Effective pharmacotherapy for major depressive disorder remains a major challenge, as more than 30% of patients are resistant to the first line of treatment (selective serotonin reuptake inhibitors) 1 . Sub-anaesthetic doses of ketamine, a noncompetitive N-methyl-d-aspartate receptor antagonist 2,3 , provide rapid and long-lasting antidepressant effects in these patients [4][5][6] , but the molecular mechanism of these effects remains unclear 7,8 . Ketamine has been proposed to exert its antidepressant effects through its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) 9 . The antidepressant effects of ketamine and (2R,6R)-HNK in rodents require activation of the mTORC1 kinase 10,11 . mTORC1 controls various neuronal functions 12 , particularly through cap-dependent initiation of mRNA translation via the phosphorylation and inactivation of eukaryotic initiation factor 4E-binding proteins (4E-BPs) 13 . Here we show that 4E-BP1 and 4E-BP2 are key effectors of the antidepressant activity of ketamine and (2R,6R)-HNK, and that ketamine-induced hippocampal synaptic plasticity depends on 4E-BP2 and, to a lesser extent, 4E-BP1. It has been hypothesized that ketamine activates mTORC1-4E-BP signalling in pyramidal excitatory cells of the cortex 8,14 . To test this hypothesis, we studied the behavioural response to ketamine and (2R,6R)-HNK in mice lacking 4E-BPs in either excitatory or inhibitory neurons. The antidepressant activity of the drugs is mediated by 4E-BP2 in excitatory neurons, and 4E-BP1 and 4E-BP2 in inhibitory neurons. Notably, genetic deletion of 4E-BP2 in inhibitory neurons induced a reduction in baseline immobility in the forced swim test, mimicking an antidepressant effect. Deletion of 4E-BP2 specifically in inhibitory neurons also prevented the ketamineinduced increase in hippocampal excitatory neurotransmission, and this effect concurred with the inability of ketamine to induce a long-lasting decrease in inhibitory neurotransmission. Overall, our data show that 4E-BPs are central to the antidepressant activity of ketamine.A single sub-anaesthetic dose of ketamine elicits a rapid (within hours) and sustained (up to seven days) antidepressant response in patients with treatment-resistant major depressive disorder (MDD) [4][5][6] , serving as the basis for the approval of the enantiomer (S)-ketamine (esketamine) by the FDA for treatment of MDD. Ketamine may exert its antidepressant effects via one of its metabolites, (2R,6R)-HNK 9 , which may act as an inhibitor of NMDA (N-methyl-d-aspartate) receptors at certain concentrations 9,15,16 . Ketamine and (2R,6R)-HNK activate mTORC1 signalling and protein synthesis in the prefrontal cortex (PFC) and hippocampus (HPC) 7,10,11,[17][18][19][20] . Furthermore, in rodents, the antidepressant response to ketamine and (2R,6R)-HNK is blocked by infusion of rapamycin, an allosteric inhibitor of mTORC1, into the PFC 10,11 . mTORC1 affects cellular functions as diverse as nucleotide and lipid synthesis, glucose metabolism, autophagy, lysosome biogenesis, proteasome as...

Highlights d Searchable astroglial translatome database of male and female mice during development d Astroglia show early (P1-P7) and late (P14-adult) developmental phenotypes d Astroglia show sex differences in gene expression patterns during development

150 WORDS) Astroglial cells are emerging as key players in the development and homeostatic maintenance of neurons and neuronal networks. Astroglial cell functions are critical to neuronal migration and maturation, myelination, and synapse dynamics, however little is known about astroglial phenotypic changes over development. Furthermore, astroglial cells express steroid hormone receptors and show rapid responses to hormonal manipulations, however, despite important sex differences in telencephalic regions such as the cortex and hippocampus few studies have examined sex differences in astroglial cells in development and outside of the hypothalamus and amygdala. To phenotype cortical astroglial cells across postnatal development while considering potential sex differences, we used translating ribosome affinity purification together withRNAsequencing (TRAPseq) and immunohistochemistry to phenotype the entire astroglial translatome in males and females at key developmental time points: P1, P4, P7, P14, P35 and in adulthood. We found that although astroglia show few sex differences in adulthood, they show significant sex differences in developmental gene expression patterns between p7 and P35, that suggest sex differences in developmental functions. We also found two distinctive, non-overlapping, astroglial phenotypes between early (P1-P7) and late development (P14-Adult). Together these data clearly delineate and phenotype astroglia across development and identify sex differences in astroglial developmental programs that could have an important impact on the construction and maintenance of neuronal networks and related behavioural phenotypes.

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The neuroplasticity hypothesis of depression proposes that major depressive disorders are related to decreased hippocampal and cortical neural plasticity, which is reversed by antidepressant treatment. Astroglial cells have emerged as key mediators of neural plasticity and are involved in the cause and treatment of depression and anxiety-like behaviors. One of the ways that astroglia modulate neuroplasticity is through the formation and maintenance of perineuronal nets (PNNs). Perineuronal nets are important extracellular matrix components that respond to stress and are implicated in anxiety-like behaviors. Normally, astroglial cells continuously turnover PNNs by degrading and donating PNN proteins; however, chronic stress slows PNN protein degradation and increases cortical PNN expression overall. In this report, we used weighted gene co-expression network analysis and eigengene analysis to further delineate the pathways and key regulators involved in the astroglial-PNN relationship following chronic stress. Our analyses indicate that chronic variable stress induces the expression of PNNs through inhibition of trophic pathways and key transcription factors in astroglial cells. These data further support the integral role of astroglial cells in the neuroplasticity hypothesis of depression through their modulation of anxiety-like behaviors and PNNs.