Depression is a leading cause of disabilities around the world, and the underlying mechanisms involved in its pathophysiology are broad and complex. Exposure to chronic stress is a risk factor for developing depressive-symptoms and contributes to cellular and molecular changes precipitating the emergence of symptoms. In the brain, excitatory neurons, inhibitory interneurons and supporting astroglial cells are all sensitive to chronic stress exposure and are known to be impaired in depression. Using an animal model of chronic stress, we assessed the impact of variable durations of chronic stress on the emergence of behavioral deficits and associated molecular changes in the prefrontal cortex (PFC), brain region highly sensitive to stress and impaired in depression. Mice were exposed to up to 35 days of chronic restraint stress and were assessed weekly on behavioral tests measuring anxiety and anhedonia. PFC Protein and RNA levels of specific markers of excitatory, inhibitory synapses and astroglia were quantified using western blot and qPCR, respectively. Correlation and integrative network analyses were used to investigated the impact of chronic stress on the different compartments. Results showed that chronic stress induces anxiety-like behaviors within 7 days, while anhedonia-like behaviors were observed only after 35 days. At the molecular level, alterations of many markers were observed, in particular with longer exposure to chronic stress. Finally, correlation analyses and integrative network analyses revealed that male and female mice react differently to chronic stress exposure and that some markers seem to be more correlated to behaviors deficits in males than in females. Our study demonstrate that chronic induces a dynamic changes that can be observed at the behavioral and molecular levels, and that male and female mice, while exhibiting similar symptoms, have different underlying pathologies.

Chronic stress is a major risk factor for developing depressive disorders and animal models of stress recapitulate behavioral, cellular and molecular changes that are observed in human depression. Individuals exposed to chronic stress, or patients with MDD experience mood and cognitive dysfunctions. This is in part due to neuronal shrinkage in brain regions involved in several cognitive functions such as the prefrontal cortex (PFC) and the hippocampus (HPC). Also in the context of depression and chronic stress, expression levels and function of the main inhibitory neurotransmitter GABA are reduced. Thus far, drugs targeting this GABA deficit have failed to produce beneficial effects due to broad activity at various GABA receptor subunits, including the α1-subunit, resulting in broad side effects. However, refined and selective activity at the α2/3/5-subunit is hypothesized to exert beneficial effect, devoid of side effects.Here, we show that GL-II-73 and GL-I-54 exert positive allosteric modulation at the α5, and α2/3/5-contianing GABAA receptors respectively, and that they are effective both independently and in combination. Using unpredictable chronic mild stress (UCMS) experiments in male and female C57BL/6 mice (n=12 per group), we showed that acute and chronic administration of a GL-II-73/GL-I-54 racemic mixture (termed “GL-RM”) reduced anxiety-like phenotypes and reversed a working memory deficit in UCMS exposed mice. Brains from animals receiving chronic treatment were collected and stained using a Golgi staining technique. Using stereological approaches, neuronal morphology was reconstructed and dendritic length, spine count and spine density were assessed in pyramidal neurons of the PFC and hippocampus. Chronic GL-RM rescued spine density depletions caused by UCMS at apical and basal dendrites (PFC and CA1). Interestingly, spine densities in both brain regions were correlated to cognitive performance, confirming ameliorative benefits of GL-RM.Together, results support the value of selectively targeting GABAA receptors, excluding the α1-subunit, to overcome chronic stress-induced mood symptoms and cognitive deficits, as well as detriments in neuronal morphology. This study confirm results that were observed in old mice, using a α5-selective positive allosteric modulator, and reinforce the concept that the α2/3/5-containing GABAA receptor are suitable targets for the treatment of stress-induced disorders.