Astrocyte dystrophy in ageing brain parallels impaired synaptic plasticity

Little is known about age-dependent changes in structure and function of astrocytes and of the impact of these on the cognitive decline in the senescent brain. The prevalent view on the age-dependent increase in reactive astrogliosis and astrocytic hypertrophy requires scrutiny and detailed analysis. Using two-photon microscopy in conjunction with 3D reconstruction, Sholl and volume fraction analysis, we demonstrate a significant reduction in the number and the length of astrocytic processes, in astrocytic territorial domains andin astrocyte-to-astrocyte coupling in the aged brain. Probing physiology of astrocytes with patch clamp, and Ca2+ imaging revealed deficits in K+ and glutamate clearance and spatiotemporal reorganisation of Ca2+ events in old astrocytes. These changes paralleled impaired synaptic long-term potentiation (LTP) in hippocampal CA1 in old mice. Our findings may explain the astroglial mechanisms of age-dependent decline in learning and memory.The research was supported by the Russian Science Foundation grant 20‐14‐00241

Modulation of excitatory synaptic transmission to hippocampal interneurons by metabotropic glutamate receptors.

The hippocampus is a medial temporal lobe structure implicated both in consolidation of experience into long-term memory and generation of epileptiform discharges. Information processing in the hippocampus occurs through interactions between glutamatergic granule cells and pyramidal neurons, and a smaller number of GABAergic inhibitory interneurons. Excitatory connections onto interneurons are a relatively poorly characterised class of synapse, yet they have a central role in mediating the recruitment of inhibitory drive within the hippocampus. This thesis describes investigation of modulation of excitatory synaptic transmission to interneurons in area CA1 of the hippocampus by metabotropic glutamate receptors (mGluRs). mGluRs are G protein-coupled heptahelical transmembrane receptors, which exert powerful modulatory effects upon synaptic transmission and neuronal excitability. Use of patch clamp electrophysiology in acute brain slices allowed synaptic responses elicited by stimulation of afferent inputs to be recorded in single neurons within a functionally intact network. The selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was found to acutely depress glutamatergic transmission to stratum radiatum interneurons in the rat hippocampal CA1 subfield. Both mGluRI and mGluR5 subtypes contributed to this phenomenon. DHPG-evoked depression was consistently accompanied by an elevation in paired-pulse ratio, implying a presynaptic mechanism of expression. However, it was also attenuated by blocking G protein and Ca2+ signalling within the postsynaptic neuron, arguing for a postsynaptic site of induction. The DHPG-evoked depression was unaffected by antagonists of GABAB and CB1 endocannabinoid receptors but was occluded when presynaptic P/Q-type Ca2+ channels were blocked. A heterosynaptic depression was observed in a test pathway when a high-frequency tetanus was delivered to an independent conditioning pathway. This depression was reversible and abolished by group I mGluR antagonists. Group I mGluRs thus provide a mechanism for population activity in glutamatergic synapses to influence synaptic excitation of cortical interneurons

