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1.
Neuron ; 99(3): 555-563.e5, 2018 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-30017392

RESUMEN

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.


Asunto(s)
Potenciales de Acción/fisiología , Ritmo Circadiano/fisiología , Neuronas del Núcleo Supraquiasmático/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuropéptidos/metabolismo , Técnicas de Cultivo de Órganos , Núcleo Supraquiasmático/metabolismo
2.
eNeuro ; 4(3)2017.
Artículo en Inglés | MEDLINE | ID: mdl-28560311

RESUMEN

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.


Asunto(s)
Ritmo Circadiano/fisiología , Actividad Motora/fisiología , Neuronas/metabolismo , Proteínas Circadianas Period/metabolismo , Canales de Potasio Shal/metabolismo , Núcleo Supraquiasmático/metabolismo , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Proteínas Circadianas Period/genética , Fotoperiodo , Interferencia de ARN , Canales de Potasio Shal/genética , Técnicas de Cultivo de Tejidos
3.
J Biol Rhythms ; 31(1): 57-67, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26712166

RESUMEN

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.


Asunto(s)
Neuronas/fisiología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/fisiología , Péptido Intestinal Vasoactivo/metabolismo , Potenciales de Acción , Animales , Ritmo Circadiano , Ratones , Fotoperiodo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/genética
4.
J Biol Rhythms ; 30(5): 396-407, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26152125

RESUMEN

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.


Asunto(s)
Ritmo Circadiano/fisiología , Canal de Potasio Kv1.4/fisiología , Proteínas Circadianas Period/metabolismo , Canales de Potasio Shal/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Ritmo Circadiano/genética , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Canal de Potasio Kv1.4/genética , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Masculino , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Actividad Motora/genética , Actividad Motora/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Proteínas Circadianas Period/genética , Canales de Potasio Shal/genética , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Técnicas de Cultivo de Tejidos
5.
Neuron ; 84(2): 311-23, 2014 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-25374357

RESUMEN

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.


Asunto(s)
Diferenciación Celular/fisiología , Cuerpo Estriado/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , MicroARNs/metabolismo , Neostriado/citología , Neuronas/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Factores de Transcripción/metabolismo
6.
Sleep ; 36(12): 1839-48, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24293758

RESUMEN

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.


Asunto(s)
Neuronas GABAérgicas/fisiología , Prosencéfalo/fisiología , Canales de Potasio Shab/fisiología , Sueño/fisiología , Vigilia/fisiología , Animales , Electroencefalografía , Electromiografía , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Parvalbúminas/fisiología , Prosencéfalo/citología , Proteínas Proto-Oncogénicas c-fos/fisiología , Canales de Potasio Shab/genética , Sueño/genética , Privación de Sueño/fisiopatología , Vigilia/genética
7.
J Comp Neurol ; 518(21): 4298-310, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20853508

RESUMEN

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.


Asunto(s)
Banda Diagonal de Broca , Neuronas/metabolismo , Área Preóptica , Canales de Potasio Shab/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Banda Diagonal de Broca/citología , Banda Diagonal de Broca/metabolismo , Femenino , Técnicas de Sustitución del Gen , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Área Preóptica/citología , Área Preóptica/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
8.
J Biol Chem ; 285(20): 15048-55, 2010 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-20202934

RESUMEN

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.


Asunto(s)
Encéfalo/metabolismo , Neuronas/metabolismo , Canales de Potasio Shab/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , Inmunohistoquímica , Inmunoprecipitación , Datos de Secuencia Molecular , Mutación , Ratas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Canales de Potasio Shab/química , Canales de Potasio Shab/genética
9.
J Orofac Pain ; 21(1): 7-18, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17312637

RESUMEN

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.


Asunto(s)
Artritis Experimental/fisiopatología , Condicionamiento Operante/fisiología , Conducta Alimentaria/fisiología , Trastornos de la Articulación Temporomandibular/fisiopatología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/patología , Conducta Animal/fisiología , Colorantes , Dermatitis/patología , Dermatitis/fisiopatología , Modelos Animales de Enfermedad , Azul de Evans , Conducta Exploratoria/fisiología , Adyuvante de Freund/administración & dosificación , Aseo Animal/fisiología , Indometacina/uso terapéutico , Masculino , Músculo Masetero/patología , Músculo Masetero/fisiopatología , Miositis/patología , Miositis/fisiopatología , Premedicación , Ratas , Trastornos de la Articulación Temporomandibular/patología , Factores de Tiempo
10.
Am J Physiol Regul Integr Comp Physiol ; 291(2): R343-8, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16469833

RESUMEN

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.


Asunto(s)
Estrógenos/farmacología , Inflamación/etiología , Inflamación/fisiopatología , Trastornos de la Articulación Temporomandibular/inmunología , Testosterona/farmacología , Animales , Femenino , Terapia de Reemplazo de Hormonas , Inflamación/inducido químicamente , Masculino , Orquiectomía , Ovariectomía , Ratas , Caracteres Sexuales , Articulación Temporomandibular , Trastornos de la Articulación Temporomandibular/inducido químicamente
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