Project Summary/Abstract This proposal aims to delineate the electrical and molecular diversity of the suprachiasmatic nucleus (SCN) and provide new evidence for receptor-expressing subtypes of SCN neurons using novel nanowire arrays that allow single cell recording at 1024 contacts simultaneously. The SCN is among the most robust and far-reaching networks of the brain. Located in the ventral hypothalamus, the SCN integrates light input to keep a 24-hour rhythm that informs time-of-day function and transcription in nearly every cell and organ system in the body. Because of its discrete structure, robust molecular and electrical rhythm, and quantifiable behavioral output, the SCN is an ideal network to study to understand the fundamental mechanisms in neural networks overall. The goal of this proposal is to understand SCN function at a single-cell and whole-network level, using newly developed technology that affords long-term electrophysiological recordings. In the mentored phase, the first experiments will demonstrate use nanowire arrays for long-term, intracellular-like recordings in dispersed SCN primary neurons, which have a circadian firing pattern. I will determine firing characteristics, period, and peptide responsiveness of the individual cells on the array with 1024 contacts. Then, we will examine the effect of disruption of the molecular clock on firing patterns in these neurons. Finally, we will harvest these single SCN neurons for RNA sequencing to associate firing properties with the transcriptome, and compare to published datasets. The goal of these experiments is to characterize the functional and molecular properties of individual SCN neurons, and to gain training in SCN culture, transcriptomics, and large-scale electrophysiology analysis. In the independent phase, the experiments will use the nanowire arrays for long-term recordings in SCN slices from wildtype and transgenic animals. The transcriptional network of the SCN is maintained in slice preparations, and has a unique spatiotemporal pattern of the molecular clock; it is unknown if firing follows a similar pattern. I will use the arrays to create a functional map of SCN neuron firing types, phases, and properties, and illustrate the effect of receptor-expressing population silencing using optogenetics. We will continue this approach with SCN slices from mutant mice with conditional clock disruptions in receptor-expressing neurons, and ultimately characterize the behavior of these animals. The goal of these experiments is to understand the phase and network relationship among these neurons using electrophysiological data, and how discrete receptor- expressing populations contribute to the SCN network and behavior. Ultimately, the data from these studies would be used together to interrogate how the transcriptome of a single SCN neuron informs its firing patterns both within and outside of the SCN network, and how these individual oscillators unite to form the SCN network. Together, these experiments will advance our understanding of neural networks and the cellular composition of this critical brain region.

Project Narrative Disrupted circadian rhythms, such as in shift work or genetic disorders, can result in obesity, cancer, or infertility. The proposed research will use novel electrophysiological and molecular methods to describe the contribution of individual neurons in the development and maintenance of a daily rhythm throughout the body, and how disruption of this system contributes to disease.