Optical induction of presynaptic plasticity using synaptoPAC

A major topic in neuroscience is the cellular basis of learning and memory. Memories are stored in neuronal engrams, co-active neurons that are synaptically connected. Synaptic plasticity, due to its ability to alter synaptic weight within a neuronal network, has been hypothesised to play a role in information storage. Despite decades of research, little is known about presynaptic plasticity, a form of plasticity defined as activity-dependent modulation of neurotransmitter release. Remarkably, the specific contribution of presynaptic plasticity to behaviour is unknown, which is in part due to a lack of methods that allow its specific in vivo manipulation. In order to overcome these limitations, we have engineered and characterised synaptoPAC, a novel optogenetic tool that allows induction of presynaptic plasticity. SynaptoPAC is designed as the fusion of the photoactivated adenylyl cyclase bPAC to the presynaptic vesicle protein synaptophysin. The design allows an increase of cyclic adenosine monophosphate (cAMP) in presynaptic terminals using light. Elevated presynaptic cAMP was previously demonstrated to cause an increase in release probability and induce presynaptic potentiation at specific synapses. With immunofluorescence imaging of cultured neurons we demonstrated that synaptoPAC is enriched at presynaptic terminals, as indicated by its co-localization with the synaptic vesicle protein VGLUT1. We verified the light-driven increase of cAMP by synaptoPAC by performing electrophysiological whole-cell recordings of ND7/23 cells co-expressing synaptoPAC and the cAMP-gated channel SthK. In whole-cell recordings of autaptic hippocampal cultures expressing synaptoPAC, we observed an increase of neurotransmitter release during light stimulation as well as concomitant changes in short-term plasticity properties in granule cells, but not in other cell types. In vivo expression of synaptoPAC in the dentate gyrus of the hippocampus and subsequent field excitatory postsynaptic potential (fEPSP) recordings in acute brain slices enabled us to demonstrate optically induced long-term plasticity in mossy fibre-CA3 synapses. Interestingly, activation of the tool did not cause increase in the amplitude of Schaffer collateral-CA1 synapse fEPSPs in in vitro recordings, indicating that synaptoPAC can induce potentiation only in synapses that are already predisposed to presynaptic plasticity. Further investigations in hippocampal slice preparations of short-term plasticity in mossy fibre synapses confirmed the presynaptic nature of the optical potentiation. Our results establish synaptoPAC as a valid tool that can be used to answer questions regarding the role of presynaptic plasticity in the brain and increase understanding of diseases characterised by impairments of this kind of plasticity.Ein wichtiges Thema der Neurowissenschaften ist die zelluläre Grundlage von Lernen und Gedächtnis. Gedächtnisspuren werden in neuronalen Engrammen gespeichert, dies sind koaktive Neurone, welche synaptisch miteinander verbunden sind. Es wird angenommen, dass synaptische Plastizität aufgrund ihrer Eigenschaft, synaptische Gewichtungen innerhalb eines neuronalen Netzwerks zu verändern, eine Rolle bei der Informationsspeicherung spielt. Trotz jahrzehntelanger Forschung ist wenig über präsynaptische Plastizität bekannt, eine Form von Plastizität, die als aktivitätsabhängige Modulation der Neurotransmitterfreisetzung definiert wird. Bemerkenswerter Weise ist kein spezifischer Beitrag der präsynaptischen Plastizität zu Verhalten bekannt, hauptsächlich auf Grund eines Mangels an Methoden, die ihre spezifische Manipulation in vivo erlauben. Um diese Limitierungen zu überwinden, entwickelten und charakterisierten wir synaptoPAC, ein neues optogenetisches Werkzeug, das eine lichtvermittelte Induktion präsynaptischer Plastizität ermöglicht. SynaptoPAC wurde als Fusion der photoaktivierten Adenylylzyklase bPAC mit dem präsynaptischen Vesikelprotein Synaptophysin konzipiert. Dieses Design ermöglicht eine Erhöhung von cyclischem Adenosinmonophosphat (cAMP) in präsynaptischen Terminalen mittels Licht. Es wurde bereits gezeigt, dass erhöhtes präsynaptisches cAMP an bestimmten Synapsen eine Zunahme der Freisetzungswahrscheinlichkeit bewirkt und eine präsynaptische Potenzierung induziert. Mittels immunfluoreszenzmikroskopischer Bildgebung von kultivierten Neuronen zeigten wir eine Anreicherung von synaptoPAC in präsynaptischen Terminalen, was durch eine Ko- Lokalisation mit dem synaptischen Vesikelprotein VGLUT1 indiziert wurde. Wir verifizierten die lichtgesteuerte Erhöhung von cAMP durch synaptoPAC mittels elektrophysiologischer Ganzzellableitungen an ND7/23 Zellen, welche synatoPAC und den cAMP-gesteuerten SthK Kanal koexprimierten. Bei Ganzzellableitungen von synaptoPAC exprimierenden, autaptischen Kulturen hippokampaler Neurone beobachteten wir eine Zunahme der Transmitterfreisetzung während einer Stimulation mit Licht spezifisch in Körnerzellen, aber nicht in anderen Zelltypen, und eine damit einhergehende Veränderungen der Kurzzeitplastizität. In vivo Expression von synaptoPAC im Gyrus Dentatus und anschließende Feldpotenzialmessungen von exzitatorischen postsynaptischen Potenzialen in akuten Hirnschnitten ermöglichte uns, eine optisch induzierte Langzeitpotenizierung an Moosfaser-CA3 Synapsen zu demonstrieren. Interessanterweise bewirkte die Aktivierung dieses Werkzeugs keine Verstärkung der Transmitterfreisetzung in Schaffer Kollateral-CA1 Synapsen in in-vitro Messungen, was darauf hindeutet, dass synaptoPAC nur in Synapsen, die bereits für präsynaptische Plastizität prädisponiert sind, eine Potenzierung induzieren kann. Weitere Untersuchungen an hippocampalen Schnittpräparationen zur Veränderung der Kurzzeitplastizität durch synaptoPAC in Moosfasern bestätigten den präsynaptischen Charakter der optischen Potenzierung. Unsere Ergebnisse etablieren synaptoPAC als valides Werkzeug, das zur Beantwortung offener Forschungsfragen zur Rolle der präsynaptischen Plastizität im Gehirn eingesetzt werden kann, und unser Verständnis von Krankheiten verbessern könnte, welche durch eine Beeinträchtigungen dieser Art von neuronaler Plastizität gekennzeichnet sind

‘Astrocytic cradle’ controls extracellular potassium and glutamate during synaptic transmission

It is widely recognised that astrocytes are able to shape synaptic transmission by restricting glutamate transients to the synaptic cleft. In this thesis, I demonstrate that during synaptic transmission K+ efflux through postsynaptic NMDA receptors depolarises the astrocytic membrane and thus slows down glial glutamate uptake. This effect involves the rectifying K+ channels (Kir4.1), predominantly located at perisynaptic astrocytic processes (PAPs). Genetic upregulation of this channel subtype in astrocytes does not affect glutamate transporters efficiency but curtails increase in presynaptic glutamate release probability during extracellular K+ rises. Thus, activity-dependent accumulation of extracellular K+ can boost glutamate release from the presynaptic site while decreasing astroglial glutamate uptake. Both factors occasion increased extrasynaptic glutamate escape and therefore inter-synaptic crosstalk in the hippocampus

Ultrastructure-function properties of recycling synaptic vesicles in acute hippocampal slices

Synaptic vesicles are the substrate of neurotransmission in most nerve terminals in the central nervous system. These small membrane spheres fuse with the synaptic membrane in an activity-dependent manner and release neurotransmitter into the synaptic cleft. Subsequently, vesicles are reclaimed through endocytosis prior to reuse. This recycling process is key to supporting ongoing signalling in the brain. While substantial effort has gone into defining basic characteristics of vesicle recycling, for example elucidating the timing of vesicle turnover, key questions remain unanswered. An important area with significant knowledge deficits relates to the relationship between vesicle function and ultrastructural organisation in the terminal. The aim of this thesis is to address this issue, exploiting new methodologies which provide novel insights into function-structure relationships of vesicle populations in acute brain slices. Specifically, this study considers organisational principles of three defined vesicle pools as well as examining the impact of an established plasticity protocol on pool properties. The first results chapter, Chapter 3, outlines and validates the novel protocol used for fluorescently labelling functional recycling vesicle populations in acute rat brain slices using the vesicle-labelling dye FM1-43 and new antibody based probes (syt1-Oyster, CypHer5E). Reporter-labelling and release properties are compared to similar approaches using cultured neurons. We conclude that this approach provides a more physiologically relevant method to study the functional properties of cells than used previously in cultured neurons. Chapter 4 outlines experiments utilising the capability of FM 1-43 to be photoconverted to an electron-dense form to allow a defined vesicle population, the readily releasable pool (RRP), to be characterised ultrastructurally. The RRP is arguably the most significant pool class, released first in response to an activity train. Functional assays and time-stamped electron microscopy are used to define basic properties of this pool, including its size, functional release kinetics, and temporal organisation. Specifically, the results demonstrate that retrieved vesicles are close to the active zone after stimulation, but mixed randomly in the terminal volume over 20 min. These findings address fundamental questions about vesicle reuse, the composition of future vesicle pools, and thus the mechanism of ongoing signalling in the brain. The same approach was used in Chapter 5 to examine the influence of Long Term Depression (LTD) on pool function and ultrastructure. LTD was induced in presynaptic terminals in CA1 via Schaffer collateral activation, and the following effects were observed: 1) a change in release kinetics; 2) a reduction in the total recycling pool size; and 3) no change in the composition of the docked pool. These findings demonstrate that there is a presynaptic component to LTD and that vesicle recruitment into the recycling pool appears to be an important possible substrate. However, the results suggest that such changes appear to be selective for specific pool subsets. Overall, work in this chapter offers new insights into fundamental principles supporting synaptic plasticity. Chapter 6 expands on previous studies which have demonstrated that recycling vesicles are constitutively shared between neighbours. This sharing of a ‘superpool’ of vesicles has implications for the ability of synapses to adapt to changes in input weighting. In this chapter, the methods outlined above, as well as a new 3D EM technology, are used to define the size, positional organisation, and clustering properties of this pool in native hippocampal slice system. The findings in this chapter reveal that extrasynaptic vesicles appear to show a greater degree of motility than vesicles which remain in the intrasynaptic cluster, perhaps implying differential interactions with structural proteins in the synapse. Characterising the superpool is increasingly relevant, as it is now implicated in models of plasticity and disease. Taken together, these results show that the ultrastructural arrangement of recycling vesicles is highly activity-dependent, and that the cytoarchitecture plays a large role in determining the functionality of individual vesicles and synapses

