Protocol for in vivo analysis of pre- and post-synaptic protein function in mice

Summary: Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins.For complete details on the use and execution of this protocol, please refer to Cramer et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Palmitoylation of BMPR1a regulates neural stem cell fate

Neural stem cells (NSCs) generate neurons and glial cells throughout embryonic and postnatal brain development. The role of S-palmitoylation (also referred to as S-acylation), a reversible posttranslational lipid modification of proteins, in regulating the fate and activity of NSCs remains largely unknown. We used an unbiased screening approach to identify proteins that are S-acylated in mouse NSCs and showed that bone morphogenic protein receptor 1a (BMPR1a), a core mediator of BMP signaling, is palmitoylated. Genetic manipulation of S-acylated sites affects the localization and trafficking of BMPR1a and leads to altered BMP signaling. Strikingly, defective palmitoylation of BMPR1a modulates NSC function within the mouse brain, resulting in enhanced oligodendrogenesis. Thus, we identified a mechanism regulating the behavior of NSCs and provided the framework to characterize dynamic posttranslational lipid modifications of proteins in the context of NSC biology

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

Abstract Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation