From Fast Oscillations to Circadian Rhythms: Coupling at Multiscale Frequency Bands in the Rodent Subcortical Visual System.

The subcortical visual system (SVS) is a unique collection of brain structures localised in the thalamus, hypothalamus and midbrain. The SVS receives ambient light inputs from retinal ganglion cells and integrates this signal with internal homeostatic demands to influence physiology. During this processing, a multitude of oscillatory frequency bands coalesces, with some originating from the retinas, while others are intrinsically generated in the SVS. Collectively, these rhythms are further modulated by the day and night cycle. The multiplexing of these diverse frequency bands (from circadian to infra-slow and gamma oscillations) makes the SVS an interesting system to study coupling at multiscale frequencies. We review the functional organisation of the SVS, and the various frequencies generated and processed by its neurons. We propose a perspective on how these different frequency bands couple with one another to synchronise the activity of the SVS to control physiology and behaviour.

Bright daytime light enhances circadian amplitude in a diurnal mammal.

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light-dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light-dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health.

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons.

There is no consensus on the best inhibitory optogenetic tool. Since Gi/o signalling is a native mechanism of neuronal inhibition, we asked whether Lamprey Parapinopsin ("Lamplight"), a Gi/o-coupled bistable animal opsin, could be used for optogenetic silencing. We show that short (405 nm) and long (525 nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, respectively, and that combining these wavelengths can be used to achieve intermediate levels of activity. These properties can be applied to produce switchable neuronal hyperpolarisation and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. Expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, with 405 and 525 nm stimuli producing responses of opposite sign in the output neurons of the retina. We conclude that bistable animal opsins can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and reversible.

Perforated Multi-Electrode Array Recording in Hypothalamic Brain Slices.

The ability to record ensemble action potential (AP) discharge frequencies from large populations of neurons over extended periods of time in vitro offers clear advantages in neuroscience and circadian biology research. Here, we provide an overview of a step-by-step method to perform multisite extracellular AP activity recordings in suprachiasmatic and mediobasal hypothalamic nuclei brain slices, using a state-of-the-art perforated multielectrode array system. Further, we describe in detail a setup architecture which systematically delivers stable, high-quality recordings with excellent anatomical accuracy and consistency. We also provide some procedural, technical, and methodological troubleshooting notes and examples of good quality recordings.

Circadian VIPergic Neurons of the Suprachiasmatic Nuclei Sculpt the Sleep-Wake Cycle.

Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of day-nighttime-when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep but have no effect upon daytime sleep. We propose that mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24-h metronome: specific populations sculpt critical features of the sleep-wake cycle.

PACAP Neurons in the Ventromedial Hypothalamic Nucleus Are Glucose Inhibited and Their Selective Activation Induces Hyperglycaemia.

Background: Glucose-sensing neurons are located in several parts of the brain, but are concentrated in the ventromedial nucleus of the hypothalamus (VMH). The importance of these VMH neurons in glucose homeostasis is well-established, however, little is known about their individual identity. In the present study, we identified a distinct glucose-sensing population in the VMH and explored its place in the glucose-regulatory network. Methods: Using patch-clamp electrophysiology on Pacap-cre::EYFP cells, we explored the glucose-sensing ability of the pituitary adenylate cyclase-activating peptide (PACAP) neurons both inside and outside the VMH. We also mapped the efferent projections of these neurons using anterograde and retrograde tracing techniques. Finally, to test the functionality of PACAPVMH in vivo, we used DREADD technology and measured systemic responses. Results: We demonstrate that PACAP neurons inside (PACAPVMH), but not outside the VMH are intrinsically glucose inhibited (GI). Anatomical tracing techniques show that PACAPVMH neurons project to several areas that can influence autonomic output. In vivo, chemogenetic stimulation of these neurons inhibits insulin secretion leading to reduced glucose tolerance, implicating their role in systemic glucose regulation. Conclusion: These findings are important as they identify, for the first time, a specific VMH neuronal population involved in glucose homeostasis. Identifying the different glucose-sensing populations in the VMH will help piece together the different arms of glucose regulation providing vital information regarding central responses to glucose metabolic disorders including hypoglycaemia.

