The suprachiasmatic nucleus (SCN) is the master circadian pacemaker driving daily rhythms in mammalian physiology and behavior. SCN neurons utilize a transcription/translation feedback loop to generate circadian changes in electrical activity. Although we have known that SCN neurons fire during the day and are silent at night since 1982 and considerable evidence implicates subthreshold K+ conductance(s), the critical K+ conductance(s) have not been identified. In recent studies focused on testing the hypothesis that subthreshold, A-type (IA) voltage-gated K+ (Kv) channels are involved, we found that mice lacking Kv4.2 (Kv4.2-/-) or Kv1.4 (Kv1.4-/-) pore-forming (?) subunits have markedly shorter circadian periods of locomotor (wheel running) activity than wild-type (WT) mice. Using in vitro extracellular microelectrode recordings, we found that the periods of circadian rhythms in firing are similarly shortened in SCN neurons lacking either Kv4.2 or Kv1.4. Initial experiments here (aim 1) will determine if Kv4.2 and Kv1.4 are the only Kv ? subunits contributing to the IA channels that modulate SCN excitability and reveal the effects the combined loss Kv4.2 and Kv1.4 on rhythms in SCN firing and locomotor activity. The goal of aim 2 is to determine if the shorter period of circadian firing in SCN neurons lacking Kv4.2 or Kv1.4 reflects the functioning of IA channels in the synchronization (i.e., network properties) or the cell-autonomous regulation of SCN neuron excitability.
This aim will, for the first time, establish whether the critical K+ conductance(s) in different SCN cell types are distinct. A long-standing debate in the field is whether daily changes in membrane potential are required for the generation of circadian rhythms in gene expression.
Aim 3 will test directly the hypothesis that Kv4.2- and Kv1.4-encoded IA channel mediated changes in excitability also modulate the period and amplitude of circadian changes in gene expression. Finally, the observation that the cyclic changes in SCN neuron firing and locomotor activity persist (albeit with a shorter period) in the absence of Kv1.4 or Kv4.2 indicates that other K+ conductances regulate the daily oscillations in SCN neuron membrane potentials.
In aim 4, we will exploit a novel, high-throughput quantitative Taqman-based RT-PCR based method to quantify the expression levels of multiple K+ channel subunits simultaneously, as a function of circadian time, and to identify the subthreshold K+ conductance(s) that mediates the daily depolarizations and hyperpolarizations in the membrane potentials of SCN neurons. These studies will provide fundamentally important new insights into the roles of specific K+ conductances in regulating/modulating daily rhythms in the excitability of SCN neurons. In addition to guiding further investigations into the molecular, cellular and systemic mechanisms linking daily rhythms in neuronal excitability, gene expression and behavior, these insights will translate to advances in understanding the regulation and dysregulation of circadian rhythms and to the development of novel therapeutic strategies to benefit individuals suffering genetic and environmentally-induced disruptions in circadian rhythms.
Daily rhythms in behavior, physiology and cognitive performance are driven by circadian clocks in the brain. This project examines the role of specific potassium channels in the generation and synchronization of circadian oscillators using real-time molecular, physiological and behavioral assays. The findings will be relevant to normal daily health and to current health problems arising from disrupted daily rhythms including cardiac arrhythmias, some forms of depression, and obesity.
|Victor, Matheus B; Richner, Michelle; Hermanstyne, Tracey O et al. (2014) Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts. Neuron 84:311-23|