Summary It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo . We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We found that both typical and fast-acting antidepressants bind to a cholesterol interaction motif in the BDNF receptor TRKB, a known mediator of neuronal plasticity and antidepressant responses. Cholesterol stabilized a cross-shaped configuration of TRKB transmembrane domain dimers and prolonged TRKB cell surface expression and activation by BDNF. Mutation of the TRKB cholesterol interaction site or cholesterol depletion by pravastatin impaired BDNF-mediated plasticity and cellular and behavioral responses to antidepressants in vitro and in vivo . We suggest that binding to and facilitation of TRKB activity is the common mechanism for antidepressant action, and propose a framework for how molecular effects of antidepressants are translated into clinical mood recovery.

Primary neurons from rodent brain hippocampus and cortex have served as important tools in biomedical research over the years. However, protocols for the preparation of primary neurons vary, which often lead to conflicting results. This report provides a robust and reliable protocol for the production of primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-neuronal cells. The neurons were grown in serum-free media and maintained for several weeks without any additional feeder cells. The neuronal cultures maintained according to this protocol differentiate and by 3 weeks develop extensive axonal and dendritic branching. The cultures produced by this method show excellent reproducibility and can be used for histological, molecular and biochemical methods.

7Primary neurons from rodent brain hippocampus and cortex has served as great tools in biomedical 8 research over the years. However, protocols for the preparation of primary neurons vary, which often 9 leads to conflicting results. This report provides a robust and reliable protocol for the production of 10 primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-11 neuronal cells. The neurons were grown in serum free media and maintained for several weeks 12 without any additional feeder cells. The neuronal cultures maintained according to this protocol 13 differentiate and by three weeks develop extensive axonal and dendritic branching. The cultures 14 produced by this method show excellent reproducibility and can be used for histological, molecular 15 and biochemical methods. 16 17

Neurotrophins and their receptors have highly conserved evolutionary lineage in vertebrates including zebrafish. The NTRK2 receptor has two isoforms in zebrafish, Ntrk2a and Ntrk2b. The spatio-temporal expression pattern of bdnf and ntrk2b in the zebrafish brain was studied using in situ hybridization. The robust and corresponding expression pattern of ntrk2b to bdnf suggests that ntrk2b is the key receptor for bdnf in the zebrafish brain, unlike its duplicate isoform ntrk2a. To study ntrk2b function, two different genetic strategies, the TILLING mutant and morpholino oligonucleotides (MO), were used. Specific subsets of the dopaminergic and serotonergic neuronal populations were affected in the mutants and morphants. The mutant showed anxiety- like behavior both in larval and adult stages. Our results consistently indicate that BDNF/NTRK2 signaling has a significant role in the development and maintenance of aminergic neuronal populations. Therefore, the ntrk2b-deficient zebrafish is well suited to study mechanisms relevant for psychiatric disorders attributed to a dysfunctional monoaminergic system.