Suprachiasmatic Nucleus Neurons Research Articles

System-level time computation and representation in the suprachiasmatic nucleus revealed by large-scale calcium imaging and machine learning.

The suprachiasmatic nucleus (SCN) is the mammalian central circadian pacemaker with heterogeneous neurons acting in concert while each neuron harbors a self-sustained molecular clockwork. Nevertheless, how system-level SCN signals encode time of the day remains enigmatic. Here we show that population-level Ca2+ signals predict hourly time, via a group decision-making mechanism coupled with a spatially modular time feature representation in the SCN. Specifically, we developed a high-speed dual-view two-photon microscope for volumetric Ca2+ imaging of up to 9000 GABAergic neurons in adult SCN slices, and leveraged machine learning methods to capture emergent properties from multiscale Ca2+ signals as a whole. We achieved hourly time prediction by polling random cohorts of SCN neurons, reaching 99.0% accuracy at a cohort size of 900. Further, we revealed that functional neuron subtypes identified by contrastive learning tend to aggregate separately in the SCN space, giving rise to bilaterally symmetrical ripple-like modular patterns. Individual modules represent distinctive time features, such that a module-specifically learned time predictor can also accurately decode hourly time from random polling of the same module. These findings open a new paradigm in deciphering the design principle of the biological clock at the system level.

Increased glutamatergic neurotransmission between the retinohypothalamic tract and the suprachiasmatic nucleus of old mice.

Circadian rhythms synchronize to light through the retinohypothalamic tract (RHT), which is a bundle of axons coming from melanopsin retinal ganglion cells, whose synaptic terminals release glutamate to the ventral suprachiasmatic nucleus (SCN). Activation of AMPA-kainate and NMDA postsynaptic receptors elicits the increase in intracellular calcium required for triggering the signaling cascade that ends in phase shifts. During aging, there is a decline in the synchronization of circadian rhythms to light. With electrophysiological (whole-cell patch-clamp) and immunohistochemical assays, in this work, we studied pre- and postsynaptic properties between the RHT and ventral SCN neurons in young adult (P90-120) and old (P540-650) C57BL/6J mice. Incremental stimulation intensities (applied on the optic chiasm) induced much lesser AMPA-kainate postsynaptic responses in old animals, implying a lower recruitment of RHT fibers. Conversely, a higher proportion of old SCN neurons exhibited synaptic facilitation,and variance-mean analysis indicated an increase in the probability of release in RHT terminals. Moreover, both spontaneous and miniature postsynaptic events displayed larger amplitudes in neurons from aged mice, whereas analysis of the NMDA and AMPA-kainate components (evoked by RHT electrical stimulation) disclosed no difference between the two ages studied. Immunohistochemistry revealed a bigger size in the puncta of vGluT2, GluN2B, and GluN2A of elderly animals, and the number of immunopositive particles was increased, but that of PSD-95 was reduced. All these synaptic adaptations could be part of compensatory mechanisms in the glutamatergic signaling to ameliorate the loss of RHT terminals in old animals.

Circadian rhythm mechanism in the suprachiasmatic nucleus and its relation to the olfactory system.

Animals need sleep, and the suprachiasmatic nucleus, the center of the circadian rhythm, plays an important role in determining the timing of sleep. The main input to the suprachiasmatic nucleus is the retinohypothalamic tract, with additional inputs from the intergeniculate leaflet pathway, the serotonergic afferent from the raphe, and other hypothalamic regions. Within the suprachiasmatic nucleus, two of the major subtypes are vasoactive intestinal polypeptide (VIP)-positive neurons and arginine-vasopressin (AVP)-positive neurons. VIP neurons are important for light entrainment and synchronization of suprachiasmatic nucleus neurons, whereas AVP neurons are important for circadian period determination. Output targets of the suprachiasmatic nucleus include the hypothalamus (subparaventricular zone, paraventricular hypothalamic nucleus, preoptic area, and medial hypothalamus), the thalamus (paraventricular thalamic nuclei), and lateral septum. The suprachiasmatic nucleus also sends information through several brain regions to the pineal gland. The olfactory bulb is thought to be able to generate a circadian rhythm without the suprachiasmatic nucleus. Some reports indicate that circadian rhythms of the olfactory bulb and olfactory cortex exist in the absence of the suprachiasmatic nucleus, but another report claims the influence of the suprachiasmatic nucleus. The regulation of circadian rhythms by sensory inputs other than light stimuli, including olfaction, has not been well studied and further progress is expected.

