Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.
Bibliographical noteFunding Information:
We thank Dr. Jens Vanselow, Stephanie Lamer and Alvaro Ciudad for supporting the mass spectrometric studies and analysis. We thank Prof. Eric Allemand and Dr. Fabrice Ango for providing gephyrin isoform constructs. And we thank Dr. Katharina Hemmen, Dr. Hanna Heil, Mike Friedrich and Jürgen Pinnecker for their guidance in 3D image analysis and light microscopy. This work was supported by the Deutsche Forschungsgemeinschaft (DFG MA6957/1‐1) to H.M.M.; Agence Nationale de la Recherche (InVivoNanoSpin, ANR‐20‐CE11‐0002‐02) to C.G.S.; Deutsche Forschungsgemeinschaft (Project ID 424778381 – TRR 295), Heisenberg professorship and project funds to P.T. (TO 1124); PhD fellowship from Fundação para a Ciência e a Tecnologia to S.L.R. Open Access funding enabled and organized by Projekt DEAL.
© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.
- Fluorescent Probes
- Super-Resolution Microscopy