Molecular features of glycine-mediated neurotransmission in rat brain

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2015

Glycine, an inhibitory neurotransmitter in central nervous system, binds to its high affinity post-synaptic glycine receptors (GlyR). GlyR are pentameric channels, composed of several subunits (α1, α2, α3 and β). Termination of glycine-mediated synaptic activity occurs through removal of neurotransmitter from extracellular space, and is mediated by two glycine transporters (GlyT), GlyT1 and GlyT2. It is widely accepted that GlyT1 is mainly expressed in astrocytes, while GlyT2 is predominantly expressed in glycinergic pre-synaptic nerve terminals. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that activates its high affinity tropomyosin-related kinase B (TrkB) receptor, which includes full length (TrkB-FL) and truncated (TrkB-T1/T2) isoforms. Throughout this work the key players of glycinergic neurotransmission were studied in the three components which comprise the tripartite synapse in rat brain. GlyR localization and subunit composition were assessed in the post-synaptic neurons, GlyT2 expression was evaluated in pre-synaptic nerve terminals and GlyT1 and GlyT2 expression and localization was examined in astrocytes. In order to evaluate if GlyT are functional, [3 H]glycine uptake experiments were performed with selective inhibitors, Org-24598 and ALX-1393, for GlyT1 and GlyT2, respectively. Additionally, the BDNF effect upon glycine uptake mediated by GlyT in rat cortical cultured astrocytes and in hippocampal pre-synaptic nerve terminals was analyzed. Moreover, the signaling pathways involved in the BDNF effect were evaluated through a pharmacological approach. Immunofluorescence assays in brain slices showed a predominance of extrasynaptic GlyR and an alteration in synaptic GlyR composition. At P7, post-synaptic receptors are mainly GlyR α2/β. In mature hippocampus (P21) synaptic GlyR decrease and are composed by α1/β subunits. Furthermore, extrasynaptic α2/α3-containing GlyR become predominant. It was also demonstrated that hippocampal pre-synaptic nerve terminals express GlyT2. Quantitative PCR indicated that GlyT1 and GlyT2 transcripts are expressed in cultured astrocytes and immunofluorescence analysis corroborated these results since GlyT1 and GlyT2 were detected in astrocytes, both in culture and in brain slices. By [3 H]glycine uptake assays, GlyT2 in pre-synaptic nerve terminals and both GlyT1 and GlyT2 in astrocytes, were shown to be functional. xx It was also reported that BDNF decreases glycine uptake, mediated by GlyT, causing similar Km values and a lower Vmax, in both astrocytes and pre-synaptic nerve terminals. In astrocytes BDNF acts through TrkB-T1 receptors, promoting GlyT internalization through a Rho-GTPase-dependent mechanism. In pre-synaptic nerve terminals the BDNF effect is due to the activation of the TrkB-FL receptors and the subsequent intracellular cascades, namely PLC, Akt and MAPK pathways, leading to the inhibition of GlyT2 insertion in the plasma membrane. This work irrefutably confirms the occurrence of glycinergic synapses in the brain. It was shown the predominance of extrasynaptic GlyR, which suggests a role for slow tonic glycinergic neurotransmission, as well as the expression of functional GlyT1 and GlyT2 in astrocytes and GlyT2 in nerve terminals. In addition, it was described that GlyT1 and GlyT2 are modulated by BDNF. In conclusion, this work provides new insights about glycinergic neurotransmission in brain.

Fundação para a Ciência e a Tecnologia (FCT)