GABA-mediated repulsive coupling between circadian clock neurons in the SCN encodes seasonal time

Abstract

The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABA A -mediated synaptic input. We show that increasing daylength changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABA A signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation-inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.day-length encoding | repulsive coupling | SCN | GABA | chloride T he physiological and behavioral rhythms of all life on earth are bound to the Earth's rotational cycle of ∼24 h. This fundamental rhythm is also affected by the planet's slanted rotational axis, which causes seasonal variations in the length of the day. How life has adapted to anticipate this yearly rhythm is still debated.The suprachiasmatic nucleus (SCN), the central circadian (∼24 h) pacemaker in mammals, consists of ∼10,000 "clock" neurons. These single-cell clocks maintain endogenous rhythms by autoregulatory feedback loops of genes including period (Per) and brain and muscle Arnt-like 1 (Bmal1) (1). Although it was speculated that seasonal rhythms might be encoded in a single cell (2), single-cell oscillations remain similar regardless of the seasonal time that the SCN tissue encodes (3). Seasonal timing is thus proposed to be encoded in the network of the SCN (4-7) through spatial reorganization of the relative phases of clocks within individual cells (8-10). Subsets of SCN clocks form phase clusters (11) that map approximately to dorsal (shell, D-SCN) and ventral (core, V-SCN) regions of the SCN. When measured through a luciferase reporter monitoring oscillations in the Bmal1 gene, the D-SCN and V-SCN clusters show a phase gap, ...

Functional network inference of the suprachiasmatic nucleus

“…It has been shown that excitatory GABA action drives reentrainment of the shell region in response to light phase shifts (17). GABA action was examined in depth in recent collaborative works (16,26); these studies demonstrated the encoding of day length through the GABA pathway, by the Cl − -dependent excitatory (core, phase-attractive) or inhibitory (shell, phase-repulsive) effects of slow-scale (tonic) GABA. Visually, the core regions identified in our networks overlap with the regions of excitatory GABA action (26).…”

“…GABA action was examined in depth in recent collaborative works (16,26); these studies demonstrated the encoding of day length through the GABA pathway, by the Cl − -dependent excitatory (core, phase-attractive) or inhibitory (shell, phase-repulsive) effects of slow-scale (tonic) GABA. Visually, the core regions identified in our networks overlap with the regions of excitatory GABA action (26). In future studies, our TTX-based assay could be combined with VIP/GABA/glutamate agonist or antagonist application and repeated to identify the molecular mechanisms responsible for the identified connections.…”

“…Prominent modeling studies of the past decade have assumed a wide variety of network structures: nearest neighbor (15,20), small-world (21), or mean-field (22,23), or combinations of these depending on coupling pathway (16), pointing to the high degree of uncertainty regarding the general connectivity of the SCN. There has been significant recent interest in attempting to elucidate the network structure and mechanisms driving synchrony the SCN, commonly through light-driven desynchronization assays (19,(24)(25)(26). These methods have the advantage of reducing the SCN into large phase clusters of neurons, whose behavior can be easily tracked and modeled with reduced approaches (26).…”

“…There has been significant recent interest in attempting to elucidate the network structure and mechanisms driving synchrony the SCN, commonly through light-driven desynchronization assays (19,(24)(25)(26). These methods have the advantage of reducing the SCN into large phase clusters of neurons, whose behavior can be easily tracked and modeled with reduced approaches (26). This approach has had great successes in reconciling the roles of GABA and VIP (17,16,19,26).…”

“…These methods have the advantage of reducing the SCN into large phase clusters of neurons, whose behavior can be easily tracked and modeled with reduced approaches (26). This approach has had great successes in reconciling the roles of GABA and VIP (17,16,19,26). A significant obstacle in developing a mechanistic understanding of synchronization in the SCN is the lack of single-cell resolution in these studies, preventing observation of the dynamics within these clusters.…”