Neuronal excitability is controlled by inhibitory transmission, which, in the brain, is mostly controlled by the gamma-aminobutyric acid (GABA). Accordingly, to a great extent excitability control involves control of the GABAergic transmission. The latter has two main components: 1) a phasic component, whereby the GABA is released synchronically with the action potentials that enter the GABAergic nerve terminals and 2) a tonic component independent from the presynaptic action potential and which corresponds to continuous inhibition mediated by the GABA at synaptic and extrasynaptic levels. Tonic inhibition depends mainly on the activity of GABA transporters – GATs – which remove extracellular GABA. The modulation of phasic inhibition or tonic inhibition has distinct consequences on neuronal excitability.

The hippocampus is an area in the brain with well-known cytoarchitecture and neuronal connections, and which has inhibitory neurons that (I) project to other inhibitory neurons (synapses I-I) and inhibitory neurons that project to excitatory neurons (synapses I-E), in addition to excitatory neurons projecting over other excitatory neurons (synapses E-E). The modulation of the two inhibitory synapses I-I and I-E have predictably opposing consequences in excitability control.

Adenosine is a neuromodulator released by the majority of cells, including neurons and astrocytes. It can be released as such or be formed outside the cells from ATP hydrolysis. Its neuroprotection and anticonvulsive effects have been mostly attributed to the inhibition of excitatory transmission. Conversely, a lot less is known about adenosine’s control of inhibitory transmission, and of GABAergic transmission in particular.

With this project, which has been funded by Fundação para a Ciência e Tecnologia (Science and Technology Research Council) since February 2011, we intend to clarify the influence of adenosine on hippocampal GABAergic transmission. We will focus on the transmission between two inhibitory neurons (synapses I-E) and between one inhibitory neuron and one excitatory neuron (synapses I-E). Astrocyte modulation, particularly their capacity to transport GABA and thus influence tonic and/or phasic inhibition will also be investigated. Accordingly, we will record postsynaptic inhibitory currents in interneurons (synapses I-I) and pyramidal cells (synapses I-E) in slices of the hippocampus. We will also ascertain whether the modulation exerted by high affinity A1 and A2A adenosine receptors occurs at pre or postsynaptic level by measuring the changes in inhibitory currents evoked by afferent stimulation, in inhibitory currents evoked by direct stimulation of GABAA receptors, and in spontaneous miniature currents. Tonic inhibition will be determined by measuring changes in the injection current that is necessary to keep the membrane potential at a pre-set value. We will also assess if adenosine receptors affect the plasticity of inhibitory synapses. We will use primary cultures of astrocytes to establish if GABA transporters GAT1 and GAT3 are modulated by activation of adenosine A1 and A2A receptors. Astrocytes signal by means of calcium waves which are triggered by activation of ATP receptors of the P2Y subtype. We will also ascertain the extent to which these receptors affect GABA transportation in astrocytes, to better understand the multiple processes used by adenosine or its precursors to control excitability.

Besides the Leading Researcher (Ana M Sebastião), four PhD students (Diogo Rombo, Raquel Dias, Sandra Vaz, and Sofia Cristóvão-Ferreira), and two Master degree students (Joaquim Jacob and Andreia Silva) are involved in this project. The necessary methodologies and equipment to carry out the project, namely equipment for patch clamp recordings in hippocampus slices, for imaging calcium activity of astrocytes, for measuring transport (release and recapture) of neurotransmitters using isolated nerve terminals already exist in the Neuroscience Unit of the Institute of Molecular Medicine and in the Institute of Pharmacology and Neurosciences of the Faculty of Medicine of the University of Lisbon.

Data already collected demonstrate that adenosine A1 receptors restrain inhibitory currents in pyramidal neurons (synapses I-E) and in a subpopulation of interneurons (synapses I-I), and that this adenosine action is exerted by inhibiting the activation of extrasynaptic GABAA receptors; according to these observations, adenosine, via receptors A1, attenuates the tonic inhibition exerted by GABA on excitatory pyramidal neurons (Rombo et al., in progress). Adenosine A1 receptors also inhibit excitatory transmission to excitatory neurons (synapses E-E) and this effect is also quite well known at a pre-synaptic level (on this matter, see Sebastião and Ribeiro, 2009). The direct inhibitory effect of adenosine A1 receptors on AMPA receptors (the receptors that convey glutamate-mediated rapid excitatory transmission) has also been described by members of the research team (Dias et al, 2010). With regard to the modulation of AMPA receptors by adenosine, it is important to underline that these receptors are also affected by another subtype of adenosine receptors – receptors A2A – which have an opposing effect to that of A1 receptors, and facilitate the insertion of AMPA receptors into the membrane of the postsynaptic neuron (Dias et al., 2010).

This action, together with the facilitation of the role of neurotrophic factors, namely the facilitation of neurotrophic factors receptors translocation into the microdomains of the neuronal membrane in situations of high neural activity (Assaife-Lopes et al., 2010), helps strengthen excitatory synapses (E-E). This research unit is currently planning the forthcoming assessment of inhibitory synapses plasticity control processes, since this plasticity depends heavily on endocannabinoids, and, as we have noted recently (Sousa et al., 2011), adenosine receptors, particularly subtype A1 receptors, modulate the actions of cannabinoids.

During this ongoing project, we have also noted that subtype A1 and A2A adenosine receptors modulate astrocyte GABA transport. Surprisingly, we have seen that adenosine receptors in astrocytes form A1-A1-A2A-A2A heteromers, and that the functional unit is the tetramer coupled to canonical transduction systems of receptors A1 and A2A. This functional unit internalizes as a whole when there is super activation of any one of the components of the functional unit, which suggests that astrocytes have tight “security systems” with regard to control of GABA recapture (Cristovão-Ferreira et al, in progress). Equally exciting preliminary results point to a close connection between astrocyte calcium signalling and GABA recapture by the former, showing common aspects between two main functions of these cells (master degree dissertations of Joaquim Jacob and Andreia Vaz, in progress).

By identifying the synapses where adenosine acts, the receptors where adenosine operates, ascertaining if this action occurs at a pre or postsynaptic level, and whether it affects predominantly phasic or tonic inhibition, while determining the involvement of astrocytes in this process, the present project will offer a deeper understanding of the way neurons and astrocytes control inhibition. Accordingly, we hope it will contribute towards the creation of more suitable strategies to control excessive excitability in pathologies.

Ana M. Sebastião
Institute of Pharmacology and Neurosciences of the Faculty of Medicine, and Neuroscience Unit of the Institute of Molecular Medicine, University of Lisbon.
anaseb@fm.ul.pt

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References

Assaife-Lopes N, Sousa VC, Pereira DB, Ribeiro JA, Chao MV, Sebastião AM (2010). Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation. J Neurosci, 23, 8468-8480. (ver artigo aqui)

Dias RB, Ribeiro JA, Sebastião AM (2010). Hippocampus, ePub ahead of print (August 2010) (ver artigo aqui)

Sebastião AM, Ribeiro JA (2009) In: Handbook of Experimental Pharmacology, 193, 471-534. (ver artigo aqui)

Sousa VC, Assaife-Lopes N, Ribeiro, JA, Pratt JA, Brett RR, Sebastião AM (2011) Regulation of hippocampal cannabinoid CB1 receptor actions by adenosine A1 receptors and chronic caffeine administration: implications for the effects of 9-tetrahydrocannabinol on spatial memory. Neuropsychopharmacology, 36, 472-487. (ver artigo aqui)