Phenotyping of light-activated neurons in the mouse SCN based on the expression of FOS and EGR1.

Light-sensitive neurons are located in the ventral and central core of the suprachiasmatic nucleus (SCN), whereas stably oscillating clock neurons are found mainly in the dorsal shell. Signals between the SCN core and shell are believed to play an important role in light entrainment. Core neurons express vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), and Neuroglobin (Ngb), whereas the shell neurons express vasopressin (AVP), prokineticin 2, and the VIP type 2 (VPAC2) receptor. In rodents, light has a phase-shifting capacity at night, which induces rapid and transient expression of the EGR1 and FOS in the SCN. Methods: The present study used immunohistochemical staining of FOS, EGR1, and phenotypical markers of SCN neurons (VIP, AVP, Ngb) to identify subtypes/populations of light-responsive neurons at early night. Results: Double immunohistochemistry and cell counting were used to evaluate the number of SCN neurons expressing FOS and EGR1 in the SCN. The number of neurons expressing either EGR1 or FOS was higher than the total number of neurons co-storing EGR1 and FOS. Of the total number of light-responsive cells, 42% expressed only EGR1, 43% expressed only FOS, and 15% expressed both EGR1 and FOS. Light-responsive VIP neurons represented only 31% of all VIP neurons, and EGR1 represents the largest group of light-responsive VIP neurons (18%). VIP neurons expressing only FOS represented 1% of the total light-responsive VIP neurons. 81% of the Ngb neurons in the mouse SCN were light-responsive, and of these neurons expressing only EGR1 after light stimulation represented 44%, whereas 24% expressed FOS. Although most light-responsive neurons are found in the core of the SCN, 29% of the AVP neurons in the shell were light-responsive, of which 8% expressed EGR1, 10% expressed FOS, and 11% co-expressed both EGR1 and FOS after light stimulation. Discussion: Our analysis revealed cell-specific differences in light responsiveness between different peptidergic and Ngb-expressing neurons in different compartments of the mouse SCN, indicating that light activates diverse neuronal networks in the SCN, some of which participate in photoentrainment.

Alpha-synuclein expression in oxytocin neurons of young and old bovine brains.

Understanding of central nervous system mechanisms underlying age-related infertility remains limited. Fibril α-synuclein, distinct from its monomeric form, is implicated in age-related diseases. Notably, fibril α-synuclein spreads among neurons, similar to prions, from damaged old neurons in cortex and hippocampus to healthy neurons. However, less is known whether α-synuclein propagates into oxytocin neurons, which play crucial roles in reproduction. We compared α-synuclein expression in the oxytocin neurons in suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), paraventricular hypothalamic nucleus (PVN), and posterior pituitary (PP) gland of healthy heifers and aged cows to determine its role in age-related infertility. We analyzed mRNA and protein expression, along with Congo red histochemistry and fluorescent immunohistochemistry for oxytocin and α-synuclein, followed by confocal microscopy with Congo red staining. Both mRNA and protein expressions of α-synuclein were confirmed in the bovine cortex, hippocampus, SCN, SON, PVN, and PP tissues. Significant differences in α-synuclein mRNA expressions were observed in the cortex and hippocampus between young heifers and old cows. Western blots showed five bands of α-synuclein, probably reflecting monomers, dimers, and oligomers, in the cortex, hippocampus, SCN, SON, PVN, and PP tissues, and there were significant differences in some bands between the young heifers and old cows. Bright-field and polarized light microscopy did not detect obvious amyloid deposition in the aged hypothalami; however, higher-sensitive confocal microscopy unveiled strong positive signals for Congo red and α-synuclein in oxytocin neurons in the aged hypothalami. α-synuclein was expressed in oxytocin neurons, and some differences were observed between young and old hypothalami.

