Firing Rate Of Suprachiasmatic Nucleus Neurons Research Articles

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.

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.

Agomelatine affects rat suprachiasmatic nucleus neurons via melatonin and serotonin receptors

AimsThe hypothalamic suprachiasmatic nucleus (SCN), which functions as a circadian pacemaker in mammals, is influenced by melatonin and serotonin. Agomelatine, which acts as an antidepressant and can synchronize disturbed circadian rhythms, displays a unique mechanism of action involving both melatonergic agonist and 5-HT2C antagonist properties. This study investigated the dose-dependent effects of agomelatine, melatonin and a selective 5-HT2C receptor antagonist, S32006, on SCN neurons in an in vitro slice preparation. Main methodsBrain slices containing the SCN were prepared from male Wistar rats and maintained in a recording chamber. Changes in firing rates of SCN neurons were recorded after perfusion of drugs. Key findingsSCN firing rates were dose-dependently suppressed by 19.2–80.9% following perfusion of 0.04–0.32mM agomelatine (p<0.001, IC50=0.14mM). Perfusion with melatonin (0.4–3.2mM) resulted in 16.6–62.5% dose-dependent reductions in firing rates (at least p<0.01, IC50=1.59mM) and of the duration of suppression. A selective melatonin receptor antagonist (S22153 at 0.32mM) and a 5-HT2c receptor agonist (Ro60-0175) reduced the suppressive effects of 0.16mM agomelatine by 35% and 50.2%, respectively. A 5-HT2C receptor antagonist (S32006; 0.03–0.12mM) significantly decreased SCN firing rates (19.6–91.8%; at least p<0.05, IC50=0.05mM). Co-perfusion of S32006 (0.06mM) with a 5-HT2C agonist (Ro60-0175; 0.003mM) reduced suppressions evoked by S32006 alone by ~72.1%. SignificanceThese results are consistent with the hypothesis that agomelatine acts directly on the SCN via both agonist effects at melatonergic receptors and antagonist effects at 5-HT2C receptors, which parallel its mechanisms of action as an antidepressant.

Rhythmic Trafficking of TRPV2 in the Suprachiasmatic Nucleus is Regulated by Prokineticin 2 Signaling.

The mammalian circadian clock is composed of single-cell oscillators. Neurochemical and electrical signaling among these oscillators is important for the normal expression of circadian rhythms. Prokineticin 2 (PK2), encoding a cysteine-rich secreted protein, has been shown to be a critical signaling molecule for the regulation of circadian rhythms. PK2 expression in the suprachiasmatic nucleus (SCN) is highly rhythmic, peaking during the day and being essentially absent during the night. Mice with disrupted PK2 gene or its receptor PKR2 display greatly reduced rhythmicity of broad circadian parameters such as locomotor activity, body temperature and sleep/wake patterns. PK2 has been shown to increase the firing rate of SCN neurons, with unknown molecular mechanisms. Here we report that TRPV2, an ion channel belonging to the family of TRP, is co-expressed with PKR2 in the SCN neurons. Further, TRPV2 protein, but not TRPV2 mRNA, was shown to oscillate in the SCN in a PK2-dependent manner. Functional studies revealed that TRPV2 enhanced signaling of PKR2 in calcium mobilization or ion current conductance, likely via the increased trafficking of TRPV2 to the cell surface. Taken together, these results indicate that TRPV2 is likely part of the downstream signaling of PK2 in the regulation of the circadian rhythms.

