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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.

Neuroimaging and genetics studies have advanced our understanding of the neurobiology of sleep and its disorders. However, individual studies usually have limitations to identifying consistent and reproducible effects, including modest sample sizes, heterogeneous clinical characteristics and varied methodologies. These issues call for a large-scale multi-centre effort in sleep research, in order to increase the number of samples, and harmonize the methods of data collection, preprocessing and analysis using pre-registered well-established protocols. The Enhancing NeuroImaging Genetics through Meta-Analysis (ENIGMA) consortium provides a powerful col-How to cite this article: Tahmasian M, Aleman A, Andreassen OA, et al. ENIGMA-Sleep: Challenges, opportunities, and the road map.

Study Objectives Suggested neural correlates of insomnia disorder have been hard to replicate. Even the most consistent finding, altered white matter microstructure in the anterior limb of the internal capsule, is based on handful studies. The urge for replicable targets to understand the underlying mechanisms of insomnia made us study white matter fractional anisotropy (FA) across three samples of cases and controls. Methods 3-Tesla MRI diffusion tensor imaging data of three independent samples were combined for analysis, resulting in n = 137 participants, of whom 73 were diagnosed with insomnia disorder and 64 were matched controls without sleep complaints. Insomnia severity was measured with the Insomnia Severity Index (ISI). White matter microstructure was assessed with FA. White matter tracts were skeletonized and analyzed using tract-based spatial statistics. We performed a region-of-interest analysis using linear mixed-effect models to evaluate case–control differences in internal capsule FA as well as associations between internal capsule FA and insomnia severity. Results FA in the right limb of the anterior internal capsule was lower in insomnia disorder than in controls (β = −9.76e−3; SE = 4.17e−3, p = .034). In the entire sample, a higher ISI score was associated with a lower FA value of the right internal capsule (β = −8.05e− 4 FA/ISI point, SE = 2.60e− 4, p = .008). Ancillary whole brain voxel-wise analyses showed no significant group difference or association with insomnia severity after correction for multiple comparisons. Conclusions The internal capsule shows small but consistent insomnia-related alterations. The findings support a circuit-based approach to underlying mechanisms since this tract connects many brain areas previously implicated in insomnia.

Insomnia poses a high risk for depression. Brain mechanisms of sleep and mood improvement following cognitive behavioral therapy for insomnia remain elusive. This longitudinal study evaluated whether: 1) individual differences in baseline brain white matter microstructure predict improvements and 2) intervention affects brain white matter microstructure. People meeting the Diagnostic and Statistical Manual of Mental Disorders-5 criteria for Insomnia Disorder (n=117) participated in a randomized controlled trial comparing 6 weeks of no treatment with therapist-guided digital cognitive behavioral therapy for insomnia, circadian rhythm support, or their combination (cognitive behavioral therapy for insomnia + circadian rhythm support). Insomnia severity index and inventory of depressive symptomatology – self report were assessed at baseline and followed up at week 7, 26, 39 and 52. Diffusion weighted magnetic resonance images were acquired at baseline and week 7. Skeletonized white matter tracts, fractional anisotropy and mean diffusivity, were quantified both tract-wise and voxel-wise using tract-based spatial statistics. Analyses used linear and mixed-effect models while correcting for multiple testing using false discovery rate and Bonferroni for correlated endpoint measures. Our results show: 1) tract-wise lower fractional anisotropy in the left retrolenticular part of the internal capsule at baseline predicted both worse progression of depressive symptoms in untreated participants as well as more improvement in treated participants (fractional anisotropy x any intervention, pFDR=0.053, pcorr=0.045). 2) Only the cognitive behavioral therapy for insomnia + circadian rhythm support intervention induced a trend-level mean diffusivity decrease in the right superior corona radiata (pFDR = 0.128, pcorr = 0.108), and individuals with a stronger mean diffusivity decrease showed a stronger alleviation of insomnia (R = 0.20, p = 0.035). In summary, individual differences in risk and treatment-supported resilience of depression involve white matter microstructure. Future studies could target the role of the left retrolenticular part of the internal capsule and right superior corona radiata and the brain areas they connect.

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