In vivo knockdown of astroglial glutamate transporters GLT-1 and GLAST increases excitatory neurotransmission in mouse infralimbic cortex: Relevance for depressive-like phenotypes

Abstract

Alterations of energy metabolism and of astrocyte number/function in ventral anterior cingulate cortex (vACC) have been reported in major depressive disorder (MDD) patients and may contribute to MDD pathophysiology. We recently developed a mouse model of MDD mimicking these alterations. We knocked down the astroglial glutamate transporters GLAST and GLT-1 in infralimbic cortex (IL, rodent equivalent of vACC) using small interfering RNA (siRNA). GLAST and GLT-1 siRNA microinfusion in IL evoked a depressive-like phenotype, associated with a reduced serotonergic function and reduced forebrain BDNF expression. Neither effect occurred after siRNA application in the adjacent prelimbic cortex (PrL), thus emphasizing the critical role of vACC/IL in MDD pathogenesis. Here we examined the cellular/network basis of the changes induced in IL using intracellular recordings of layer V pyramidal neurons from mice microinjected with siRNA 24 h before. We analyzed (i) the electrophysiological characteristics of neurons; (ii) the synaptic transmission properties, by monitoring miniature, spontaneous and evoked EPSCs, and (iii) the gliotransmission, by monitoring slow inward currents (SICs), mediated by astrocytic glutamate release and activation of extra-synaptic NMDA receptors. GLT-1 and GLAST knockdown led to a more depolarized membrane potential and increased action potential firing rate of layer V pyramidal neurons, and enhanced excitatory synaptic transmission, as shown by the enhanced amplitude/frequency of spontaneous EPSCs. Gliotransmission was also increased, as indicated by the enhanced SIC amplitude/frequency. Hence, the depressive-like phenotype is associated with IL hyperactivity, likely leading to an excessive top-down inhibitory control of serotonergic activity through IL-midbrain descending pathways.

Introduction

Major depressive disorder (MDD) is a severe mental disorder with a very large socioeconomic impact worldwide (Vos et al., 2016). This situation is attributable to the high prevalence of MDD and to the slow clinical action and limited efficacy of monoamine-based antidepressants.

Although the pathophysiology of MDD remains largely unknown, neuroimaging studies reported an enhanced energy metabolism in ventral areas of the anterior cingulate (vACC) of MDD patients (Mayberg, 2009; Savitz and Drevets, 2009). This area shows a large reciprocal connectivity with many other cortical and subcortical areas, including the brainstem monoamine cell groups (Croenewegen and Uylings, 2000). In particular, layer V pyramidal neurons in the medial prefrontal cortex (mPFC) project extensively to the serotonergic dorsal raphe nucleus (DR) (Gabbott et al., 2005), and their stimulation at physiological rates markedly inhibits the activity of most serotonergic neurons via local GABA_A inputs and 5-HT_1A autoreceptors (Celada et al., 2001). Therefore, an enhanced vACC activity may induce downstream activity changes of subcortical areas involved in emotional processing, including DR, leading to MDD symptoms (Artigas, 2015). Interestingly, fast-acting antidepressant strategies such as ketamine and deep brain stimulation regulate neuronal activity in vACC (Fuchikami et al., 2015; Mayberg 2009)

Astrocytes are emerging as key players in synaptic function, controlling extracellular levels of ions and neurotransmitters, responding to them, and releasing gliotransmittters that regulate synaptic transmission, plasticity (Perea and Araque 2007; Perea et al., 2014), and animal behavior (Oliveira et al., 2015). The astroglial glutamate transporters GLT-1 and GLAST take up most synaptic glutamate from central excitatory synapses (Danbolt et al., 1992; Petr et al., 2015), thereby exerting a tight direct control of neuronal excitability. Astrocytes have been proposed as an early contributor to the underlying pathogenesis of MDD (Sanacora and Banasr 2013) and alterations in astrocyte number/function have been reported in MDD patients and suicide victims (Banasr and Duman, 2008; Rajkowska and Stockmeier, 2013). Hence, the functional hyperactivity observed in the vACC of MDD patients may result from a disturbed astrocyte-based glutamate homeostasis.

In an attempt to mimic glial dysfunction in MDD, we developed a mouse model of MDD through the acute RNAi-induced knockdown of GLT-1 or GLAST expression in infralimbic cortex (IL, rodent equivalent of vACC). The local microinfusion of small interfering RNA (siRNA) targeting GLT-1 or GLAST into mouse IL evoked a moderate, long-lasting (≥7 days) decrease of GLAST/GLT-1 expression and induced a robust depressive-like phenotype as soon as 1 day after siRNA administration, associated to a reduced 5-HT release and a reduced BDMF expression (Fullana et al., 2019). The behavioral phenotype was reversed by classical (citalopram) and fast-acting (ketamine) antidepressant treatments. Remarkably, no behavioral/neurochemical effects were observed when GLT-1 or GLAST were knocked down in the adjacent prelimbic cortex (PrL) (Fullana et al., 2019), which emphasizes the critical role of IL-based circuits in emotional control and stress resilience.

We carried out the present electrophysiological study in order to better understand the cellular basis of the model described above, under the working hypothesis that GLAST/GLT-1 knockdown in IL would enhance excitatory activity in layer V pyramidal neurons, that project extensively to raphe serotonergic neurons (Gabbott et al., 2005)and that markedly inhibit their activity (Celada et al., 2001).