effect remained after subchronic treatment. Matching the results of the experiment in vivo, the docking study indicated an interaction between FTL and NOS enzyme and NMDA receptor. In conclusion, FTL was found to have an antidepressant-like effect mediated by the NMDA receptor/NO/cGMP pathway. / Frutalina (FTL), uma lectina α-D-galactose ligante, obtida de sementes de Artocarpus incisa L., tem apresentado várias atividades biológicas, como ação na modulação de alvos moleculares e reversão de neurotoxicidade in vitro, porém, há pouca evidência de seu efeito sobre doenças no Sistema Nervoso Central (SNC). Diante disto, este estudo avaliou os efeitos neurocomportamentais da FTL em camundongos. Os camundongos (n = 6 / grupo) foram tratados com FTL (0,25; 0,5 ou 1 mg/kg; i.p.) ou Veículo (NaCl 0,9 % 10 mL/kg; i.p.; Controle) e submetidos aos testes da placa perfurada, labirinto em cruz elevado, campo aberto, suspensão pela cauda ou natação forçada. Num segundo conjunto de experimentos, a ioimbina (1 mg/kg), cetamina (0,1 mg/kg), L-NAME (10 mg/kg) ou azul de metileno (10 mg/kg) foram administrados (i.p.) 15 min antes da FTL (0,5 mg/kg) e os animais foram submetidos ao teste de natação forçada. Verificou-se também se o efeito de FTL no teste de natação forçada era dependente da integridade estrutural e capacidade de interação a carboidratos. A fim de avaliar o efeito subcrônico da FTL, os camundongos receberam FTL (0,5 mg/kg) ou veículo durante 7 dias e submetidos ao teste de natação forçada no primeiro e último dia de tratamento. Foi realizado docking molecular da FTL com NOS e receptor NMDA. Os resultados mostraram que não houve alterações neurocomportamentais dos camundongos nos testes de placa perfurada e campo aberto. FTL na dose mais baixa (0,25 mg/kg) aumentou o número de entradas nos braços fechados no teste do labirinto em cruz elevado, permitindo sugerir um possível efeito do tipo ansiogênico. FTL reduziu o tempo de imobilidade nos testes de suspensão pela cauda (0,25 e 0,5 mg/kg; p <0,05) e natação forçada (0,25 mg/kg, p <0,05 e 0,5 mg/kg; p <0,01) apresentando um efeito antidepressivo-símile. A redução da imobilidade provocada pela FTL foi prevenida pela Cetamina (antagonista de receptores NMDA), L-NAME (inibidor não-seletivo da NOS) e azul de metileno (inibidor da cGMP), mas não pela Ioimbina (antagonista α2-adrenérgico). A desnaturação da FTL, bem como a sua associação à galactose, também preveniu o efeito antidepressivo-símile da lectina. O efeito antidepressivo-símile da FTL permaneceu o mesmo após tratamento subcrônico (7 dias) e não houve alteração no peso dos animais. Corroborando com os resultados in vivo, os estudos de docking molecular demonstraram que a FTL interage com a enzima NOS e receptor NMDA. Nossos resultados demonstraram que a FTL possui efeito antidepressivo-símile mediado por receptores NMDA e via L-Arginina/NO/cGMP, além de ser dependente de sua integridade estrutural e capacidade de ligação a α-D-galactose.