<p>Stress is both an underlying and exacerbating factor in a wide range of neuropsychiatric disorders, from anxiety to major depressive disorder. Such stress-related behaviours are thought to be modulated, in part, by the receptors for the stress hormone cortisol. These receptors, namely the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), are highly conserved transcription factors that bind as homodimers to response elements in target genes. Although it has been shown that MR and GR can act as heterodimers as well, little is known regarding the physiological relevance of MR/GR heterodimerization. Here we tested the hypothesis that MR/GR heterodimerization is essential to mediate the behavioural effects of cortisol. For this purpose, we established mutant MRs and GRs that could selectively homo- or heterodimerize and expressed these receptors in zebrafish from an MR/GR double knockout line. Subsequent behavioural analysis revealed that cortisol induced hyperactivity, but only under conditions of MR/GR heterodimerization. To investigate the molecular targets of these heterodimers, we next performed RNA-sequencing, which showed that MR/GR heterodimers regulated the expression of genes involved in glutamatergic signalling. This was confirmed by CRISPR/Cas9-mediated gene editing of a response element in the gene encoding the metabotropic glutamate receptor 3, which abolished the cortisol-induced hyperactivity. Furthermore, pharmacological treatments showed that besides glutamatergic signalling, GABAergic and serotonergic signalling are involved as well. Taken together, this study establishes the physiological relevance of MR/GR heterodimerization, which appears to play a central role in eliciting behavioural alterations upon stress, by altering the activity of crucial neurotransmitter systems.</p>