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. 2017 Feb:1:2470547017720459.
doi: 10.1177/2470547017720459. Epub 2017 Aug 3.

Characterization of GABAergic marker expression in the chronic unpredictable stress model of depression

Mounira Banasr  1   2   3 , Ashley Lepack  1 , Corey Fee  2 , Vanja Duric  1   4 , Jaime Maldonado-Aviles  1 , Ralph DiLeone  1 , Etienne Sibille  2   3 , Ronald S Duman  1 , Gerard Sanacora  1
Affiliations

Characterization of GABAergic marker expression in the chronic unpredictable stress model of depression

Mounira Banasr et al. Chronic Stress (Thousand Oaks). 2017 Feb.

Abstract

Evidence continues to build suggesting that the GABAergic neurotransmitter system is altered in brains of patients with major depressive disorder. However, there is little information available related to the extent of these changes or the potential mechanisms associated with these alterations. As stress is a well-established precipitant to depressive episodes, we sought to explore the impact of chronic stress on GABAergic interneurons. Using western blot analyses and quantitative real-time PCR (qPCR) we assessed the effects of five-weeks of chronic unpredictable stress (CUS) exposure on the expression of GABA-synthesizing enzymes (GAD65 and GAD67), calcium-binding proteins (calbindin (CB), parvalbumin (PV) and calretinin (CR)), and neuropeptides co-expressed in GABAergic neurons (somatostatin (SST), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and cholecystokinin (CCK)) in the prefrontal cortex (PFC) and hippocampus (HPC) of rats. We also investigated the effects of corticosterone (CORT) and dexamethasone (DEX) exposure on these markers in vitro in primary cortical and hippocampal cultures. We found that CUS induced significant reductions of GAD67 protein levels in both the PFC and HPC of CUS-exposed rats, but did not detect changes in GAD65 protein expression. Similar protein expression changes were found in vitro in cortical neurons. In addition, our results provide clear evidence of reduced markers of interneuron population(s), namely SST and NPY, in the PFC, suggesting these cell types may be selectively vulnerable to chronic stress. Together, this work highlights that chronic stress induces regional and cell type-selective effects on GABAergic interneurons in rats. These findings provide additional supporting evidence that stress-induced GABA neuron dysfunction and cell vulnerability play critical roles in the pathophysiology of stress-related illnesses, including major depressive disorder.

Keywords: GABA; GAD67; calcium-binding proteins; chronic stress; depression; neuropeptides.

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Conflict of interest statement

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dr. Sanacora has received consulting fees form Allergan, Alkermes, BioHaven Pharmaceuticals Holding company, Janssen, Merck, Sage pharmaceuticals, Takeda, Taisho Pharmaceuticals, and Vistagen therapeutics over the last 24 months. He has also received additional research contracts from AstraZeneca, Bristol-Myers Squibb, Eli Lilly & Co., Johnson & Johnson, Hoffman La-Roche, Merck & Co., Naurex, and Servier over the last 24 months. Free medication was provided to Dr. Sanacora for an NIH sponsored study by Sanofi-Aventis. In addition, he holds shares in BioHaven Pharmaceuticals Holding Company and is a co-inventor on a patent “Glutamate agents in the treatment of mental disorders” Patent number: 8778979. ES is co-inventor on a US provisional patent application that covers compounds modulating the function of GABA neurons.

Figures

Figure 1.
Figure 1.
Effects of chronic unpredictable stress (CUS) on GAD65 and GAD67 protein expression. (a) GAD67 and (b) GAD65 protein expression following CUS compared to home cage controls (HCC) in the PFC. (c) GAD67 and (d) GAD65 protein expression in the HPC. Levels of proteins were normalized to GAPDH. For each marker, a representative immunoblot and its respective GAPDH blot are illustrated. Results are expressed as fold change compared to HCC and displayed as means ± SEM. *p < 0.05.
Figure 2.
Figure 2.
Effects of CUS on calcium-binding proteins. For each marker, a representative immunoblot and its respective GAPDH blot are illustrated. (a) CB, (b) PV, and (c) CR protein expression following CUS compared to HCC in the PFC. (d) Levels of CB, (e) PV, (f) and CR protein expression in the HPC following CUS compared to HCC. Levels of proteins were normalized to GAPDH. Results are expressed as fold change compared to HCC and displayed as means ± SEM. *p < 0.05.
Figure 3.
Figure 3.
Effects of CUS on neuropeptides specific to GABAergic interneurons. (a) mRNA levels of SST, (b) NPY, (c) VIP, and (d) CCK following CUS compared to HCC in the PFC. (e) mRNA levels of SST, (f) NPY, (g) VIP, and (h) CCK after CUS exposure compared to HCC in the HPC. Gene expression was normalized to GAPDH. Results are expressed as fold change compared to HCC and displayed as means ± SEM. *p < 0.05.
Figure 4.
Figure 4.
Effects of corticosterone or dexamethasone on GABAergic neurons in vitro. Illustration of (a) DAPI-positive cells from cortical primary neuronal culture, where (b) cells were labeled with GAD67 using immunocytochemistry and visualized with fluorescence microscopy to show co-localization of both markers (merge, c). (d) GAD67 and (e) GAD65 protein expression following 72 h exposure to various doses of corticosterone (CORT) or dexamethasone (DEX), compared to DMSO. For GAD67, a representative immunoblot and its respective GAPDH blot are illustrated. Levels of proteins were normalized to GAPDH. Results are expressed as fold change compared to vehicle treatment and displayed as means ± SEM. *p < 0.05.
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