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.

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 rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K + conductance(s) driving these daily rhythms in repetitive firing rates, 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, 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 nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1 -/- , Kv12.2 -/- , Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

Understanding Circadian Mechanisms of Sudden Cardiac Death: A Report From the National Heart, Lung, and Blood Institute Workshop, Part 1: Basic and Translational Aspects.

Sudden cardiac death (SCD), the unexpected death due to acquired or genetic cardiovascular disease, follows distinct 24-hour patterns in occurrence. These 24-hour patterns likely reflect daily changes in arrhythmogenic triggers and the myocardial substrate caused by day/night rhythms in behavior, the environment, and endogenous circadian mechanisms. To better address fundamental questions regarding the circadian mechanisms, the National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death. We present a 2-part report of findings from this workshop. Part 1 summarizes the workshop and serves to identify research gaps and opportunities in the areas of basic and translational research. Among the gaps was the lack of standardization in animal studies for reporting environmental conditions (eg, timing of experiments relative to the light dark cycle or animal housing temperatures) that can impair rigor and reproducibility. Workshop participants also pointed to uncertainty regarding the importance of maintaining normal circadian rhythmic synchrony and the potential pathological impact of desynchrony on SCD risk. One related question raised was whether circadian mechanisms can be targeted to reduce SCD risk. Finally, the experts underscored the need for studies aimed at determining the physiological importance of circadian clocks in the many different cell types important to normal heart function and SCD. Addressing these gaps could lead to new therapeutic approaches/molecular targets that can mitigate the risk of SCD not only at certain times but over the entire 24-hour period.

Understanding Circadian Mechanisms of Sudden Cardiac Death: A Report From the National Heart, Lung, and Blood Institute Workshop, Part 2: Population and Clinical Considerations.

Sudden cardiac death (SCD) is the sudden, unexpected death due to abrupt loss of heart function secondary to cardiovascular disease. In certain populations living with cardiovascular disease, SCD follows a distinct 24-hour pattern in occurrence, suggesting day/night rhythms in behavior, the environment, and endogenous circadian rhythms result in daily spans of increased vulnerability. The National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death to identify fundamental questions regarding the role of the circadian rhythms in SCD. Part 2 summarizes research gaps and opportunities in the areas of population and clinical research identified in the workshop. Established research supports a complex interaction between circadian rhythms and physiological responses that increase the risk for SCD. Moreover, these physiological responses themselves are influenced by several biological variables, including the type of cardiovascular disease, sex, age, and genetics, as well as environmental factors. The emergence of new noninvasive biotechnological tools that continuously measure key cardiovascular variables, as well as the identification of biomarkers to assess circadian rhythms, hold promise for generating large-scale human data sets that will delineate which subsets of individuals are most vulnerable to SCD. Additionally, these data will improve our understanding of how people who suffer from circadian disruptions develop cardiovascular diseases that increase the risk for SCD. Emerging strategies to identify new biomarkers that can quantify circadian health (eg, environmental, behavioral, and internal misalignment) may lead to new interventions and therapeutic targets to prevent the progression of cardiovascular diseases that cause SCD.

Entrainment of Circadian Rhythms Depends on Firing Rates and Neuropeptide Release of VIP SCN Neurons.

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker, integrating environmental input to align physiological and behavioral rhythms to local time cues. Approximately 10% of SCN neurons express vasoactive intestinal polypeptide (VIP); however, it is unknown how firing activity of VIP neurons releases VIP to entrain circadian rhythms. To identify physiologically relevant firing patterns, we optically tagged VIP neurons and characterized spontaneous firing over 3 days. VIP neurons had circadian rhythms in firing rate and exhibited two classes of instantaneous firing activity. We next tested whether physiologically relevant firing affected circadian rhythms through VIP release. We found that VIP neuron stimulation with high, but not low, frequencies shifted gene expression rhythms in vitro through VIP signaling. In vivo, high-frequency VIP neuron activation rapidly entrained circadian locomotor rhythms. Thus, increases in VIP neuronal firing frequency release VIP and entrain molecular and behavioral circadian rhythms. VIDEO ABSTRACT.

Acute Knockdown of Kv4.1 Regulates Repetitive Firing Rates and Clock Gene Expression in the Suprachiasmatic Nucleus and Daily Rhythms in Locomotor Behavior.

