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Projeto de investigação
MODULATORY ROLE OF ADENOSINE AND BDNF UPON INHIBITORY SYNAPTIC TRANSMISSION AND PLASTICITY: CONSEQUENCES FOR EPILEPSY
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Extracellular alpha-synuclein oligomers modulate synaptic transmission and impair ltp via NMDA-receptor activation
Publication . Diógenes, Maria José; Dias, Raquel Baptista; Rombo, Diogo M.; Vicente Miranda, Hugo; Maiolino, Francesca; Guerreiro, Patrícia; Nasstrom, Thomas; Franquelim, Henri; Oliveira, Luís M.A.; Castanho, Miguel A. R. B.; Lannfelt, Lars; Bergstrom, Joakim; Ingelsson, Martin; Quintas, Alexandre; Sebastião, Ana M; Lopes, Luisa V.; Outeiro, Tiago
Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.
Modulatory role of adenosine upon GABAergic transmission : consequences for excitability control
Publication . Rombo, Diogo Miguel Santos, 1986-; Sebastião, Ana Maria, 1958-
Glutamatergic principal cell excitability in the hippocampus is regulated by local circuit neurons that release the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). These GABAergic interneurons exhibit vast structural, physiological and biochemical diversity, innervating both excitatory principal cells and other inhibitory interneurons. In the hippocampus, two classes of interneurons, the cholecystokinin (CCK)- and parvalbumin (PV)-containing neurons, are the most significant and abundant cell type displaying unique and complementary functions in the control of principal cells output. Hence a tuned modulation of inhibitory circuits is of great importance in the control of network hippocampal function. Adenosine, acting through high affinity A1 receptor (A1R) and A2A receptor (A2AR), is a well-recognized endogenous modulator of glutamatergic principal cells excitability. Actions mediated by A1Rs are long-known to decrease hippocampal excitability with neuroprotective effects while actions through A2ARs are associated with increased neuronal excitability and excitotoxicity. However, the role of adenosine to modulate inhibitory transmission is much less known. This work aimed to evaluate and characterize the involvement of A1Rs (Chapter 5.1, p99) and A2ARs (Chapter 5.2, p143) on inhibitory neuronal communication in CA1 hippocampus and its impact on principal cells excitability and in the control of epileptiform discharges. These main goals were achieved by performing ex vivo electrophysiology recordings (field and patch-clamp recordings) from rat and mice hippocampus. Regarding A1R-actions, it was found that tonic - mediated by GABA receptor type A (GABAAR) localized peri- and extrasynaptically - but not phasic - mediated by GABAARs located at synapses - inhibitory transmission in pyramidal cells and CCKpositive interneurons were diminished after A1R activation. The effect was dependent on a signaling cascade involving both protein kinase A (PKA) and protein kinase C (PKC) and was accompanied by decreased GABAAR δ-subunit expression. On the other hand, it was also found that A2AR-mediated increase in pyramidal cells excitability results from a direct increase of glutamatergic transmission in parallel with disinhibition of principal cells by a mechanism that involves increased GABA release from PV-positive cells to other interneurons. Also, A2AR activation or blockage respectively promotes or reduces synchronous pyramidal cell firing in hyperexcitable conditions induced by elevated extracellular potassium or following high-frequency electrical stimulation. Together the results presented in this thesis show for the first time a direct involvement of adenosine receptors in the control of inhibitory network transmission in the hippocampus. This results open new promising perspectives for the involvement of adenosine in the control of physiological hippocampal operations and maladaptive conditions.
Ex vivo model of epilepsy in organotypic slices : a new tool for drug screening
Publication . Magalhães, Daniela; Pereira, Noémia; Rombo, Diogo M.; Beltrão-Cavacas, Cláudia; Sebastião, Ana M; Valente, Cláudia A.
Background: Epilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy.
Methods: We assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation.
Results: We demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1β (IL-1β), interleukin-6, and tumor necrosis factor α, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1β quantification.
Conclusions: Thus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.
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Fundação para a Ciência e a Tecnologia
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SFRH/BD/60386/2009