Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons

• Published in: International journal of molecular sciences Vol. 22; no. 18; p. 10032
• Main Authors: Kuljis, Dika A, Micheva, Kristina D, Ray, Ajit, Wegner, Waja, Bowman, Ryan, Madison, Daniel V, Willig, Katrin I, Barth, Alison L
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 17.09.2021; MDPI
• Subjects: Animals; Binding sites; Carrier Proteins - metabolism; Chemical compounds; Electron microscopy; Female; Fibronectin; Fluorescence; Fluorophores; GABAA receptors; Genetic code; Gephyrin; Intrabodies; intrabody; Labeling; Localization; Male; Membrane Proteins - metabolism; Microscopy; Microscopy, Electrochemical, Scanning; mRNA; Neurons; Neurons - metabolism; Parvalbumin; Presynapse; Proteins; Pyramidal cells; Pyramidal Cells - metabolism; Quantitation; Quantitative analysis; Receptors, GABA-A - metabolism; Synapses; Synapses - metabolism; Synaptic density; Synaptophysin; Transgenic animals; Transgenic mice; synaptophysin; intrabody; synapses; parvalbumin; optogenetics; fluorescent protein sensors; gephyrin; correlative fluorescence and electron microscopy; light microscopy; GABAA receptors
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms221810032

Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABA Rs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions.