In the cerebral cortex, the excitability of pyramidal neurons  is mainly regulated by axo-dendritic and axo-somatic inputs from excitatory and inhibitory neurons. However, gamma-aminobutyric acid (GABA)-ergic axo-axonic synapses located at the axon initial segment (AIS) of pyramidal neurons also powerfully modulate the generation of axonal action potentials. Despite their important role in neuronal excitability and circuit function, the molecular and circuit mechanisms underlying AIS axo-axonic synapse assembly and function remain incompletely understood. Dysregulation of AIS axo-axonic synapses has been reported in patients with schizophrenia, autism spectrum disorder, and Alzheimer’s disease. Thus, defining the mechanisms of AIS axo-axonic synapse assembly may reveal how their dysregulation leads to neuropsychiatric and neurodegenerative diseases.

AIS axo-axonic synapses can be visualized by coimmunostaining pre- or postsynaptic proteins (for instance, the vesicular GABA transporter or the scaffolding protein Gephyrin) together with AIS proteins such as AnkyrinG or β4 spectrin. However, due to the high density of neurons in the cerebral cortex, it can be challenging to clearly distinguish non-AIS signals from bona fide AIS axo-axonic synapses, and whole-body knockout of components of inhibitory synapses such as Gephyrin can be lethal. in their new study titled "Hide-and-Seek genome editing reveals that Gephyrin is required for axo-axonic synapse assembly", Dr. Yuki Ogawa and his post-doctoral mentor Dr. Matthew Rasband (Baylor College of Medicine) describe a novel CRISPR-mediate genetic method called Hide and Seek that allows for the simultaneous knockout of one gene and insertion of an epitope tag into a second gene of interest. Using this creative method, they discovere that Gephyrin is required for the assembly of axo-axonic inhibitory synapses at the axon initial segment.