Reaching a cell-type specific level of understanding in depression – DEPth

To characterize cell-specific electrophysiological changes (AIM 1), we are using dual optical and electrical micro-optrode which allows single unit extracellular recordings from optically identified GLU and GABA neurons in vivo.
To identify cell-specific transcriptomic adaptations in the ACC (AIM 2), translating ribosomal affinity purification method is performed to isolate different populations from transgenic mice. Within DEPth, we are performing RNA-sequencing on ACC GLU vs. GABA neurons from depressive-like and control animals. Protein-coding genes and non-coding RNAs will be analysed and compared with our recent data set generated in whole ACC tissue. Importantly, our results will be confronted with RNA-seq of ACC post-mortem human samples from the Douglas Bell Canada Brain Bank (a facility co-directed by Pr. Turecki), including whole ACC as well as GLU specific analyses after laser capture microdissection (LCM) obtained from depressed patients and psychiatrically healthy individuals.
Based on the electrophysiological analyses and on the comparison between rodent and post mortem human transcriptomic maps, we will study the causal impact of identified gene alterations on depressive-like behaviours and on the electrophysiological properties of the ACC. We will select and manipulate 2 genes that show evidence of dysregulation across mouse/human. To determine how a given gene impacts on mood control, we will combine several approaches, such as optogenetics, local deletion and/or overexpression using viral vectors, as well as conditional knockout mice.

We first completed the behavioural characterization of the role of the Gabaergic (GABA) and Glutamatergic (GLU) neurons in the model of chronic pain induced depression (CPID). Our previous studies showed that the activation of GLU neurons in the ACC using optogenetic approach can induce depressive-like behaviours in naive animals and the blocking of this cell population can block CPID. By using our CPID model, we demonstrated that optogenetic stimulation of GABAergic neurons in the ACC blocks anxiodepressive-like behaviors induced by chronic pain, without affecting mechanical sensitivity.
In parallel, using viral-mediated translating ribosome affinity purification (TRAP) method we are sorting either GABA or GLU neurons. For instance, Dlx5/6 CRE mouse line used for the GABAergic neuron sorting. For ribosomes immunoprecipitation, CRE-dependent ribosomal protein L10 tagged with GFP is injected into the ACC of Dlx5/6 CRE mice. The obtained purified RNAs from both the whole tissue RNA and the immunoprecipitated transcripts have been sent to RNA sequencing (IGBMC). Using different bioinformatic tools, we recently finished analyzing the differential expression of individual genes for GABAergic neurons. Same strategy is now applied for GLU neurons.
To characterize cell-specific electrophysiological changes, we are using dual optical and electrical micro-optrode which allows single unit extracellular recordings from optically identified GLU and GABA neurons in vivo. These cell types are identified based on their firing response to a light stimulus (ChR2). Experiments are still ongoing. In parallel, in collaboration with Dr. Sylvain Hugel, using ex vivo whole patch clamp method, we record GLU neuronal activity after the activation of GABAergic neurons in naïve and CPID animals and our preliminary results showed impairment of inhibition in animals showing depressive-like behaviours after chronic pain.

Therefore, our already obtained results will provide a new breakthrough concerning the physiological role of ACC excitatory and inhibitory neurons, as well as the first cell type specific cortical transcriptomic deciphering of depression.
After the completion of genomic mapping in both GLU and GABA neurons from CPID mice, our results will be confronted with RNA-sequencing of ACC post-mortem human samples from the Douglas Bell Canada Brain Bank (a facility co-directed by Pr. Turecki), including whole ACC as well as GLU specific analyses after laser capture microdissection (LCM) obtained from depressed patients and psychiatrically healthy individuals. These comparison will guide us to select individual genes that similarly dysregulated in both mice and depressed patients that we are going to manipulate by combining several approaches such as the optogenetic, overexpression or downregulation to study the causal link between depression and identified genes.

We plan to start writing first paper at the end of 2020.

Depression is a recurrent mental illness predicted to become a foremost contributor to the worldwide burden of disease (WHO, 2008). The main goal of this translational research project is to reach a cell-type specific level of understanding of depression by investigating electrophysiological and molecular alterations in the glutamatergic (GLU) and GABAergic (GABA) neurons of the anterior cingulate cortex (ACC) in depression.
Depression has been related to specific structural and functional changes in brain neurocircuitry. Within this altered neurocircuitry, the anterior cingulate cortex (ACC) appears to be critical as illustrated by its functional and morphological alterations in depressed patients. In animal model, we previously reported that optogenetic activation of the ACC is sufficient to induce anxiodepressive-like behaviours in naive mice, while the lesion (Biol Psychiatry 2015) or the optogenetic inactivation (J Neuroci 2018) of the ACC prevents chronic pain-induced depression. Our recent data also evidenced molecular (Biol Psychiatry 2017) and electrophysiological alterations (J Neuroci 2018) within the ACC, which are underlying depressive-like behaviours. Therefore, the ACC appears to be a critical substrate for mood disorders and constitutes an important target for elucidating the underlying mechanisms thereof. However, one challenge in neuroscience stems from the fact that brain structures often include a variety of neuronal cell types. Characterizing physiological and molecular traits of different neuronal populations is difficult, but should be considered in order to identify: (i) how distinct cell populations within the ACC are affected in depression and (ii) new potential targets that may remain unforeseeable at the level of the whole tissue. To achieve this goal, the DEPth project will characterize cell-type specific mechanisms of depression, focusing on 2 major neuronal populations: GLU and GABA neurons. Both populations will be investigated simultaneously in validated mouse models of depression based on chronic stress and chronic pain as well as in post-mortem human brain samples from a well-characterized cohort of severely depressed subjects.
Based on the context and preliminary results, the project will be developed toward 3 objectives:
First objective of DEPth is to determine the electrophysiological alterations in ACC GLU and GABA neurons using in vivo electrophysiological recordings.
Second objective is to characterize gene expression alterations in ACC GLU and GABA neurons in both mice and post-mortem human tissue. This will be achieved using high-throughput sequencing analyses in fluorescence-activated cell sorted GLU and GABA neuronal populations from transgenic mice displaying depressive-like behaviour, and in laser capture micro-dissected neurons from post-mortem human tissue. Data from these two species and two models will be compared using well validated bioinformatic analyses in order to identify and prioritize hub genes.
Third objective is to assess in mice how the functional manipulation of selected hub genes impact gene networks, electrophysiology and behaviour.
DEPth is a project of fundamental research, developed by an independent young investigator, in relevance to the “JCJC” call. Scientifically, the project completion will provide a new breakthrough concerning the physiological role of ACC excitatory and inhibitory neurons, as well as the first cell type specific cortical transcriptomic deciphering of depression. The expertise of the principal investigator, the solid preliminary results, and the integrated scientific program ensure the immediate feasibility of the DEPth project, which will bring valuable translational information to the field of depression, and warrantee its success.