Functional Connectivity Analysis of the Mammalian Circadian Pacemaker

Daily rhythms in mammalian physiology and behavior are mediated by a circadian pacemaker within the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is composed of approximately 20,000 neurons which maintain robust daily rhythms in clock gene expression and electrical activity. Network interactions are necessary for neurons to maintain synchronous activity and produce a coherent output. To date, our understanding of the network topology and intercellular signals which modulate periodicity and synchrony remain incomplete. We utilized multielectrode array technology to record and measure correlated spontaneous electrical activity from synchronized SCN neurons and map functional network connectivity. We find that millisecond-level interactions between neurons persist over days, but vary in strength and number over circadian time. Using pharmacologic approaches, we show that these connections are primarily mediated by GABAA receptor signaling and are relatively rare—indicating that low levels of connectivity are sufficient to modulate network dynamics. To determine the role of GABAA receptor-mediated interactions in circadian time-keeping, we monitored clock gene (PERIOD2) expression using low-light imaging technologies. We find that GABAergic network interactions decrease the temporal precision of circadian gene expression in single cells. Interestingly, we find that in desynchronized networks lacking the coupling agent vasoactive intestinal polypeptide, blockade of GABAergic signaling rescues synchrony. Together these data support a model in which sparse, millisecond-level, GABAA receptor-mediated interactions introduce sufficient noise into the SCN circuit to alter precision in gene expression over hours to days, and in some cases, functionally desynchronize the circadian network.;In conjunction with our work testing the role of GABAergic signaling in SCN networks, we also find that picrotoxin, a classic GABAA receptor antagonist, decreases the period of circadian oscillations. We demonstrate that picrotoxin acts independently of GABAA receptors and other Cys-loop receptors to shorten the period of the circadian clock by specifically advancing the accumulation of PERIOD2 protein. Notably, picrotoxin's circadian target is found in several mammalian cell-types, but not Drosophila, thereby ruling out all conserved Cys-loop receptors and known regulators of PERIOD protein stability. Together, these data point to the existence of an important and novel picrotoxin-sensitive target within the mammalian circadian timing system.