Synthesis and discovery of the putative cognitive enhancer BRS-015: effect on glutamatergic transmission and synaptic plasticity

This thesis is concerned with the discovery of a novel heterocyclic compound – BRS-015, its synthesis and an analysis of its effects on excitatory synaptic transmission at a major pathway in the brain. BRS-015 is related to the natural product clausenamide, which has been shown to facilitate synaptic transmission. As such, clausenamide and related analogues may possess therapeutic potential as memory enhancing drugs, which are in urgent need of development due to the increasing numbers of patients diagnosed with memory disorders and for which there is no current effective therapy. BRS-015 was synthesized using a novel approach to the core structure of clausenamide involving an intramolecular acylal cyclisation reaction, which has not previously been reported. The first section of the thesis opens with a description of the discovery, structure and biological activity of clausenamide and discussion of previous synthetic strategies adopted by a number of research groups and attempts to classify these into the varying approaches towards the central core of clausenamide. The second section describes the structure of the rat brain and the types of processes involved in memory formation, as well as the neurophysiological assays used to investigate synaptic transmission and plasticity. The second group of chapters describes our own approach to the core of clausenamide and the synthesis of BRS-015, with a detailed discussion of the structural analysis and investigation of the intramolecular acylal cyclisation reaction used during the synthetic process. The third chapter describes the neurophysiological assays used in our investigations into the effects of BRS-015, which was tested against glutamatergic synaptic transmission and plasticity in acute rat hippocampal slices. BRS-015 was shown to reversibly enhance the amplitude of AMPA receptor mediated EPSCs recorded from CA3 pyramidal neurones and evoked by dentate stimulation. When tested in the presence of selective glutamate receptor antagonists, BRS-015 did not have this powerful enhancing effect on kainate or NMDA receptor mediated EPSCs. In addition, BRS-015 increased the amplitude of glutamate-evoked currents in CA3 pyramidal neurones and did not alter short-term synaptic plasticity but facilitated the induction of mossy fibre LTP, with little effect at associational/commissural synapses. BRS-015 has striking enhancing properties on AMPA receptor mediated synaptic transmission at mossy fibre synapses either by directly interacting with AMPA receptors or via indirect modulation, the mechanisms of which could lead to synapse strengthening

GABA_A-mediated synaptic activity in rat hippocampal neurones <i>in vitro</i> and its modulation by other neurotransmitters and second messengers.