The transcription factor VAX1 in VIP neurons of the suprachiasmatic nucleus impacts circadian rhythm generation, depressive-like behavior, and the reproductive axis in a sex-specific manner in mice.

The suprachiasmatic nucleus (SCN) within the hypothalamus is a key brain structure required to relay light information to the body and synchronize cell and tissue level rhythms and hormone release. Specific subpopulations of SCN neurons, defined by their peptide expression, regulate defined SCN output. Here we focus on the vasoactive intestinal peptide (VIP) expressing neurons of the SCN. SCN VIP neurons are known to regulate circadian rhythms and reproductive function. To specifically study SCN VIP neurons, we generated a novel knock out mouse line by conditionally deleting the SCN enriched transcription factor, Ventral Anterior Homeobox 1 (Vax1), in VIP neurons (Vax1Vip; Vax1fl/fl:VipCre). We found that Vax1Vip females presented with lengthened estrous cycles, reduced circulating estrogen, and increased depressive-like behavior. Further, Vax1Vip males and females presented with a shortened circadian period in locomotor activity and ex vivo SCN circadian period. On a molecular level, the shortening of the SCN period was driven, at least partially, by a direct regulatory role of VAX1 on the circadian clock genes Bmal1 and Per2. Interestingly, Vax1Vip females presented with increased expression of arginine vasopressin (Avp) in the paraventricular nucleus, which resulted in increased circulating corticosterone. SCN VIP and AVP neurons regulate the reproductive gonadotropin-releasing hormone (GnRH) and kisspeptin neurons. To determine how the reproductive neuroendocrine network was impacted in Vax1Vip mice, we assessed GnRH sensitivity to a kisspeptin challenge in vivo. We found that GnRH neurons in Vax1Vip females, but not males, had an increased sensitivity to kisspeptin, leading to increased luteinizing hormone release. Interestingly, Vax1Vip males showed a small, but significant increase in total sperm and a modest delay in pubertal onset. Both male and female Vax1Vip mice were fertile and generated litters comparable in size and frequency to controls. Together, these data identify VAX1 in SCN VIP neurons as a neurological overlap between circadian timekeeping, female reproduction, and depressive-like symptoms in mice, and provide novel insight into the role of SCN VIP neurons.

In-phasic cytosolic-nuclear Ca2+ rhythms in suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca2+ rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca2+-dependent kinase regulates the clock gene transcription. However, the Ca2+ regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca2+ rhythms remain unclear. In the present study, we monitored circadian-timescale Ca2+ dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca2+ rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca2+ rhythms, whereas blocking of Ca2+ release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors had a minor effect on either Ca2+ rhythms. We conclude that the in-phasic circadian Ca2+ rhythms in the cytosol and nucleus are mainly driven by Ca2+ influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca2+ rhythms directly regulate transcription in situ.

Integrated neural tracing and in-situ barcoded sequencing reveals the logic of SCN efferent circuits in regulating circadian behaviors.

