In neurons, microtubules form dense bundles and run along the length of axons and dendrites. Occasionally, dendritic microtubules can grow from the shaft directly into dendritic spines. Microtubules target dendritic spines that are undergoing activity-dependent changes, but the mechanism by which microtubules enter spines has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and local glutamate uncaging, we show that local actin remodeling at the base of a spine promotes microtubule spine targeting. Microtubule spine entry is triggered by activation of N-Methyl-D-aspartic acid (NMDA) receptors and calcium influx and requires dynamic actin remodeling. Activity-dependent translocation of the actin remodeling protein cortactin out of the spine correlates with increased microtubule targeting at a single spine level. Our data show that the structural changes in the actin cytoskeleton at the base of the spine are directly involved in microtubule entry and emphasize the importance of actin-microtubule crosstalk in orchestrating synapse function and plasticity.

Highlights d Two-photon glutamate uncaging is used to stimulate clustered excitatory spines d Spine stimulation can trigger inhibitory bouton growth onto the same dendrite d The dendrite triggers presynaptic inhibitory changes via endocannabinoid signaling d This mechanism may ensure local inhibitory control of active excitatory clusters

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Background: Speech understanding may rely not only on auditory, but also on visual information. Non-invasive functional neuroimaging techniques can expose the neural processes underlying the integration of multisensory processes required for speech understanding in humans. Nevertheless, noise (from functional MRI, fMRI) limits the usefulness in auditory experiments, and electromagnetic artifacts caused by electronic implants worn by subjects can severely distort the scans (EEG, fMRI). Therefore, we assessed audio-visual activation of temporal cortex with a silent, optical neuroimaging technique: functional near-infrared spectroscopy (fNIRS).Methods: We studied temporal cortical activation as represented by concentration changes of oxy- and deoxy-hemoglobin in four, easy-to-apply fNIRS optical channels of 33 normal-hearing adult subjects and five post-lingually deaf cochlear implant (CI) users in response to supra-threshold unisensory auditory and visual, as well as to congruent auditory-visual speech stimuli.Results: Activation effects were not visible from single fNIRS channels. However, by discounting physiological noise through reference channel subtraction (RCS), auditory, visual and audiovisual (AV) speech stimuli evoked concentration changes for all sensory modalities in both cohorts (p < 0.001). Auditory stimulation evoked larger concentration changes than visual stimuli (p < 0.001). A saturation effect was observed for the AV condition.Conclusions: Physiological, systemic noise can be removed from fNIRS signals by RCS. The observed multisensory enhancement of an auditory cortical channel can be plausibly described by a simple addition of the auditory and visual signals with saturation.

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.