The patch-clamp technique (whole-cell and outside-out configurations) has been used to characterize spontaneous ɣ-aminobutyric acid A (GABAA) receptor mediated currents in pyramidal cells of thin hippocampal slices obtained from neonatal rats. In early postnatal life, GABA is the main excitatory neurotransmitter on hippocampal pyramidal cells. The frequency distribution histogram of spontaneous GABAergic currents could be fitted by a single exponential function revealing the random nature of these events. The present results demonstrate that in tetrodotoxin (TTX) solution spontaneous GABAA receptor mediated miniature postsynaptic currents (mPSCs) were present. At -70 mV the first peak in the current amplitude distribution was 16 ± 6 pA (n =13). This value was similar to that found for GABAergic currents (14 ± 6 pA) elicited by low intensity extracellular stimulation, suggesting that this effect was due to the release of elementary units of GABA. In outside-out patches, GABA activated single-channel events of 24 and 35 pS conductance. Assuming that a postsynaptic current of 15 pA corresponds to a single quantum of GABA, one could calculate that one quantal current represents the simultaneous opening of 6 to 9 GABAA receptor channels in the postsynaptic cell. The metabotropic Glutamate Receptor (mGluR) agonist, 1 -aminocyclopentane-1,3-dicarboxylic acid (t-ACPD), induced an increase in frequency but not in amplitude of spontaneously occurring GABAergic currents; this potentiating effect was blocked by the Protein Kinase A (PKA) antagonist Rp-adenosine 3', 5'-cyclic monophosphotioate triethylamine (Rp-cAMPS), suggesting that glutamate, acting on mGluRs, is able to increase GABA release through the metabolic pathway which involves PKA. The potentiating effect of t-ACPD was not observed in TTX solution indicating that the site of action of the mGluR agonist is probably located at the somatodendritic level and not on the nerve terminals ofGABAergic intemeurones. In the presence of forskolin, which increases intracellular cyclic AMP (cAMP) levels, a rise in frequency but not in amplitude of miniature GABAA receptor mediated currents was observed, an effect that was prevented by the selective PKA antagonist Rp-cAMPS. These experiments suggest that presynaptic mGluRs localized on GABAergic interneurones may facilitate the activity of these cells and their release of GABA through cAMP-dependent PKA. Moreover, PKA may interfere directly with the mechanism of GABA release as demonstrated by its action on miniature events. The present results provide new evidence that the release of a major neurotransmitter such as GABA is up-regulated by another neurotransmitter namely Glutamate, thus demonstrating an important reinforcement of excitatory signals during an early stage of brain development

Fast-spiking parvalbumin^+ GABAergic interneurons: From cellular design to microcircuit function

The success story of fast-spiking, parvalbumin-positive (PV+) GABAergic interneurons (GABA, γ-aminobutyric acid) in the mammalian central nervous system is noteworthy. In 1995, the properties of these interneurons were completely unknown. Twenty years later, thanks to the massive use of subcellular patch-clamp techniques, simultaneous multiple-cell recording, optogenetics, in vivo measurements, and computational approaches, our knowledge about PV+ interneurons became more extensive than for several types of pyramidal neurons. These findings have implications beyond the “small world” of basic research on GABAergic cells. For example, the results provide a first proof of principle that neuroscientists might be able to close the gaps between the molecular, cellular, network, and behavioral levels, representing one of the main challenges at the present time. Furthermore, the results may form the basis for PV+ interneurons as therapeutic targets for brain disease in the future. However, much needs to be learned about the basic function of these interneurons before clinical neuroscientists will be able to use PV+ interneurons for therapeutic purposes

Modulatory role of adenosine upon GABAergic transmission : consequences for excitability control