The circadian clock coordinates rhythms in numerous physiological processes to maintain organismal homeostasis. Since the suprachiasmatic nucleus (SCN) is widely accepted as the circadian pacemaker, it is critical to understand the neural mechanisms by which rhythmic information is transferred from the SCN to peripheral clocks. Here, we present the first comprehensive map of SCN efferent connections and suggest a molecular logic underlying these projections. The SCN projects broadly to most major regions of the brain, rather than solely to the hypothalamus and thalamus. The efferent projections from different subtypes of SCN neurons vary in distance and intensity, and blocking synaptic transmission of these circuits affects circadian rhythms in locomotion and feeding to different extents. We also developed a barcoding system to integrate retrograde tracing with in-situ sequencing, allowing us to link circuit anatomy and spatial patterns of gene expression. Analyses using this system revealed that brain regions functioning downstream of the SCN receive input from multiple neuropeptidergic cell types within the SCN, and that individual SCN neurons generally project to a single downstream brain region. This map of SCN efferent connections provides a critical foundation for future investigations into the neural circuits underlying SCN-mediated rhythms in physiology. Further, our new barcoded tracing method provides a tool for revealing the molecular logic of neuronal circuits within heterogeneous brain regions.

Selective vulnerability of the suprachiasmatic nucleus in progressive Alzheimer’s Disease: A human postmortem study using spatial in‐situ proteomics

AbstractBackgroundAlzheimer’s Disease (AD) patients commonly experience sleep/wake (S/W) disturbances, such as sleep fragmentation and excessive daytime sleepiness. The S/W cycle is controlled by a complex system regulated by a master regulator, the suprachiasmatic nucleus (SCN) that controls wake‐ and sleep‐promoting nuclei. Despite its importance in orchestrating sleep architecture, little is known about the molecular changes that occur in the SCN during the progression of AD and how these changes contribute to S/W disturbances.MethodWe used postmortem brain tissue of 10 controls without AD pathology and 32 subjects with progressive AD stages. Our approach included quantitative neuropathological assessments of AVP and VIP‐expressing neurons in SCN and its adjacent hypothalamic region, the supraoptic nucleus (SON). We also used an in‐situ cell‐specific spatial proteomics platform [GeoMx Digital Spatial Profiling (DSP)] to probe levels of different pathological tau species and proteins commonly dysregulated in AD.ResultPreliminary findings reveal a significant decrease in SCN volume (∼75%) (Fig.1) and a loss of ∼58% AVP neurons and ∼41% VIP neurons in Braak stage (Braak) VI compared to Braak 0. The DSP results indicate that both neurons in the SCN have twice the burden of p‐tau levels as the SON (Fig.2‐right), and neurofibrillary tanglesAdditionally, SCN shows an increase in proteins associated with glia and immune responses (Fig.2‐left). Furthermore, an increase in microglial proteins is observed in the SCN in Braak I (Fig.3‐left). In contrast, the SON displays a milder pattern of protein dysregulation in Braak I (Fig.3‐right), consistent with its lower tau expression observed in histology and DSP.ConclusionSCN is vulnerable to AD pathology even in Braak I, which is characterized by volume loss, increased immune response protein dysregulation, and later neuronal loss, as well as p‐tau pathology. This pattern supports the idea that the SCN plays a role in S/W disturbances observed from early AD stages. Interestingly, its neighboring nucleus, SON, which also has AVP neurons, remained spared, providing opportunities for studies investigating factors related to selective vulnerability. Ongoing studies are addressing changes in the SCN and SON in intermediate AD stages.

Expression of the vesicular GABA transporter within neuromedin S+ neurons sustains behavioral circadian rhythms.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS)+ neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS-Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker.

Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons

Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons

The mouse suprachiasmatic nucleus encodes irradiance via a diverse population of neurons monotonically tuned to different ranges of intensity.