Vasoactive intestinal peptide produces long-lasting changes in neural activity in the suprachiasmatic nucleus

The neuropeptide vasoactive intestinal peptide (VIP) is expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN). While VIP is known to be important to the input and output pathways from the SCN, the physiological effects of VIP on electrical activity of SCN neurons are not well known. Here the impact of VIP on firing rate of SCN neurons was investigated in mouse slice cultures recorded during the night. The application of VIP produced an increase in electrical activity in SCN slices that lasted several hours after treatment. This is a novel mechanism by which this peptide can produce long-term changes in central nervous system physiology. The increase in action potential frequency was blocked by a VIP receptor antagonist and lost in a VIP receptor knockout mouse. In addition, inhibitors of both the Epac family of cAMP binding proteins and cAMP-dependent protein kinase (PKA) blocked the induction by VIP. The persistent increase in spike rate following VIP application was not seen in SCN neurons from mice deficient in Kv3 channel proteins and was dependent on the clock protein PER1. These findings suggest that VIP regulates the long-term firing rate of SCN neurons through a VIPR2-mediated increase in the cAMP pathway and implicate the fast delayed rectifier (FDR) potassium currents as one of the targets of this regulation.

Circadian dysfunction in a mouse model of Parkinson's disease

Many Parkinson's disease (PD) patients exhibit sleep disorders as part of their symptoms with evidence suggesting that REM sleep disorders may be intimately associated with this disease. Possible dysfunction in the circadian system in PD has received less attention, yet problems in circadian timing are common in neurodegenerative diseases. In the present study, we examined the expression of daily and circadian rhythms in the alpha-synuclein overexpressing (ASO) transgenic line. We found selective deficits in the expression of circadian rhythms of locomotor activity, including lower night-time activity and greater fragmentation in the wheel-running activity in this PD model. These alterations were prominent in young adult (3–4 mo) ASO mice and worsened progressively with age, consistent with prior reports of age-related loss of motor skills. The temporal distribution of sleep was also altered in the ASO mice compared to littermate controls. In the ASO mice, the peak/trough expression of the clock gene PERIOD2 was normal in the master pacemaker of the circadian system: the suprachiasmatic nucleus (SCN); however, the daytime firing rate of SCN neurons was reduced in the mutant mice. Together, this data raises the possibility that a weakening of circadian output is a core feature of PD. The reduction in magnitude of circadian output would be expected to have functional consequences throughout the body.

Circadian Modulation of GABA Function in the Rat Suprachiasmatic Nucleus: Excitatory Effects During the Night Phase

Gramicidin-perforated patch-clamp recordings were made from slices of the suprachiasmatic nucleus (SCN) of adult rats to characterize the role of gamma-amino butyric acid (GABA) in the circadian timing system. During the day, activation of GABA(A) receptors hyperpolarized the membrane of SCN neurons. During the night, however, activation of GABA(A) receptors either hyperpolarized or depolarized the membrane. These night-restricted depolarizations in a large subset of SCN neurons were capable of triggering spikes and thus appeared to be excitatory. The GABA(A) reversal potentials of SCN neurons revealed a significant day-night difference with more depolarized GABA(A) reversal potentials during the night than during the day. The emergence of depolarizing GABA(A)-mediated responses in a subset of SCN neurons at night can be ascribed to a depolarizing shift in GABA(A) reversal potential. The GABA(A) receptor antagonist bicuculline (12.5 microM) increased the spontaneous firing rate of all SCN neurons during the day, indicating that spontaneous GABA(A)-mediated inputs inhibited the SCN neurons during this period. The effect of bicuculline (12.5 microM) on the spontaneous firing rate of SCN neurons during the night was heterogeneous due to the mixture of depolarizing and hyperpolarizing GABA(A)-mediated inputs during this period. We conclude that GABA uniformly acts as an inhibitory transmitter during the day but excites a large subset of SCN neurons at night. This day-night modulation of GABAergic neurotransmission provides the SCN with a time-dependent gating mechanism that may counteract propagation of excitatory signals throughout the biological clock at day but promotes it at night.

Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices.

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (IH) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of IH if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of IH by Cs+ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that IH, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that IH does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of IH were modulated in a circadian manner. However, no substantial day-night differences in IH were found. We conclude that IH, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in 'rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.