Rapidly activating and inactivating A-type K+ currents (IA) encoded by Kv4.2 and Kv4.3 pore-forming (α) subunits of the Kv4 subfamily are key regulators of neuronal excitability. Previous studies have suggested a role for Kv4.1 α-subunits in regulating the firing properties of mouse suprachiasmatic nucleus (SCN) neurons. To test this, we utilized an RNA-interference strategy to knockdown Kv4.1, acutely and selectively, in the SCN. Current-clamp recordings revealed that the in vivo knockdown of Kv4.1 significantly (p < 0.0001) increased mean ± SEM repetitive firing rates in SCN neurons during the day (6.4 ± 0.5 Hz) and at night (4.3 ± 0.6 Hz), compared with nontargeted shRNA-expressing SCN neurons (day: 3.1 ± 0.5 Hz; night: 1.6 ± 0.3 Hz). IA was also significantly (p < 0.05) reduced in Kv4.1-targeted shRNA-expressing SCN neurons (day: 80.3 ± 11.8 pA/pF; night: 55.3 ± 7.7 pA/pF), compared with nontargeted shRNA-expressing (day: 121.7 ± 10.2 pA/pF; night: 120.6 ± 16.5 pA/pF) SCN neurons. The magnitude of the effect of Kv4.1-targeted shRNA expression on firing rates and IA was larger at night. In addition, Kv4.1-targeted shRNA expression significantly (p < 0.001) increased mean ± SEM nighttime input resistance (Rin; 2256 ± 166 MΩ), compared to nontargeted shRNA-expressing SCN neurons (1143 ± 93 MΩ). Additional experiments revealed that acute knockdown of Kv4.1 significantly (p < 0.01) shortened, by ∼0.5 h, the circadian period of spontaneous electrical activity, clock gene expression and locomotor activity demonstrating a physiological role for Kv4.1-encoded IA channels in regulating circadian rhythms in neuronal excitability and behavior.

Distinct Firing Properties of Vasoactive Intestinal Peptide-Expressing Neurons in the Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) regulates daily rhythms in physiology and behavior. Previous studies suggest a critical role for neurons expressing vasoactive intestinal peptide (VIP) in coordinating rhythmicity and synchronization in the SCN. Here we examined the firing properties of VIP-expressing SCN neurons in acute brain slices. Active and passive membrane properties were measured in VIP and in non-VIP neurons during the day and at night. Current-clamp recordings revealed that both VIP and non-VIP neurons were spontaneously active, with higher firing rates during the day than at night. Average firing frequencies, however, were higher in VIP neurons (3.1 ± 0.2 Hz, day and 2.4 ± 0.2 Hz, night) than in non-VIP neurons (1.8 ± 0.2 Hz, day and 0.9 ± 0.2 Hz, night), both day and night. The waveforms of individual action potentials in VIP and non-VIP neurons were also distinct. Action potential durations (APD50) were shorter in VIP neurons (3.6 ± 0.1 ms, day and 2.9 ± 0.1 ms, night) than in non-VIP neurons (4.4 ± 0.3 ms, day and 3.5 ± 0.2 ms, night) throughout the light-dark cycle. In addition, afterhyperpolarization (AHP) amplitudes were larger in VIP neurons (21 ± 0.8 mV, day and 24.9 ± 0.9 mV, night) than in non-VIP neurons (17.2 ± 1.1 mV, day and 20.5 ± 1.2 mV, night) during the day and at night. Furthermore, significant day/night differences were observed in APD50 and AHP amplitudes in both VIP and non-VIP SCN neurons, consistent with rhythmic changes in ionic conductances that contribute to shaping the firing properties of both cell types. The higher day and night firing rates of VIP neurons likely contribute to synchronizing electrical activity in the SCN.

IA Channels Encoded by Kv1.4 and Kv4.2 Regulate Circadian Period of PER2 Expression in the Suprachiasmatic Nucleus.

Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 (Kv1.4(-/-)) or Kcnd2 (Kv4.2(-/-); but not Kcnd3, Kv4.3(-/-)), voltage-gated K(+) (Kv) channel pore-forming subunits that encode subthreshold, rapidly activating, and inactivating K(+) currents (IA), have shortened (0.5 h) circadian periods in SCN firing and in locomotor activity compared with wild-type (WT) mice. In the experiments here, we used a mouse (Per2(Luc)) line engineered with a bioluminescent reporter construct, PERIOD2::LUCIFERASE (PER2::LUC), replacing the endogenous Per2 locus, to test the hypothesis that the loss of Kv1.4- or Kv4.2-encoded IA channels also modifies circadian rhythms in the expression of the clock protein PERIOD2 (PER2). We found that SCN explants from Kv1.4(-/-)Per2(Luc) and Kv4.2(-/-) Per2(Luc), but not Kv4.3(-/-)Per2(Luc), mice have significantly shorter (by approximately 0.5 h) circadian periods in PER2 rhythms, compared with explants from Per2(Luc) mice, revealing that the membrane properties of SCN neurons feedback to regulate clock (PER2) expression. The combined loss of both Kv1.4- and Kv4.2-encoded IA channels in Kv1.4(-/-)/Kv4.2(-/-)Per2(Luc) SCN explants did not result in any further alterations in PER2 rhythms. Interestingly, however, mice lacking both Kv1.4 and Kv4.2 show a striking (approximately 1.8 h) advance in their daily activity onset in a light cycle compared with WT mice, suggesting additional roles for Kv1.4- and Kv4.2-encoded IA channels in controlling the light-dependent responses of neurons within and/or outside of the SCN to regulate circadian phase of daily activity.

Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts.

The promise of using reprogrammed human neurons for disease modeling and regenerative medicine relies on the ability to induce patient-derived neurons with high efficiency and subtype specificity. We have previously shown that ectopic expression of brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), promoted direct conversion of human fibroblasts into neurons. Here we show that coexpression of miR-9/9*-124 with transcription factors enriched in the developing striatum, BCL11B (also known as CTIP2), DLX1, DLX2, and MYT1L, can guide the conversion of human postnatal and adult fibroblasts into an enriched population of neurons analogous to striatal medium spiny neurons (MSNs). When transplanted in the mouse brain, the reprogrammed human cells persisted in situ for over 6 months, exhibited membrane properties equivalent to native MSNs, and extended projections to the anatomical targets of MSNs. These findings highlight the potential of exploiting the synergism between miR-9/9*-124 and transcription factors to generate specific neuronal subtypes.

Kv2.2: a novel molecular target to study the role of basal forebrain GABAergic neurons in the sleep-wake cycle.

STUDY OBJECTIVES: The basal forebrain (BF) has been implicated as an important brain region that regulates the sleep-wake cycle of animals. Gamma-aminobutyric acidergic (GABAergic) neurons are the most predominant neuronal population within this region. However, due to the lack of specific molecular tools, the roles of the BF GABAergic neurons have not been fully elucidated. Previously, we have found high expression levels of the Kv2.2 voltage-gated potassium channel on approximately 60% of GABAergic neurons in the magnocellular preoptic area and horizontal limb of the diagonal band of Broca of the BF and therefore proposed it as a potential molecular target to study this neuronal population. In this study, we sought to determine the functional roles of the Kv2.2-expressing neurons in the regulation of the sleep-wake cycle. DESIGN: Sleep analysis between two genotypes and within each genotype before and after sleep deprivation. SETTING: Animal sleep research laboratory. PARTICIPANTS: Adult mice. Wild-type and Kv2.2 knockout mice with C57/BL6 background. INTERVENTIONS: EEG/EMG recordings from the basal state and after sleep-deprivation which was induced by mild agitation for 6 h. RESULTS: Immunostaining of a marker of neuronal activity indicates that these Kv2.2-expressing neurons appear to be preferentially active during the wake state. Therefore, we tested whether Kv2.2-expressing neurons in the BF are involved in arousal using Kv2.2-deficient mice. BF GABAergic neurons exhibited augmented expression of c-Fos. These knockout mice exhibited longer consolidated wake bouts than wild-type littermates, and that phenotype was further exacerbated by sleep deprivation. Moreover, in-depth analyses of their cortical electroencephalogram revealed a significant decrease in the delta-frequency activity during the nonrapid eye movement sleep state. CONCLUSIONS: These results revealed the significance of Kv2.2-expressing neurons in the regulation of the sleep-wake cycle.

Immunolocalization of the voltage-gated potassium channel Kv2.2 in GABAergic neurons in the basal forebrain of rats and mice.

The Kv2 voltage-gated potassium channels, Kv2.1 and Kv2.2, are important regulators of neuronal excitability in mammalian brain. It has been shown that Kv2.1 channels are expressed in virtually all neurons in the brain. However, the cellular localization of Kv2.2 has not been fully elucidated. In this article we report that Kv2.2 is highly expressed in a subset of neurons in the magnocellular preoptic nucleus (MCPO) and the horizontal limb of the diagonal band of Broca (HDB) of the basal forebrain complex, which are areas highly implicated in the regulation of cortical activity and the sleep/wake cycle. It has been shown that MCPO and HDB contain distinct populations of neurons that differ in their neurochemicals, cholinergic, glutamatergic, and gamma-aminobutyric acid (GABA)ergic neurons. Using specific immunolabeling and knockin mice in which green fluorescent protein (GFP) is expressed in GABAergic neurons, we found that Kv2.2 is abundantly expressed in a large subpopulation of the GABAergic neurons in the MCPO and HDB. These data offer Kv2.2 as a molecular target to study the role of the specific subpopulation of basal forebrain GABAergic neurons.