Neuronal oscillations on an ultra-slow timescale: daily rhythms in electrical activity and gene expression in the mammalian master circadian clockwork.

Neuronal oscillations of the brain, such as those observed in the cortices and hippocampi of behaving animals and humans, span across wide frequency bands, from slow delta waves (0.1 Hz) to ultra-fast ripples (600 Hz). Here, we focus on ultra-slow neuronal oscillators in the hypothalamic suprachiasmatic nuclei (SCN), the master daily clock that operates on interlocking transcription-translation feedback loops to produce circadian rhythms in clock gene expression with a period of near 24 h (< 0.001 Hz). This intracellular molecular clock interacts with the cell's membrane through poorly understood mechanisms to drive the daily pattern in the electrical excitability of SCN neurons, exhibiting an up-state during the day and a down-state at night. In turn, the membrane activity feeds back to regulate the oscillatory activity of clock gene programs. In this review, we emphasise the circadian processes that drive daily electrical oscillations in SCN neurons, and highlight how mathematical modelling contributes to our increasing understanding of circadian rhythm generation, synchronisation and communication within this hypothalamic region and across other brain circuits.

Circadian Disruptions in the Myshkin Mouse Model of Mania Are Independent of Deficits in Suprachiasmatic Molecular Clock Function.

BACKGROUND: Alterations in environmental light and intrinsic circadian function have strong associations with mood disorders. The neural origins underpinning these changes remain unclear, although genetic deficits in the molecular clock regularly render mice with altered mood-associated phenotypes. METHODS: A detailed circadian and light-associated behavioral characterization of the Na+/K+-ATPase α3 Myshkin (Myk/+) mouse model of mania was performed. Na+/K+-ATPase α3 does not reside within the core circadian molecular clockwork, but Myk/+ mice exhibit concomitant disruption in circadian rhythms and mood. The neural basis of this phenotype was investigated through molecular and electrophysiological dissection of the master circadian pacemaker, the suprachiasmatic nuclei (SCN). Light input and glutamatergic signaling to the SCN were concomitantly assessed through behavioral assays and calcium imaging. RESULTS: In vivo assays revealed several circadian abnormalities including lengthened period and instability of behavioral rhythms, and elevated metabolic rate. Grossly aberrant responses to light included accentuated resetting, accelerated re-entrainment, and an absence of locomotor suppression. Bioluminescent recording of circadian clock protein (PERIOD2) output from ex vivo SCN revealed no deficits in Myk/+ molecular clock function. Optic nerve crush rescued the circadian period of Myk/+ behavior, highlighting that afferent inputs are critical upstream mediators. Electrophysiological and calcium imaging SCN recordings demonstrated changes in the response to glutamatergic stimulation as well as the electrical output indicative of altered retinal input processing. CONCLUSIONS: The Myshkin model demonstrates profound circadian and light-responsive behavioral alterations independent of molecular clock disruption. Afferent light signaling drives behavioral changes and raises new mechanistic implications for circadian disruption in affective disorders.

The circadian clock: A tale of genetic-electrical interplay and synaptic integration.

Pioneering work in Drosophila uncovered the building blocks of the molecular clock, consisting of transcription-translation feedback loops (TTFLs). Subsequent experimental work demonstrated that the mammalian TTFL is localized in cells and tissues throughout the brain and body. Further research established that neuronal activity forms an essential aspect of clock function. However, how the membrane electrical activity of clock neurons of the suprachiasmatic nucleus collaborate with the TTFL to drive circadian behaviors remains mostly unknown. Intercellular communication synchronizes the individual circadian oscillators to produce a precise and coherent circadian output. Here, we briefly review significant research that is increasing our understanding of the critical interactions between the TTFL and neuronal and glial activity in the generation of circadian timing signals.

Delayed Cryptochrome Degradation Asymmetrically Alters the Daily Rhythm in Suprachiasmatic Clock Neuron Excitability.

