Results. Unilateral microinfusion of a pool of siRNA sequences targeting GLAST ar GLT- 1 in mouse IL induced a moderate (20-30%) and long-lasting (7 days) decrease in their mRNA and protein expression (p<0.0001). Intra-lL GLAST/GLT-1 siRNA infusíon also reduced glial fibrilary acidic protein (GFAP)-positive astrocyte density and increased excitatory neurotransmission in layer V pyramidal neurons, as shown by an lncreased resting membrane potential (p<0.005), lncreased evoked discharge rate (p<0.0001), slow down of evoked EPSC (p<0.003), as well as increased spontaneo EPSC (sEPSC) amplitude and frequency (p<0.0001). GLAST/GLT-1 knockdown also increased gliotransmission, as shown by the higher amplitude and frequency of slow inward currents (SICs) {p<0.0015), mediated by extra-synaptic NMDA-R. Moreover, GLAST/GLT-1 knockdown evoked a depressive-like phenotype, as assessed by the FST (p=0.0003), TST (p<0.0001) and SPT (P"'0.0004, which was reversed by the acute i.p. administration of citalopram and ketamine 30 min prior to the test. GLAST or GLT- 1 knockdown in IL markedly reduced serotonin (5-HT) release in the dorsal raphe nucleus (DR) and induced an overall reduction of brain derived neurotrophic factor {BDNF) expression in cortical and hlppocampal areas of both ipsilateral and contralateral hemlspheres. BDNF reductions were highly correlated with the reductlon of GLAST and GLT-1 mRNA expression in IL (p<0.0001. Moreover, Egr-1 (early growth response protein-1) labelling suggests that both siRNAs enhance the putative GABAergic tone onto DR 5-HT neurons, leading to an overalJ decrease of 5-HT function, likely related to the widespread reduction on BDNF expression. Remarkably, similar reductions of GLAST and GLT-1 expression in the neighbouring prelimbic cortex (Prl) did not induce a depressive-like phenotype in any of the tests examined and did not alter 5-HT release in DR Concluslon. These results show that a moderate reduction of glutamate clearance by astrocytes in IL results in very marked local and distal changes in neuronal activity, likel associated to the de ressive-like henot e evoked b GLAST GLT-1 knockdown n IL. The increased excltatory neurotransmisslon in layer V pyramidal neurons of IL, together with the reduced serotonergic function suggests that the depressive-like phenotype induced by GLAST/GLT-1 knockdown results from an excessive top-down inhibitory control of DR neurons by the IL. Hence, a focal glial change in IL translates into global change of brain activity by virtue of the descending projections from IL to DR and the subsequent attenuation of serotonergic activity.

Background. Increases in energy metabolism together with disturbances of astrocyte number/function in the ventral anterior cingulate cortex (vACC) have been suggested as important contributors to the pathophysiology of major depressive disorder (MDD). Hence, the functional hyperactivity reported in vACC may result from a reduced glutamate clearance from excitatory synapses and a subsequent increase in neuronal excitation. Astrocytes are emerging as essential players in synaptic function, controlllng extracellular levels of ions and neurotransmitters, responding to them, and releaslng gliotransmittters which regulate synaptic transmisslon and plasticity. In particular, the astroglial glutamate transporters GLT-1 and GLAST are responsible for the reuptake of nearly 98% from central glutamatergic excitatory synapses, thereby directly controlling neuronal excitability. Here we examined the functional and behavioural consequences of knocking down GLT-1 and GLAST with RNAi strategies in mouse infralimbic cortex (IL) under the working hypothesis that a functional hyperactivity in IL may result in a depressive-like phenotype. Methods. Pentobarbital-anesthetized mice (C57BL/6J) were unllaterally microinfused in IL with siRNA (small interfering RNA) targeting GLAST or GLT-1 ata single dose of 60 µg/µI; 4.2 nmol/dose). Mice were sacriflced at 1, 3 or 7 days after siRNA lnfusion. Electrophysiological recordlngs from layer V pyramidal neurons were made in cortical slices contalning the IL using whole-cell configuration of the patch-clamp technique. Behavioural assessments included the torced swim test (FST), tail suspension test (TST) and the sucrose preference test (SPT, anhedonia). Immunohistochemistry and in situ hybridization were used to assess changes in the expression of GLAST and GLT-1, and of other mRNAs/proteins (see below). Changes in 5-HT release were determined by intracerebral microdialysis in freely-moving mice. Changes induced by GLAST/GLT-1 knockdown on most variables were assessed 24 h after siRNA microinfusion. Statistical analyses were performed with N,,,8-10 animals/group and by two-tailed Student's t-test and one-way or two-way ANOVA followed by Tukey's post-hoc test as appropriate. Statistical significance has been set at the 95% confidence level.

Tesis doctoral presentada para lograr el título de Doctor por la Universidad de Barcelona, Programa Oficial de Doctorado en Biomedicina.--Calificación: Sobresaliente cum lauden