Many neurons of the mammalian master circadian oscillator in the suprachiasmatic nuclei (SCN) respond to light pulses with irradiance-dependent changes in firing. Here, we set out to better understand this irradiance coding ability by considering how the SCN tracks more continuous changes in irradiance at both population and single unit level. To this end, we recorded extracellular activity in the SCN of anaesthetised mice presented with up+down irradiance staircase stimuli covering moonlight to daylight conditions and incorporating epochs with steady light or superimposed higher frequency modulations (temporal white noise (WN) and frequency/contrast chirps). Single unit activity was extracted by spike sorting. The population response of SCN units to this stimulus was a progressive increase in firing rate at higher irradiances. This relationship was symmetrical for up vs. down phases of the ramp in the presence of white noise or chirps but exhibited hysteresis for steady light, with firing systematically higher during increasing irradiance. Single units also showed a monotonic relationship between firing and irradiance but exhibited diversity not only in response polarity (increases vs. decreases in firing), but also in the sensitivity (EC50 ) and slope of fitted functions. These data show that individual SCN neurons exhibit monotonic relationships between irradiance and firing rate but differ in the irradiance range over which they respond. This property may help the SCN to encode the large differences in irradiance found in nature using neurons with a constrained range of firing rates. KEY POINTS: Daily changes in environmental light (irradiance) entrain the suprachiasmatic nucleus (SCN) circadian clock. The mouse SCN shows graded increases in neurophysiological activity with light pulses of increasing irradiance. We show that this monotonic relationship between firing rate and irradiance is retained at population and single unit level when probed with more naturalistic staircase increases and decreases in irradiance. The irradiance response is more reliable in the presence of ongoing higher temporal frequency modulations in light intensity than under steady light. Single units varied in sensitivity allowing the population to cover a wide range of irradiances. Irradiance coding in the SCN has characteristics of a sparse code with individual neurons tracking different portions of the natural irradiance range. This property may address the challenge of encoding a 109 -fold day:night difference in irradiance within the constrained range of firing rates available to individual neurons.

Entrainment range affected by the second-order interactions between coupled neuron oscillators in the suprachiasmatic nucleus

Higher-order interactions between coupled oscillators in neural networks exhibit a series of collective behavior phenomena, especially synchronization, some of which cannot occur in pairwise interactions. Thus far, there has been little researches on whether the higher-order interactions affect other collective behaviors besides synchronization, such as the entrainment ability of the suprachiasmatic nucleus (SCN), which is a master clock located in the mammalian brain. In this article, we investigated whether the entrainment range of the SCN is affected by the second-order interactions, based on a modified Kuramoto model. The numerical simulations and theoretical analyses both showed that the entrainment range is widened by introducing the second-order interactions in the case that a part of the SCN neurons are sensitive to the light information. It was worth noting that the entrainment range depends on the different initial values of the neuronal oscillator phases, and this dependence has not been observed in previous studies of the pairwise interactions. Our findings help to understand the effect of higher-order interactions between the coupled neuron oscillators on the entrainment range of the SCN.

Aripiprazole disrupts cellular synchrony in the suprachiasmatic nucleus and enhances entrainment to environmental light-dark cycles in mice.

Many patients with psychiatric conditions, such as bipolar disorder and major depressive disorder, frequently experience disruptions in their sleep-wake cycles. Several case studies and clinical trials have shown that the administration of aripiprazole, a commonly prescribed antipsychotic drug, alleviates the symptoms of circadian sleep disorders in these patients. This improvement may be attributed to the effects of aripiprazole on the circadian central clock, specifically the hypothalamic suprachiasmatic nucleus (SCN), which regulates various circadian physiological rhythms, including the sleep-wake cycle, in mammals. To examine whether aripiprazole facilitates adaptation to changes in the light-dark cycle, we orally administered aripiprazole to mice and subjected them to jet-lag experiments. Mice receiving aripiprazole were more rapidly entrained to 6 h advanced light-dark cycles. Moreover, we examined the effect of aripiprazole on the cellular rhythms of SCN slice cultures and found that aripiprazole disrupted cellular synchronization in the SCN, thereby accelerating the damping of the SCN rhythm at the population level. Adenosine 3'5' monophosphate (cAMP) assay using a bioluminescence indicator revealed that intracellular cAMP level in the SCN increased following aripiprazole treatment. However, this increase was blocked by pre-treatment with the serotonin 1A receptor (5-HT1AR) antagonist. Based on these findings, we propose that aripiprazole modulates intracellular signaling, including 5-HT1AR-mediated cAMP signaling, and desynchronizes SCN neurons, ultimately leading to enhanced entrainment to phase advanced light-dark cycles in mice. These findings indicate that the improvement in sleep symptoms reported in patients with psychiatric disorders receiving aripiprazole may be due to modulation of the circadian clock. Our study provides novel insights into the potential clinical applications of aripiprazole in patients with various circadian sleep disorders.