Suprachiasmatic nuclei (SCN) neurons contain an intracellular molecular circadian clock and the Cryptochromes (CRY1/2), key transcriptional repressors of this molecular apparatus, are subject to post-translational modification through ubiquitination and targeting for proteosomal degradation by the ubiquitin E3 ligase complex. Loss-of-function point mutations in a component of this ligase complex, Fbxl3, delay CRY1/2 degradation, reduce circadian rhythm strength, and lengthen the circadian period by ∼2.5 h. The molecular clock drives circadian changes in the membrane properties of SCN neurons, but it is unclear how alterations in CRY1/2 stability affect SCN neurophysiology. Here we use male and female Afterhours mice which carry the circadian period lengthening loss-of-function Fbxl3Afh mutation and perform patch-clamp recordings from SCN brain slices across the projected day/night cycle. We find that the daily rhythm in membrane excitability in the ventral SCN (vSCN) was enhanced in amplitude and delayed in timing in Fbxl3Afh/Afh mice. At night, vSCN cells from Fbxl3Afh/Afh mice were more hyperpolarized, receiving more GABAergic input than their Fbxl3+/+ counterparts. Unexpectedly, the progression to daytime hyperexcited states was slowed by Afh mutation, whereas the decline to hypoexcited states was accelerated. In long-term bioluminescence recordings, GABAA receptor blockade desynchronized the Fbxl3+/+ but not the Fbxl3Afh/Afh vSCN neuronal network. Further, a neurochemical mimic of the light input pathway evoked larger shifts in molecular clock rhythms in Fbxl3Afh/Afh compared with Fbxl3+/+ SCN slices. These results reveal unanticipated consequences of delaying CRY degradation, indicating that the Afh mutation prolongs nighttime hyperpolarized states of vSCN cells through increased GABAergic synaptic transmission.SIGNIFICANCE STATEMENT The intracellular molecular clock drives changes in SCN neuronal excitability, but it is unclear how mutations affecting post-translational modification of molecular clock proteins influence the temporal expression of SCN neuronal state or intercellular communication within the SCN network. Here we show for the first time, that a mutation that prolongs the stability of key components of the intracellular clock, the cryptochrome proteins, unexpectedly increases in the expression of hypoexcited neuronal state in the ventral SCN at night and enhances hyperpolarization of ventral SCN neurons at this time. This is accompanied by increased GABAergic signaling and by enhanced responsiveness to a neurochemical mimic of the light input pathway to the SCN. Therefore, post-translational modification shapes SCN neuronal state and network properties.

Circadian regulation of mouse suprachiasmatic nuclei neuronal states shapes responses to orexin.

Our knowledge of how circadian and homeostatic brain circuits interact to temporally organize physiology and behavior is limited. Progress has been made with the determination that lateral hypothalamic orexin (OXA) neurons control arousal and appetitive states, while suprachiasmatic nuclei (SCN) neurons function as the master circadian clock. During the day, SCN neurons exhibit heterogeneity in spontaneous resting membrane potential (RMP), with some neurons becoming severely depolarized (hyperexcited) and ceasing to fire action potentials (APs), while other neurons rest at moderate RMP and fire APs. Intriguingly, the day phase is when the SCN clock is most readily influenced by arousal, but it is unclear if and how heterogeneity in the excitability state of SCN neurons shapes their response to arousal signals, such as OXA. In whole-cell recordings we show that during the day OXA recruits GABA-GABAA receptor signaling to suppress the RMP of hyperexcited silent as well as moderately hyperpolarized AP-firing SCN neurons. In the AP-firing neurons, OXA hyperpolarized and silenced these SCN cells, while in the hyperexcited silent neurons OXA suppressed the RMP of these cells and evoked either AP-firing, depolarized low-amplitude membrane oscillations, or continued silence at a reduced RMP. These results demonstrate how the resting state of SCN neurons determines their response to OXA, and illustrate that the inhibitory action of this neurochemical correlate of arousal can trigger paradoxical AP firing.

Distinct roles for GABA across multiple timescales in mammalian circadian timekeeping.

The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by-and GABA-inhibited neurons repelled from-this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function.

Intrinsic and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity.

