GENETICS OF TIMING · A Genetics Approach to the Interval Timing Mechanism

Periodic Report Summary - Genetics of Timing (A Genetics Approach to the Interval Timing Mechanism)

Time, like space, is a fundamental dimension of existence. Evolution has selected for neurobiological mechanisms that allow both human and non-human animals to respond to the temporal parameters of their environment so that they can anticipate predictable events. There are two distinct mechanisms of timing: circadian timing refers to the ability of anticipating events that recur with a period of approximately 24 hrs. The ability to calculate shorter durations, in the order of seconds to minutes, is achieved through interval timing. In effect, interval timing operates like an internal stopwatch that can estimate the time that elapses between events. Our project aims at exploring and identifying the molecular basis of the interval timing mechanism. To this end, we performed a series of experiments that investigated the ability of several mutant strains of mice with deficient Calcium/Calmodulin Kinase II (CaMKII) dependent signaling to estimate temporal durations. These strains involve the alpha CaMKII null mutants, the beta CaMKII null mutants, and the alpha CaMKII T305D point mutants. The global alpha CaMKII null mice have impaired cortical and hippocampal synaptic plasticity, as well as severe memory and learning deficits (Silva et al, 1992, Science 257:206-11; Glazewski et al, 2000, Nature Neuroscience 3:911-8). In the alpha CaMKII T305D mice, a point mutation interferes with Ca2+/Calmodulin binding, resulting in severely reduced levels of alpha/beta CaMKII in the postsynaptic density and heavily impaired plasticity (Elgersma et al, 2002, Neuron 36:493-505). Finally, the newly developed beta CaMKII null mice show deficient plasticity in several brain areas (van Woerden et al, 2009, Nature Neuroscience 12:823-5), including the hippocampus. Despite the involvement of CaMKII signaling in several learning paradigms, we found interval timing in all of the above strains to be intact. That is, their memory of the learned durations, which ranged between 3 and 42 seconds, was as accurate and precise as in the wildtype mice. Thus, it is unlikely that CaMKII signaling is involved in this type of learning. Alternatively, it is possible that the absence of the protein during the embryonic stage may induce compensatory mechanisms to rescue the plasticity involved in interval timing. Therefore, we will test the temporal memory of alpha CaMKII inducible null mice. These mice are engineered so that the gene can be deleted in adulthood. Thus, we will first train the mice to learn two criterion durations under fully functional alpha CaMKII. Then we will delete the gene and subsequently test the memory of the mutants for the learned durations. In the light of these negative results, we decided to also test the involvement of extracellular signal regulated kinase (Erk) signaling, another major signaling pathway, in interval timing. Erk is enriched in the striatum (an area shown to be important for this cognitive process) and has been implicated in several learning paradigms that rely on the proper function of this brain structure (Mazzucchelli et al, 2002, Neuron 34:807-20). To this end, we have generated several mutant strains of H- and K-ras (potent upstream activators of Erk), in which the Erk signaling pathway ranges from being severely downregulated to overactivated. If Erk signaling regulates the corticostriatal plasticity believed to be involved in interval timing, we expect the temporal memory of at least some of these mutant strains to be less precise and/or accurate. By investigating the role of two major signaling pathways, CaMKII- and Erk-signaling, our project aims at characterizing the biochemical cascades involved when the brain estimates and stores durations. Our findings will reduce our uncertainty about how this process is realized and will guide future investigations to either elaborate on the exact role of the identified signaling pathway, or focus on other biochemical cascades that contribute to this cognitive process.

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