Ultrastructure of Synaptic Connectivity within Subregions of the Suprachiasmatic Nucleus Revealed by a Genetically Encoded Tag and Serial Blockface Electron Microscopy.

The hypothalamic suprachiasmatic nucleus (SCN) is the central circadian pacemaker in vertebrates. The SCN receives photic information exclusively through melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize circadian rhythms with the environmental light cycles. The SCN is composed of two major peptidergic neuron types in the core and shell regions of the SCN. Determining how mRGCs interact with the network of synaptic connections onto and between SCN neurons is key to understand how light regulates the circadian clock and to elucidate the relevant local circuits within the SCN. To map these connections, we used a newly developed Cre-dependent electron microscopy (EM) reporter, APEX2, to label the mitochondria of mRGC axons. Serial blockface scanning electron microscopy was then used to resolve the fine 3D structure of mRGC axons and synaptic boutons in the SCN of a male mouse. The resulting maps reveal patterns of connectomic organization in the core and shell of the SCN. We show that these regions are composed of different neuronal subtypes and differ with regard to the pattern of mRGC input, as the shell receives denser mRGC synaptic input compared with the core. This finding challenges the present view that photic information coming directly from the retina is received primarily by the core region of the SCN.

Kv12-encoded K+ channels drive the day-night switch in the repetitive firing rates of SCN neurons.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

Chronic exposure to dim artificial light disrupts the daily rhythm in mitochondrial respiration in mouse suprachiasmatic nucleus

ABSTRACT Circadian rhythms of physiology, behavior, and metabolism have an endogenous 24 h period that synchronizes with environmental cycles of light/dark and food availability. Alterations in light cycles are stressful and disrupt such diurnal oscillations. Recently, we witnessed a sudden rise in studies describing the mechanisms behind the interaction between the key characteristics of mitochondrial functions, peripheral clocks, and stress responses. To our knowledge, there is no study in the suprachiasmatic nuclei (SCN) describing the dysregulated mitochondrial bioenergetics under abnormal lighting conditions, which is common in today’s modern world. Thus, we aimed to investigate the existence of daily changes in mitochondrial bioenergetics (respiratory control rate, RCR), mitochondrial abundance (mtDNA/nDNA), plasma corticosterone, and to test whether disturbances in the lighting conditions might influence such rhythms. To confirm this, mice were sacrificed, mitochondria were isolated from the suprachiasmatic nuclei in the brain and blood was collected, every 3 h at various time points zeitgeber time/circadian time, (0, 3, 6, 9, 12, 15, 18, 21, and 24 h) under 12:12 h light-dark (LD, 150 lux L: 0 lux D) cycle and chronic artificial dim lighting (LL, 5 lux: 5lux) conditions, of a 24 h period, respectively. Our results demonstrate the existence of robust daily rhythmicity in RCR, mtDNA/nDNA and plasma CORT under a normal LD cycle. However, these rhythms were significantly disrupted and clock genes expressions were dysregulated under chronic dim LL. Furthermore, mitochondrial abundance was significantly reduced during LL compared to their numbers under LD cycle. Our data demonstrate that the circadian clock regulates mitochondrial functions (RCR, number), essential for accomplishing daily energy demands and supply by the SCN neurons. Abnormal light exposure dysregulates mitochondrial functions in the SCN and may alter metabolism, resulting in obesity, diabetes, and other metabolic disorders. Therefore, properly designing lighting conditions in workplaces is essential to mitigate the adverse consequences of light on humans.

