Various types of rhythmic oscillations in the brain are associated with specific stages of sleep and wakefulness and also correlate with degree of arousal. It is hypothesized that some of those rhythms may be required for the acquisition and consolidation of memories and affect mental state, however the direct proofs of this hypothesis are still absent. One way to test the role of these oscillations is to interfere with the function of neurons producing those oscillations. There are multiple neuronal populations involved in generation and maintenance of rhythmic firing. 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 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 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.? During previous years, 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. We have completed cloning of the mouse cholinergic locus, generation of the first targeting construct for the expression of tetracycline transactivator (tTA) and creation of mice with the insertion of tTA into the cholinergic locus. Since the second construct harbors a modified tetanus toxin, it was necessary to verify that the planned modification introduced into the toxin does not interfere with its activity. To test the activity of modified toxin, we have constructed testing plasmids carrying the same modifications in the toxin structure, which will appear following its activation in the brain. We also had to clone a gene for synaptobrevin, a substrate for the toxin. We have completed functional testing of this modified toxin in cell culture confirming that it retains activity after modification.? During the last fiscal year we have generated chimeric mice that should carry tetanus toxin under the control of tet-O-driven promoter. The project will continue following successful germ line transmission of the genetic modification.