The epithalamic lateral habenula (LHb) is implicated as part of the mammalian brain's circadian system. Anatomical evidence suggests that the LHb receives extrinsic circadian timing cues from retinal ganglion cells and the master clock in the suprachiasmatic nuclei (SCN). Intriguingly, some LHb neurones contain the molecular circadian clock, but it is unclear if and how intrinsic and extrinsic circadian processes influence neuronal activity in the mouse LHb. Here, using an in vitro brain slice preparation isolating the LHb from the SCN, we show through whole-cell patch-clamp recordings that LHb neurones exhibit heterogeneity in their resting state, but the majority spontaneously fire action potentials (APs). Discharge rate of APs varied from low firing in the early day to higher firing later in the day and was absent in LHb brain slices prepared from Cry1(-/-)Cry2(-/-) mice that lack a functional molecular clock. Low amplitude circadian oscillations in the molecular circadian clock were also monitored in LHb brain slices, but were absent in Cry1(-/-)Cry2(-/-) LHb brain tissue. A putative neurochemical output signal of the SCN, prokineticin 2 (PK2), inhibited some LHb neurones by elevating the frequency of GABA release in the LHb. Using multi-electrode recordings in vivo, we found that LHb neurones sluggishly respond to retinal illumination, suggesting that they receive such information through polysynaptic processes. In summary, our results show for the first time that intrinsic circadian signals are important for regulating LHb neuronal state, while the SCN-derived signal PK2 is less influential. Moreover, we demonstrate that mouse LHb neurones have access to and can respond to visual input, but such signals are unlikely to be directly communicated to the LHb. Broadly, these findings raise the possibility that intrinsic circadian signals are likely to be influential in shaping LHb contributions to cognition and emotionality.

Acute suppressive and long-term phase modulation actions of orexin on the mammalian circadian clock.

Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brain's master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K(+) currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPY's inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain.

Daily variation in the electrophysiological activity of mouse medial habenula neurones.

Intrinsic daily or circadian rhythms arise through the outputs of the master circadian clock in the brain's suprachiasmatic nuclei (SCN) as well as circadian oscillators in other brain sites and peripheral tissues. SCN neurones contain an intracellular molecular clock that drives these neurones to exhibit pronounced day-night differences in their electrical properties. The epithalamic medial habenula (MHb) expresses clock genes, but little is known about the bioelectric properties of mouse MHb neurones and their potential circadian characteristics. Therefore, in this study we used a brain slice preparation containing the MHb to determine the basic electrical properties of mouse MHb neurones with whole-cell patch clamp electrophysiology, and investigated whether these vary across the day-night cycle. MHb neurones (n = 230) showed heterogeneity in electrophysiological state, ranging from highly depolarised cells (∼ -25 to -30 mV) that are silent with no membrane activity or display depolarised low-amplitude membrane oscillations, to neurones that were moderately hyperpolarised (∼40 mV) and spontaneously discharging action potentials. These electrical states were largely intrinsically regulated and were influenced by the activation of small-conductance calcium-activated potassium channels. When considered as one population, MHb neurones showed significant circadian variation in their spontaneous firing rate and resting membrane potential. However, in recordings of MHb neurones from mice lacking the core molecular circadian clock, these temporal differences in MHb activity were absent, indicating that circadian clock signals actively regulate the timing of MHb neuronal states. These observations add to the extracellularly recorded rhythms seen in other brain areas and establish that circadian mechanisms can influence the membrane properties of neurones in extra-SCN sites. Collectively, the results of this study indicate that the MHb may function as an intrinsic secondary circadian oscillator in the brain, which can shape daily information flow in key brain processes, such as reward and addiction.

Causes and consequences of hyperexcitation in central clock neurons.

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 electrical silencing in the mammalian circadian clock.

Neurons in the brain's suprachiasmatic nuclei (SCNs), which control the timing of daily rhythms, are thought to encode time of day by changing their firing frequency, with high rates during the day and lower rates at night. Some SCN neurons express a key clock gene, period 1 (per1). We found that during the day, neurons containing per1 sustain an electrically excited state and do not fire, whereas non-per1 neurons show the previously reported daily variation in firing activity. Using a combined experimental and theoretical approach, we explain how ionic currents lead to the unusual electrophysiological behaviors of per1 cells, which unlike other mammalian brain cells can survive and function at depolarized states.