Among these groups, cholinergic neurons are considered the key modulators of the oscillatory activities. In the past, the functional role of cholinergic neurons has been studied by the elimination of these neurons with immunotoxins, however this irreversible elimination of neurons brings about irreversible changes compromising interpretation of behavioral experiments. To directly test the role of oscillations in learning, memory and mood, we will reversibly inactivate cholinergic neurons in the mouse brain using regulated expression of the light chain of tetanus toxin. This toxin does not kill neurons, but prevent secretion of neurotransmitter by cleaving synaptobrevin, which is required for the docking of synaptic vesicles. Once the expression of the toxin is turned off, neurons should recover their functions. We will test the role of rhythmic oscillations at different stages of memory formation, consolidation and retrieval taking advantage of the reversibility of the system. In vivo recording and analysis of neuronal activity will be performed by Dr. Buzsaki at Rutgers University. We have completed the design of the scheme for reversible genetic inactivation of cholinergic neurons. The scheme includes generation of 2 lines of genetically modified mice. The first line will express tetracycline transactivator in the cholinergic neurons. It will be produced by targeting cholinergic locus with the construct harboring a gene for tetracycline transactivator. The second line will carry modified inactive tetanus toxin, which could only be activated only in the brain following a withdrawal of doxycycline from mouse diet. During the past fiscal year, we have completed a generation of mice with the tetracyclin transactivator gene expressed from the cholinergic locus promoter. Specifically, as the last step, we have successfully excised a selectable marker used during targeting of the locus. We were continuing work on making a targeting construct containing the tetanus toxin gene.
