Figure 4

Specific activation of neural ensembles that encode memory. A. The two studies utilized the same c-fos promoter-tTA mouse line as shown in Figure 3. B. The first study used tTA to induce hM3Dq expression under the control of doxycycline. C. Initially, the mice were exposed to context A (CtxA) in the absence of doxycycline. The resulting neuronal hM3Dq expression is associated with cells that responded upon exposure to CtxA. Next, the mice were fear conditioned in context B (CtxB) and injected with CNO to activate neurons that express hM3Dq and recapitulate the neuronal activity pattern that was present during the earlier exposure to CtxA. In the final test session, mice were exposed to CtxB with or without CNO injection. Interestingly, only CNO injected mice were able to exhibit memory recall (by displaying freezing behavior). D. This study utilized a combined approach consisting of c-fos promoter-tTA mice and an AAV vector that carries a gene expression cassette for ChR2 under the control of TetO. E. First, the mice were habituated to CtxA and light stimulation was applied. The mice were under doxycycline administration at this stage. The mice did not freeze during this stage, confirming that light stimulation alone does not induce freezing. Next, doxycycline is withdrawn to promote ChR2 expression under the control of the c-fos promoter. Then, the mice were fear conditioned in CtxB. This procedure promotes ChR2 expression in neurons that were specifically activated by the contextual fear conditioning protocol in CtxB. Finally, the mice were placed back in CtxA under doxycycline administration and ChR2 expressing neuronal ensembles were activated by light stimulation. The mice displayed freezing only when light was administered (but not by exposure to CtxA alone). Importantly, mice that were not fear-conditioned also did not show freezing in the presence of light.