Effects of NALCN-Encoded Na+ Leak Currents on the Repetitive Firing Properties of SCN Neurons Depend on K+-Driven Rhythmic Changes in Input Resistance.

Neurons in the suprachiasmatic nucleus (SCN) generate circadian changes in the rates of spontaneous action potential firing that regulate and synchronize daily rhythms in physiology and behavior. Considerable evidence suggests that daily rhythms in the repetitive firing rates (higher during the day than at night) of SCN neurons are mediated by changes in subthreshold potassium (K+) conductance(s). An alternative "bicycle" model for circadian regulation of membrane excitability in clock neurons, however, suggests that an increase in NALCN-encoded sodium (Na+) leak conductance underlies daytime increases in firing rates. The experiments reported here explored the role of Na+ leak currents in regulating daytime and nighttime repetitive firing rates in identified adult male and female mouse SCN neurons: vasoactive intestinal peptide-expressing (VIP+), neuromedin S-expressing (NMS+) and gastrin-releasing peptide-expressing (GRP+) cells. Whole-cell recordings from VIP+, NMS+, and GRP+ neurons in acute SCN slices revealed that Na+ leak current amplitudes/densities are similar during the day and at night, but have a larger impact on membrane potentials in daytime neurons. Additional experiments, using an in vivo conditional knockout approach, demonstrated that NALCN-encoded Na+ currents selectively regulate daytime repetitive firing rates of adult SCN neurons. Dynamic clamp-mediated manipulation revealed that the effects of NALCN-encoded Na+ currents on the repetitive firing rates of SCN neurons depend on K+ current-driven changes in input resistances. Together, these findings demonstrate that NALCN-encoded Na+ leak channels contribute to regulating daily rhythms in the excitability of SCN neurons by a mechanism that depends on K+ current-mediated rhythmic changes in intrinsic membrane properties.SIGNIFICANCE STATEMENT Elucidating the ionic mechanisms responsible for generating daily rhythms in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, is an important step toward understanding how the molecular clock controls circadian rhythms in physiology and behavior. While numerous studies have focused on identifying subthreshold K+ channel(s) that mediate day-night changes in the firing rates of SCN neurons, a role for Na+ leak currents has also been suggested. The results of the experiments presented here demonstrate that NALCN-encoded Na+ leak currents differentially modulate daily rhythms in the daytime/nighttime repetitive firing rates of SCN neurons as a consequence of rhythmic changes in subthreshold K+ currents.

Rhythmic cilia changes support SCN neuron coherence in circadian clock.

The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.

Free running period affected by network structures of suprachiasmatic nucleus neurons exposed to constant light

Exposed to the natural light-dark cycle, 24 h rhythms exist in behavioral and physiological processes of living beings. Interestingly, under constant darkness or constant light, living beings can maintain a robust endogenous rhythm with a free running period (FRP) close to 24 h. In mammals, the circadian rhythm is coordinated by a master clock located in the suprachiasmatic nucleus (SCN) of the brain, which is composed of about twenty thousand self-oscillating neurons. These SCN neurons form a heterogenous network to output a robust rhythm. Thus far, the exact network topology of the SCN neurons is unknown. In this article, we examine the effect of the SCN network structure on the FRP when exposed to constant light by a Poincaré model. Four typical network structures are considered, including a nearest-neighbor coupled network, a Newman–Watts small world network, an ErdÖs–Rényi random network and a Barabási–Albert (BA) scale free network. The results show that the FRP is longest in the BA network, because the BA network is characterized by the most heterogeneous structure among these four types of networks. These findings are not affected by the average node degree of the SCN network or the value of relaxation rate of the SCN neuronal oscillators. Our findings contribute to the understanding of how the network structure of the SCN neurons influences the FRP.