• Calcium and BK Potassium Channel Regulation of Circadian Rhythms in the Suprachiasmatic Nucleus

      Plante, Amber; Meredith, Andrea L.; 0000-0002-1010-2348 (2021)
      Mammalian circadian rhythms are driven by a network of neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN exhibits daily (24-hour) rhythms in spontaneous action potential (AP) firing rate that encodes a time-of-day signal that coordinates the timing of circadian physiological and behavioral processes. Large-conductance Ca2+-activated K+ (BK) channels have a major role in driving the diurnal patterns of spontaneous firing in SCN neurons. BK K+ currents are larger at night, correlating with reduced neuronal excitability. The diurnal variation in BK current in the SCN is required for setting the day-night difference in firing frequency. BK currents undergo multi-level regulation by genetic and posttranslational mechanisms as well as functional coupling to Ca2+ channels. Intracellular Ca2+ (Ca2+i) is required for BK channel activation and previous studies have shown BK current is predominantly coupled to two types of Ca2+ sources in the SCN: L-type Ca2+ channels (LTCCs), and ryanodine receptors (RyRs). Circadian rhythms in Ca2+i have also been identified in SCN neurons. However, the Ca2+ channels involved in generating both AP and Ca2+i rhythms have not been clearly identified. First, to determine which Ca2+ channels are involved in AP rhythms, this study measured the impact of Ca2+ channel agonists and antagonists on the circadian parameters of spontaneous AP activity from organotypic SCN slice cultures grown on multi-electrode arrays. Next, to determine which Ca2+ channels are involved in Ca2+i rhythms, this study tested the effects of the same Ca2+ channel pharmacology on the circadian parameters of Ca2+i measured from SCN slice cultures transfected with a fluorescent Ca2+ sensor. Lastly, this investigated a potential mechanism by which LTCCs contribute to firing rate in SCN neurons by examining their ability to activate BK channels under controlled conditions. This study provides insight into the roles of specific Ca2+ sources in neural coding of the circadian time signal in the SCN.
    • Dural Afferent Mechanisms of Migraine Pain

      Harriott, Andrea; Gold, Michael S., Ph.D. (2009)
      Compelling evidence indicates that dural afferent sensitization initiates migraine pain. Furthermore, mechanisms of dural afferent sensitization may be unique given that ion channels underlying afferent excitability differ greatly depending on the target of innervation. Triptans, serotonin 1B/1D receptor (5HT1B/1DR) agonists, are selectively used for migraine pain which raises the possibility that their selectivity reflects modulation of unique mechanisms of dural afferent sensitization. The specific hypotheses tested in my thesis were that: 1) triptan selectivity reflects differential 5HT1DR distribution, 2) dural afferents have unique electrophysiological properties, and 3) triptan selectivity reflects modulation of these unique properties. Female Sprague Dawley rats were used for all experiments. The density of 5HT1DR was quantified with Western blot and localized with immunohistochemistry. Acutely dissociated dural afferents were examined using patch clamp recordings and compared to temporalis muscle afferents (TM) in the absence and presence of inflammatory mediators (IM): (μM) prostaglandin E2 (1), bradykinin (10), and histamine (1); and sumatriptan. There were four major observations in this thesis : 1) The 5HT1DR is differentially distributed in nerve fibers innervating peripheral tissues such that the density is highest in tissues known to produce migraine-like pain (i.e. circle of Willis and dura) than in structures in which triptans have no efficacy (i.e. TM), 2) dural afferents have marked differences in the ion channel mechanisms mediating passive and active electrophysiological properties in the absence and presence of IM including an increase in Na+ current, a decrease in Ca2+-dependent K+ current, a decrease in voltage gated Ca2+ current, and an increase in Ca2+ dependent Cl- current, and 3) prolonged sumatriptan incubation with 1µM sumatriptan prevented dural afferent sensitization via K+ current modulation. Taken together, the importance of dural afferent signaling in migraine pathogenesis provide a rationale utilizing ionic mechanisms involved in dural afferent sensitization as targets of novel anti-migraine therapies.
    • Mechanisms of Diurnal Regulation of the BK current in the Brain's Central Clock

      Whitt, Joshua Patrick; Meredith, Andrea L. (2016)
      The large conductance Ca2+- and voltage-activated potassium (BK) channel is activated via two important cellular signaling mechanisms: increases in intracellular Ca2+ and membrane depolarization. Though the BK channel is encoded via a single gene, Kcnma1, currents produced by this channel exhibit a large degree of phenotypic variability across different cell types, indicating regulation of the BK channel can influence BK current properties. Furthermore, regulation of current properties, as opposed to changing expression of the channel, may act as a mechanism through which excitability can be modulated in various neurons. In order to determine whether regulation of BK current properties can influence neuronal excitability, we utilized the suprachiasmatic nucleus (SCN), as a model system. The SCN is the brain's central circadian oscillator, encoding time-of-day information through changes in neuronal excitability. Increased excitability is found during the day, promoting faster firing rates, while at night excitability is dampened, slowing firing rate. This diurnal change in firing rate is due in part to an increase in BK channel protein found in the SCN at night. While regulation of expression of the BK channel gene can account for part of the diurnal difference in excitability of SCN neurons, we hypothesized that daily regulation of BK current properties in the SCN via β-subunit association, alternative splicing, and control of Ca2+ sources limits a role for BK currents in setting firing frequency during the day. Using patch-clamp electrophysiolgy we demonstrate that BK current properties in the SCN are diurnally regulated, with smaller BK currents during the day found to be due to BK channel inactivation, Ca2+ source regulation and alternative splicing, rather than simply a decrease in BK channel expression. We also found loss of these mechanisms produces an increase in BK current and results in a decrease in firing rate during the day in SCN neurons. These results support the hypothesis that BK current regulation is just as important as regulation of BK protein expression in setting a diurnal difference in firing rate in the SCN. Furthermore, these results provide a framework through which modulation of current properties may impact firing in other pacemaking neurons.