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2015Glutamatergic 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.A transmissão glutamatérgica no hipocampo é continuamente controlada por neurónios inibitórios, denominados interneurónios, que libertam o neurotransmissor ácido gama-aminobutírico (GABA). Estas células apresentam uma grande diversidade anatómica, fisiológica e bioquímica, estando descritos mais de vinte e um tipos diferentes de interneurónios no hipocampo. Estes são capazes de comunicar quer com células principais excitatórias (denominadas células piramidais), quer com outros interneurónios inibitórios, com resultados diferentes para a excitabilidade do sistema. A inibição de células piramidais leva a uma diminuição direta da sua excitabilidade; ao passo que a inibição de outros interneurónios pode resultar na desinibição das células principais e consequente aumento da excitabilidade. Desta grande variedade de interneurónios, destacam-se duas grandes classes que correspondem às duas populações de interneurónios mais importantes e abundantes no hipocampo – os neurónios que expressam colecistocinina (CCK) e os neurónios que expressam parvalbumina (PV). As funções de cada uma destas populações no hipocampo são únicas e complementares no controlo da atividade das redes neuronais. Desta forma, um controlo rigoroso destes circuitos inibitórios é de extrema importância na regulação das funções do hipocampo. A adenosina é um neuromodulador ubíquo do sistema nervoso central que atua através de dois grandes tipos de recetores de alta afinidade – os recetores A1 (A1R) e os recetores A2A (A2AR). Os primeiros têm ações principalmente inibitórias da excitabilidade neuronal, e portanto estão normalmente associados a funções neuroprotetoras, enquanto os segundos atuam no sentido de aumentar a excitabilidade no hipocampo e induzir excitotoxicidade. Enquanto que a função da adenosina no controlo da transmissão excitatória glutamatérgica tem vindo a ser caracterizada há várias décadas, o papel da adenosina na modulação da transmissão inibitória tem sido muito menos explorada. O trabalho apresentado nesta tese tem como objetivo a caracterização das ações dos A1Rs (Capítulo 5.1, p99) e dos A2ARs (Capítulo 5.2, p143) na comunicação neuronal inibitória no hipocampo bem como tentar perceber quais as consequências que uma possível modulação a este nível tem na excitabilidade das células piramidais e no desenvolvimento de atividade do tipo epiléptica. Para responder a estas questões foi planeado e executado um trabalho experimental que envolveu o registo da atividade elétrica neuronal no hipocampo de ratos e ratinhos através de técnicas eletrofisiológicas ex vivo (nomeadamente registos extracelulares e registos de patch-clamp). Relativamente às ações dos A1Rs, foi demonstrado que apenas um tipo de respostas inibitórias, denominadas por respostas tónicas, são afetadas pela ativação dos A1Rs, levando à sua diminuição. Este tipo de resposta tónica tem caraterísticas lentas e prolongadas no tempo e é mediada principalmente por recetores ionotrópicos do GABA do tipo A (GABAAR) que estão localizados em porções peri- e extrasináticas dos neurónios. Pelo contrário, as respostas habitualmente rápidas e concertadas no tempo, denominadas por respostas fásicas, e que são mediadas por recetores localizados nas sinapses, não parecem ser afetadas pela ativação dos A1Rs. Curiosamente, estas ações ocorrem seletivamente em neurónios excitatórios piramidais e numa subpopulação de interneurónios que expressam o neuropéptido CCK. O efeito dos A1Rs na diminuição das respostas tónicas está associado a uma cascata de sinalização intracelular que envolve as proteínas cinase A (PKA) e C (PKC) e é acompanhado pela diminuição da expressão de GABAARs que contêm a subunidade δ, habitualmente implicada nas respostas tónicas. Neste trabalho foi também demonstrado que a adenosina, através dos A2ARs, também influencia a transmissão inibitória no hipocampo. De facto, os efeitos da ativação dos A2ARs levam a um aumento da excitabilidade das células piramidais, que pode ser explicado pela ação destes recetores em dois locais: (1) a ativação dos A2ARs aumentam diretamente as respostas glutamatérgicas sobre as células piramidais; (2) simultaneamente, os A2ARs vão desinibir as células principais através de um mecanismo que envolve o aumento da libertação de GABA dos terminais sinápticos de neurónios que expressam PV e que contactam com outros neurónios inibitórios. Estas ações moduladoras têm implicações importantes em modelos de hiperexcitabilidade neuronal induzida pelo aumento das concentrações extracelulares de potássio, na medida em que a ativação ou inibição dos A2ARs leva a um exacerbação ou diminuição, respetivamente, desta hiperatividade neuronal sincronizada. No seu conjunto, os resultados apresentados nesta tese revelam, pela primeira vez, o envolvimento dos recetores de adenosina na modulação da transmissão neuronal inibitória no hipocampo. Estes resultados poderão abrir novas e promissoras perspetivas relativamente ao envolvimento da adenosina no controlo das funções do hipocampo em condições fisiológicas e patológicas.Network of European Neuroscience Schools; Medical Research Counci