The master mammalian circadian clock, located in the suprachiasmatic nucleus (SCN) maintains the proper phase relationship between circadian clocks located in tissues throughout the body and entrains the circadian system to the environment. The SCN is composed of individual neuronal oscillators linked by intercellular communication into a neural network that generates a robust and precise rhythm. The long-term goal of our research is to understand the intercellular signaling mechanisms that couple SCN neurons into a neural network that generates circadian rhythms. GABA is the most abundant neurotransmitter in the SCN, where it regulates light-induced phase shifts, synchronization of the dorsal and ventral SCN, synchronization of the circadian phase of individual SCN neurons, and the sensitivity of the circadian clock to light-entraining signals. The strength and functional consequences of GABA(A) receptor activation (whether inhibitory or excitatory) are dynamically regulated by the circadian clock. GABA activates postsynaptic GABA(A) receptors located at structurally specialized synapses, which mediates fast interneuronal communication and extrasynaptic receptors which mediate a tonic GABA current. However, it remains unknown how these two GABA currents regulate the activity and synchrony of the SCN neural network. We hypothesize that two membrane transporter families play critical roles in the regulation of the circadian activity of GABA neurotransmission in the SCN. The GABA transporters GAT-1 and GAT-3 regulate the amount and duration of neurotransmitter GABA in the synaptic cleft and extrasynaptic space. We recently demonstrated that in the SCN the GATs are only expressed in astrocytes suggesting that astrocytes play a vital role in regulating the physiological actions of GABA in the SCN network. The chloride cotransporters of the sodium-potassium- chloride (NKCC) and potassium-chloride (KCC) families control the intracellular Cl- concentration and the polarity and magnitude of the GABA(A) receptor-mediated current. In the adult SCN, GABA may serve as both an inhibitory and excitatory neurotransmitter. We propose that the circadian clock uses WNK/SPAK kinases and Ca2+- and cyclic AMP-activated kinases to regulate the activity of the Cl- cotransporters. The goal of this application is to understand better the regulation of GABAergic signaling and how GABA-mediated signaling contributes to the generation of circadian timing signals in the suprachiasmatic nucleus. To accomplish this goal, we will use single cell electrophysiological and imaging techniques together with transgenic mouse models to study GABAergic neurotransmission identified populations of SCN neurons and examine the roles that synaptic and tonic GABAergic neurotransmission and astrocytes play in synchronizing the circadian clocks of individual SCN neurons. We will also kinase signaling pathways in the circadian control of the polarity of GABA(A) receptor-mediated neurotransmission in the SCN and determine the role of the NKCC and KCC families of chloride cotransporters in regulating the synchronization of SCN neuronal oscillators.
Humans are rhythmic creatures living in a society that has rigid schedules for work, school, and social activities often requiring them to work when their endogenous circadian clock is signaling it is time to sleep. Significant negative health outcomes are linked to alterations in the timing of the endogenous clock and the onset of sleep or activity. This proposal will examine the neurological mechanisms responsible for biological timing and identify possible targets for the therapeutic intervention of circadian based disorders.
