Summary
Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling [1]. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors [2, 3]. Here we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In response to comments from reviewers, we have revised the preprint as follows. 1. We have modified text to reference accessible GluA2-containing AMPARs. 2. We have removed natively derived as requested. 3. We have specified percentage of dextran cryoprotectant as 20%. 4. We have labelled the stratum radiatum and stratum lacunosum-moleculare in Figure 3B. 5. We have added units to Y-axes for Figures 1C, 2B, 5B, and 5D-E. 6. We have added band annotation to Supplemental Figure 1. 7. We have added scale bars to Supplemental Figure 3. 8. We have corrected the video file.
