In a very elegant study published last week in Science, Mark Mayford and colleagues use a synthetic bistable genetic switch to visualize the activity of neurons during associative learning in mice (Reijmers et al, 2007). The reporter system (called TetTag) has two components (see drawing, adapted from Reijmers et al, 2007): 1) the tTA transactivator (tetR-VP16) is placed under the control of the immediate-early gene fos; 2) a tetO-regulated bidirectional promoter drives the expression of both a tau-LacZ reporter and a mutated tetracycline-insensitive version of the tet transactivator, *tTA (tTAH100Y).
Strong neuronal activity activates the fos promoter and stimulates production of tTA. Under permissive conditions, that is, in absence of doxycycline, tTA will activate the “toggle switch”. Even after putting the mice back on doxycycline, the positive feedback maintains the switch active, apparently for up to five days after the initial stimulation. Temporary removal of doxycycline defines thus a time window during which active neurons can be permanently “tagged” (at least over a time scale of several days).
The authors use the TetTag system in transgenic mice to visualize neurons activated during the learning phase of fear conditioning, a behavioral task in which mice learn to associate a given stimulus (eg the “context” represented by the test cage) with a noxious shock. In the case of contextual fear conditioning, the memory of the learned fear response depends on the basolateral amygdala and can be assayed by exposing the mice to the stimulus several days after training and measuring their “freezing” behavior. By enabling TetTag activity during the learning phase only, neurons involved in the learning process are permanently tagged. Three days later, retrieval is tested by exposing the mice to the training cage and immunostaining of the immediate-early gene zif268 (egr1) is used as surrogate measure of neuronal activity during the retrieval phase.
Reijmers and colleagues observe that a significant fraction (12%) of the LacZ-tagged “learning” neurons are also “memory” neurons which are reactivated during retrieval. As the authors write, “reactivated neurons seem to be a likely component of a stable engram or memory trace for conditioned fear”. With a clever application of an memory extinction protocol, they further show that the number of reactivated neurons correlates with the strength of the learned association.
It will be interesting in the future to know more on the quantitative and dynamical characteristics of the TetTag system. But is likely that it will be a useful reporter system in the brain and possibly in other systems as well or in developmental studies. I find it also very nice to see that such a simple switch coupled to an immediate-early gene is already a sufficient device to keep a long-term trace of past neuronal activity. Would it not be nice to identify endogenous neuronal multistable genetic circuits coupled to electrical activity that could explain long-term changes of neuronal properties after given stimuli (as is drug addiction)?