Formation of heteromeric Kv2 channels in mammalian brain neurons.

The formation of heteromeric tetramers is a common feature of voltage-gated potassium (Kv) channels. This results in the generation of a variety of tetrameric Kv channels that exhibit distinct biophysical and biochemical characteristics. Kv2 delayed rectifier channels are, however, unique exceptions. It has been previously shown that mammalian Kv2.1 and Kv2.2 are localized in distinct domains of neuronal membranes and are not capable of forming heteromeric channels with each other (Hwang, P. M., Glatt, C. E., Bredt, D. S., Yellen, G., and Snyder, S. H. (1992) Neuron 8, 473-481). In this study, we report a novel form of rat Kv2.2, Kv2.2(long), which has not been previously recognized. Our data indicate that Kv2.2(long) is the predominant form of Kv2.2 expressed in cortical pyramidal neurons. In contrast to the previous findings, we also found that rat Kv2.1 and Kv2.2(long) are colocalized in the somata and proximal dendrites of cortical pyramidal neurons and are capable of forming functional heteromeric delayed rectifier channels. Our results suggest that the delayed rectifier currents, which regulate action potential firing, are encoded by heteromeric Kv2 channels in cortical neurons.

An operant conditioning model to assess changes in feeding behavior associated with temporomandibular joint inflammation in the rat.

AIMS: To develop and validate a model in which to assess a loss of function associated with temporomandibular joint (TMJ) inflammation in awake, freely moving rats. METHODS: The dependent variable in the model was the time between food rewards (pellets), or interfeeding interval (IFI). IFI was quantified after rats were trained to "bar-press" for food. To validate use of the IFI as a surrogate for temporomandibular disorder (TMD) pain, we determined the impact of several manipulations, including changes in pellet size, the presence and severity of inflammation of the TMJ, masseter muscle, or skin (induced with complete Freund's adjuvant [CFA]), and the influence of preadministration of the non-steroidal anti-inflammatory drug indomethacin (4 mg/kg). Furthermore, in order to determine whether a change in IFI reflected an increase in the time rats spent eating, rats were videotaped, and the amount of time spent eating, grooming, and exploring was analyzed. RESULTS: Inflammation of the TMJ or masseter muscle resulted in significant dose- and pellet size-dependent increases in the IFI. Inflammation of the skin overlying the TMJ had no effect on IFI. Pre-administration of indomethacin reversed the inflammation-induced shift in the IFI. An inflammation-induced increase in IFI was associated with an increase in feeding time. CONCLUSIONS: Our model constitutes a relatively fast and sensitive method with which to assess changes in feeding behavior associated with TMJ inflammation. Only 2 days of training are required to obtain a stable baseline IFI. It is possible to detect changes in IFI as small as 40% with 12 rats per group.

Testosterone and estrogen have opposing actions on inflammation-induced plasma extravasation in the rat temporomandibular joint.

The present study was designed to test the hypothesis that estrogen exacerbates inflammation of the temporomandibular joint (TMJ). Evans blue dye was used to quantify plasma extravasation (PE) around the rat TMJ. In an initial set of experiments, TMJ PE was compared in naïve intact male and female rats, as well as in both groups after complete Freund's adjuvant (CFA)-induced inflammation of the TMJ. In contrast to our hypothesis, TMJ PE was significantly greater in both naïve and CFA-inflamed male rats than in females. To determine whether these differences were due to gonadal hormones, four additional groups of rats were studied: gonadectomized (Gx) males and females, Gx males with chronic testosterone (T) replacement, and Gx females with chronic estrogen (E) replacement. The sex difference in baseline TMJ PE appeared to reflect the actions of T. However, in the presence of TMJ inflammation, T augmented TMJ PE in males, while E attenuated TMJ PE in females. Changes in PE were also assessed in the contralateral TMJ. Results from this analysis indicated that there is a transient contralateral increase in TMJ PE in females but not males. Given that there is an inverse relationship between PE and joint damage, our results suggest that testosterone may mitigate, but estrogen may exacerbate, TMJ damage, particularly in the presence of overt inflammation. Importantly, our results may help explain both the higher prevalence and severity of temporomandibular disorder pain in females than males.