Due to the very recent development of in vivo widefield fluorescence imaging using a miniaturized mi- croscope, neuronal networks can now be observed in real time during responses to environmental input, goal- directed behaviors, and abnormal function with unprecedented anatomical and phenotypic specificity. The cir- cadian neural network in the hypothalamic suprachiasmatic nucleus (SCN) is an ideal model system in which to apply these tools. The SCN network is unique in that it receives direct monosynaptic sensory input from the eyes, encodes light onset and daylength into its network function, and provides neuronal output that regulates the timing of most physiological functions and behavior. However, a major limitation in investigations of SCN physiology to-date has been the need to section the brain, destroying most of the network, and divorcing it from its sensory inputs or behavioral outputs. The ability to study the intact SCN network with functional visual afferents and behavioral efferents, would represent an unprecedented leap forward for the field. We propose to develop a toolkit for the real-time imaging with cell-specific optogenetic control of SCN neurophysiological activity. This requires optimization via systematic testing of genetic constructs, viral vectors, imaging devices, mouse models, and surgical procedures. Once optimized, we will use these powerful approaches to study a critically important feature of circadian biology which was previously unaddressable: the network processing of light input that leads to shifts of the circadian behavioral rhythm.
The circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) of the mammalian brain is a compact neural network which can be studied as a simplified model of the entire central nervous system. A major limitation in investigations of SCN physiology to-date has been the need to section the brain, destroying most of the network, and divorcing it from its sensory inputs or behavioral outputs. We propose to develop a toolkit for in vivo real-time Ca2+ imaging with cell-specific optogenetic control of SCN neurophysiological activ- ity, in order to study the cell-type-specific responses of SCN neurons to photic input in awake, behaving mice.
