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Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high-resolution two-photon microscopy in organotypic hippocampal slices from GAD65-GFP mice of both sexes to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons, we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilized boutons rapidly recruited synaptic vesicles, followed by accumulation of postsynaptic gephyrin and were functional after 24 h, as determined by electrophysiology and immunohistochemistry. Inhibitory boutons are only sensitive to Sema4D at a specific stage during synapse formation and sensitivity to Sema4D is regulated by network activity. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurs through rapid remodeling of the actin cytoskeleton. This could be mimicked by the actin-depolymerizing drug latrunculin B or by reducing ROCK activity. We discovered that the intracellular signaling cascade requires activation of the receptor tyrosine kinase MET, which is a well known autism risk factor. By using a viral approach to reduce MET levels specifically in inhibitory neurons, we found that their axons are no longer sensitive to Sema4D signaling. Together, our data yield important insights into the molecular pathway underlying activitydependent Sema4D-induced synapse formation and reveal a novel role for presynaptic MET at inhibitory synapses.

Background: Major depressive disorder is among the most burdening and costly chronic health hazards. Since its prognosis is poor and treatment effectiveness is moderate at best, prevention would be the strategy of first choice. Insomnia may be the best modifiable risk factor. Insomnia is highly prevalent (4-10%) and meta-analysis estimates ±13% of people with insomnia to develop depression within a year. Among people with insomnia, recent work identified three subtypes with a particularly high lifetime risk of depression. The current randomized controlled trial (RCT) evaluates the effects of internet-guided Cognitive Behavioral Therapy for Insomnia (CBT-I), Chronobiological Therapy (CT), and their combination on insomnia and the development of depressive symptoms. Methods:We aim to include 120 participants with Insomnia Disorder (ID) of one of the three subtypes that are more prone to develop depression. In a two by two factorial repeated measures design, participants will be randomized to CBT-I, CT, CBT-I + CT or treatment as usual, and followed up for one year. The primary outcome is the change, relative to baseline, of the severity of depressive symptoms integrated over four follow-ups spanning one year. Secondary outcome measures include a diagnosis of major depressive disorder, insomnia severity, sleep diaries, actigraphy, cost-effectiveness, and brain structure and function.(Continued on next page)

HighlightsPeople with insomnia show widespread brain structural hyperconnectivity.The right angular gyrus is central to the structural connectivity alterations.Connectivity of this angular gyrus subnetwork correlates with reactive hyperarousal.Brain structural hyperconnectivity may mark vulnerability to insomnia.