¶ Co-first authors
The authors have declared that no competing interests exist.
Conceived and designed the experiments: COD MDCB RPI CNA HDP DBF. Performed the experiments: COD MDCB RPI. Analyzed the data: COD MDCB RPI CNA HDP DBF. Contributed reagents/materials/analysis tools: COD MDCB RPI CNA HDP DBF. Wrote the paper: COD MDCB RPI CNA HDP DBF.
Hyperexcited states, including depolarization block and depolarized low amplitude membrane oscillations (DLAMOs), have been observed in neurons of the suprachiasmatic nuclei (SCN), the site of the central mammalian circadian (∼24-hour) clock. The causes and consequences of this hyperexcitation have not yet been determined. Here, we explore how individual ionic currents contribute to these hyperexcited states, and how hyperexcitation can then influence molecular circadian timekeeping within SCN neurons. We developed a mathematical model of the electrical activity of SCN neurons, and experimentally verified its prediction that DLAMOs depend on post-synaptic L-type calcium current. The model predicts that hyperexcited states cause high intracellular calcium concentrations, which could trigger transcription of clock genes. The model also predicts that circadian control of certain ionic currents can induce hyperexcited states. Putting it all together into an integrative model, we show how membrane potential and calcium concentration provide a fast feedback that can enhance rhythmicity of the intracellular circadian clock. This work puts forward a novel role for electrical activity in circadian timekeeping, and suggests that hyperexcited states provide a general mechanism for linking membrane electrical dynamics to transcription activation in the nucleus.
Daily rhythms in the behavior and physiology of mammals are coordinated by a group of neurons that constitute the central circadian (∼24-hour) clock. Clock neurons contain molecular feedback loops that lead to rhythmic expression of clock-related genes. Much progress has been made in the past two decades to understand the genetic basis of the molecular circadian clock. However, the relationship between the molecular clock and the primary output of clock neurons—their electrical activity—remains unclear. Here, we explore this relationship using computational modeling of an unusual electrical state that clock neurons enter at a certain time of day. We predict that this state causes high concentration of calcium ions inside clock neurons, which activates transcription of clock genes. We demonstrate that this additional feedback promotes 24-hour gene expression rhythms. Thus, we propose that electrical activity is not just an output of the clock, but also part of the core circadian timekeeping mechanism that plays an important role in health and disease.
The conventional theory of neuronal information processing is based on action potential (AP) firing
In the SCN, which function as the central mammalian circadian (∼24-hour) pacemaker
Our approach uses mathematical modeling in combination with experimental validation. We find that DLAMOs are caused by the interplay of L-type calcium and calcium-activated potassium (KCa) currents. During depolarized states, we predict that intracellular calcium concentration reaches high (but physiological) levels. We propose that these daily elevated calcium levels activate clock gene transcription during the day, which in turn increases the expression of KCa and potassium leak currents to hyperpolarize the membrane at dusk and night. We show that this additional feedback loop between membrane excitability and gene expression can promote rhythmicity of the intracellular circadian clock.
Our new computational model of a SCN neuron extends the model of Belle et al.
A subpopulation of SCN neurons, specifically those expressing detectable levels of the
We tested this hypothesis by simulating a neuron in a state producing spontaneous DLAMOs (
The fact that DLAMOs require L-type calcium current suggests that they may be sensitive to factors affecting calcium homeostasis in SCN neurons, since inactivation of L-type current is primarily calcium-dependent
To better understand the ionic currents underlying the electrical behaviors of SCN neurons, we first considered the contribution of sodium (
A key difference between TTXLAMOs and DLAMOs was the mean calcium current. In TTXLAMOs, the mean calcium current was small and at times near zero (
Next, we compared the intracellular calcium levels predicted by the model during quiescence (−65 mV RMP), extended AP firing, and DLAMOs. The model predicts that DLAMOs induce a much greater increase in steady-state intracellular calcium (Δ
Elevation of intracellular calcium levels may play an important role in the rhythmic gene expression that constitutes the molecular circadian clocks within SCN neurons. A major phase-shifting and entrainment pathway for these clocks involves CREB-dependent activation of
To explore the relationship between hyperexcitation and the intracellular circadian clock, we integrated our model of SCN neuron excitability with a simple model of gene regulation based on the Goodwin oscillator
We also note that the proposed mechanism of signaling from membrane to gene transcription within a single cell via depolarized states does not necessarily require AP firing. In simulations of our extended model, ∼24-hour oscillations in cytosolic calcium and gene expression persist in the presence of TTX (
Previous studies have shown higher levels of
While the basic mechanism of circadian timekeeping in mammalian cells is a transcriptional-translational negative feedback loop, electrical activity has sometimes been considered part of the core timekeeping mechanism
This hypothesis is consistent with and extends previously published data. In cerebellar granule cell cultures,
Mathematical modeling is an established tool for understanding the complex interaction of neuronal ion channels and calcium dynamics
Several experimental studies have used TTX to assess the role of sodium-dependent APs on circadian rhythmicity. Infusion of TTX into the SCN of freely moving rats disrupts behavioral rhythms but not internal circadian timekeeping
Mizrak et al.
We extended the computational model of a SCN neuron from Belle et al.
The dynamics of the gating variables
The
Intracellular calcium dynamics are extremely complex and involve many different mechanisms, such as buffering, uptake into and release from intracellular stores, and extrusion through membrane pumps. However, because in SCN neurons many of the details of these mechanisms have not been measured experimentally, we chose to use a very simple model of calcium dynamics that could be fitted directly to experimental data from these neurons. Our model represents all calcium handling mechanisms with a single term for the removal of free calcium ions from the cytosol, as in Booth et al.
Our basic model of gene regulation (
The extended gene regulation model (
In both the basic gene regulation model and the extended version, we assume that the CRE and the E-box interact multiplicatively
All differential equations are expressed in millisecond time units and all simulations were performed using the
We carried out targeted whole-cell electrophysiology in SCN neurons from fourteen male and female mice (∼2–3 months old) heterozygous for
Simultaneous electrophysiological recordings and calcium imaging were performed using three male C57BL/6 mice (heterozygous for
One-parameter bifurcation diagram of the model's behavior as a function of KCa conductance. For a given basal calcium concentration in the shell (here
(TIF)
Two-parameter bifurcation diagram showing the location of the Hopf bifurcation point in
(TIF)
One-parameter bifurcation diagram of the model's behavior as a function of basal shell calcium for low KCa conductance (
(TIF)
Two-parameter bifurcation diagram showing the location of the saddle-node bifurcation points in
(TIF)
Membrane excitability promotes gene expression rhythms in the absence of sodium-dependent AP firing. Simulations of the extended gene regulation model of
(TIF)
Visualization of calcium dynamics. Model fires APs (top left) upon release from a hyperpolarizing current (Iapp = −5 pA), leading to an influx of calcium current (bottom left) and increases in calcium concentration in the shell (top right) and cytosolic (bottom right) compartments.
(TIF)
We thank Daniel DeWoskin for a critical reading of the manuscript.