Circadian rhythm generation in the cultured suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is involved in the generation and entrainment of circadian rhythms. The results of a series of experiments in long-term cultured organotypic SCN slices suggest that (1) some but not all SCN neurons display circadian rhythmicity in their extracelluar discharges. To the extent they could be studied, these neurons did not usually show synchronized high/low levels of activity, (2) simultaneous daily depolarization of these neurons (K + pulses) to some extent influenced the distribution of the firing rate of SCN neurons around the time of expected daily pulses; (3) extracelluar Ca ++ and synaptic input is required for the pacemaker activity of the SCN. We conclude that the mammalian biological clock is a heterogeneous neuronal system in which the circadian pacemaker rhythm is generated and entrained via complex interactions among SCN neurons.

Phase advances of circadian rhythms in somatostatin depleted rats: effects of cysteamine on rhythms of locomotor activity and electrical discharge of the suprachiasmatic nucleus.

Somatostatin is synthesized in the suprachiasmatic nucleus (SCN), a circadian pacemaker in mammals. To explore the functional significance of somatostatin in the circadian system, we examined rhythms of rat locomotor activity and electrical firing rate of SCN neurons in the brain slice after temporal depletion of somatostatin levels in the SCN. Intraperitoneal administration of cysteamine (200 mg/kg), a somatostatin depletor, significantly reduced somatostatin level in the in vivo SCN 5 min after injection and kept low level as long as 3 to 4 days. This administration, on the other hand, induced significant phase advances of about 51 min in the subsequent free-running rhythm of locomotor activity of the rat. A marked phase advance in the circadian rhythm of firing rate in the SCN was also observed after administration of cysteamine in coronal hypothalamic slices. These persistent phase shifts after administration of a somatostatin depletor may suggest that the change of somatostatin level in the SCN have a feedback influence on the circadian pacemaker.

Development of neuronal activity in the rat suprachiasmatic nucleus

The development of function was studied in the rat suprachiasmatic nucleus (SCN) by analyzing the rate and pattern of neuronal discharge in vitro from embryonic day 22 (E22) to postnatal day 14 (P14), and comparing these with adult SCN. The firing rate of SCN neurons on E22 is low but there is a clear circadian rhythm with subjective day values (1.6 ± 0.2 impulses/s) significantly higher than those for subjective night (0.9 ± 0.2 impulses/s). At E22 most neurons show an irregular firing pattern. The firing rate of SCN neurons gradually increases from E22 to P14 with emergence of patterns of firing of individual neurons that approximate those of adult SCN. In calcium-free medium the firing rate of SCN neurons at P1 is reduced and the circadian rhythm in firing rate is abolished. These results demonstrate that SCN neurons exhibit a circadian rhythm in firing rate early in development, largely prior to the formation of synaptic contacts within the SCN, and indicate that expression of the rhythm requires the presence of calcium ions.

The role of calcium ions in circadian rhythm of suprachiasmatic nucleus neuron activity in rat hypothalamic slices.

The role of calcium ions in maintaining the circadian rhythm of suprachiasmatic nucleus (SCN) neuron activity was investigated using rat hypothalamic slice preparations. In normal Krebs solution, the firing rate of SCN neurons was higher in the light period than in the dark period. In Ca2+-free Krebs solution, SCN neuron activity was low during all periods and did not show diurnal rhythm. These results suggest that the disappearance of circadian rhythmic change of SCN neuron activity in Ca2+-free Krebs solution may be due to the disappearance of synaptic transmission in the SCN.

Development of the circadian rhythm of neuronal activity in suprachiasmatic nucleus of rat hypothalamic slices

Development of the circadian rhythm of neuronal activity in the suprachiasmatic nucleus (SCN) was studied in rat hypothalamic slices. The firing rate of SCN neurons of 7- and 11-day-old rat pups was low during all of the day, whereas that of 14- and 21-day-old pups was high during daytime and low during nighttime. The present results suggest that the circadian rhythm of SCN neuronal activity is established between 11 and 14 days of age, corresponding closely to the time of onset of the other various hormonal and behavioral circadian rhythms.