The suprachiasmatic nucleus (SCN) in the mammalian brain contains the master circadian clock. Individual SCN neurons are competent circadian oscillators and constitute a neural network that stabilizes and enhances the generation of circadian timing signals. The SCN neural network is regulated by the molecular clock and shows significant plasticity with changes in day length altering clock gene expression and responses to entraining signals. Neurotransmission mediated by γ- aminobutyric acid (GABA) is a key functional component of the SCN network, since virtually all SCN neurons utilize GABA as a neurotransmitter and express ionotropic GABAA receptors. Additionally, more than 50% of synapses onto SCN neurons are GABAergic. GABA shifts the phase of the circadian clock, synchronizes dorsal and ventral SCN neurons, and modulates the action potential firing frequency of individual SCN neurons. The strength of GABAergic neurotransmission is regulated by the intracellular Cl- concentration. In the adult brain, activation of GABAA receptors inhibits a majority of SCN neurons during the day, while at night the majority of GABAA responses are excitatory. The day-night difference in GABAA receptor-mediated synaptic activity results from dynamic regulation of the intracellular Cl- concentration. The sodium-potassium-chloride (NKCC1) cotransporter responsible for increasing intracellular Cl- and the potassium-chloride (KCC) family of cotransporters that reduce intracellular Cl- are heterogeneously expressed throughout the SCN. We hypothesize that the circadian variation in GABA neurotransmission depends on the type and activity of the cotransporters expressed. In this application we will examine the role that dynamic circadian regulation of the intracellular Cl- concentration plays in mediating the activity of the SCN neural network and the response of the circadian clock to phase shifting stimuli. We will combine electrophysiological and cell-imaging techniques to study the role of NKCC1 and KCCs in the circadian regulation of the strength and direction of GABAA receptor-mediated neurotransmission in individual SCN neurons.
The Specific Aims of the proposal are: 1) Determine, in individual SCN neurons, whether the direction of the GABA-activated chloride current underlies the shift in GABA response from inhibitory to excitatory during a circadian cycle. 2) Investigate the role of the NKCC1 and KCC cotransporters in regulating the activity of the SCN neural network. 3) Examine the role of the NKCC1 cotransporter in the regulation of the intracellular Cl- concentration and GABA activity in SCN neurons. 4) Examine the role of different KCC cotransporters in regulating the intracellular Clconcentration and GABA responses of SCN neurons.
Humans live in a complex 24 hour a day society where desynchrony between circadian clocks and the environment contribute to an increased vulnerability to a variety of diseases. The current application aims to identify the mechanisms of the generation of circadian timing signals and strategies leading to mitigation or correction of circadian based disorders.
