PSAM ion channels/PSEM agonists. Cell type-specific tools are crucial for establishing causal relationships between neuron activity and animal behavior. We developed a modular toolbox of ion channels for cell-type-selectively activating or inhibiting neurons by combining synthetic chemistry and protein engineering. Ultrapotent, selective brain-penetrant chemical ligands (PSEMs) were synthesized and tailored for engineered ligand binding domains (Pharmacologically Selective Actuator Modules, PSAMs). These PSAMs and their selective agonists could be mixed-and-matched with different ion channel domains to create designer ligand-gated ion channels (LGICs) with readily tuned electrical properties. These chimeric ion channels show excellent performance for cell-type-selective activation or inhibition of neurons in vivo.
Synaptic silencers. We examined the mechanism of the hM4Di/CNO DREADD system and found that it was only a modest inhibitor of neuronal electrical activity, but it was actually a powerful silencer of synaptic release. We modified the system to target it selectively to synaptic release sites (hM4Dnrxn) so that the effect on synaptic silencing can be achieved using targeted intracranial CNO injections. This tool is useful for loss-of-function circuit manipulations on molecularly defined cell types and does not appear to affect action potential transmission to downstream areas. For comparison PSAM/PSEM silencer or optogenetic silencers can be used to inhibit axonal projections, but they do so by blocking action potential propagation which also affects every site downstream of the targeted area.
Cell-type selective ion channel pharmacology. We used an enzymatic unmasking strategy to target the use-dependent NMDA-R ion channel antagonist, MK801 to molecularly defined cell types. Rendering MK801 inert with a stable ester modification (to form a reagent called CM-MK801), the masked molecule could be liberated in neurons transgenically expressing pig liver esterase (PLE). We found that we could use this tool in brain slices to block NMDA-Rs selectively in PLE-expressing neurons but not neurons that lacked PLE.
Cre-dependent viral vectors
We developed highly compact Cre-dependent viral vectors using the flip-excision (FLEX) switch that is now used for cell type-specific transgene expression by hundreds of labs. This also allowed the first example of long-range functional synaptic circuit mapping from molecularly defined neurons.
Plasmids are available from Addgene.com
These vectors are prone to recombination. This is a well known issue with these AAV vectors and is due to the inverted terminal repeats (ITRs) required for rAAV production. To minimize recombination, we propagate these plasmids in Stbl2 cells from Invitrogen. Also, to minimize recombination, cells should be cultured at 30 ºC.
Note that these cultures will grow slowly (20 h for minipreps). Better yields and culture times are obtained with 2xYT as the media. This is strongly recommended.
Because recombination may still happen occasionally, we do a panel of restriction digestions to assess whether the ITRs are in tact. Separate digestions with PvuII, Sma1, and SnaB1 should be performed. The expected patterns can be calculated from the attached sequence available on addgene.com.
Viruses for Cre-dependent optogentics based on FLEX switch available from University of Pennsylvania Vector Core.