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

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.

Protocol for the culturing of primary hippocampal mouse neurons for functional in vitro studies.

Primary hippocampal cultures grown from genetically modified mice provide a simplified context to study molecular mechanisms underlying neuronal development, synaptogenesis, and synapse plasticity in vitro. Here, we describe a simple protocol for culturing hippocampal neurons from P0 to P2 mice and a strategy for inducing alterations in synaptic strength at inhibitory and excitatory synapses in vitro. We also describe approaches for immunofluorescent labeling, image acquisition, and quantification of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al.1.

Adamtsl3 mediates DCC signaling to selectively promote GABAergic synapse function.

The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.