simultaneous-electrophysiological-recording-calcium-imaging

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Oregon Health & Science University

Presynaptic GABA(B) receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABA(B)-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by omega-conotoxin GVIA (< or = 72%), mibefradil (< or = 52%), and omega-agatoxin TK (< or = 15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP-sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.

Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels.

Many postsynaptic neurons release a retrograde transmitter that modulates presynaptic neurotransmitter release. In the suprachiasmatic nucleus (SCN), retrograde signaling is suggested by the presence of dendritic dense-core vesicles. Whole-cell voltage-clamp recordings were made from rat SCN neurons to determine whether a retrograde messenger could modulate the activity of afferent gamma-aminobutyric acid (GABA)ergic inputs. The frequency and amplitude of spontaneous GABAergic currents was significantly reduced in a subpopulation of SCN neurons (eight out of 13) following a postsynaptic depolarization. Similarly, a postsynaptic depolarization significantly reduced the amplitude of evoked GABAergic currents during both day and night recordings. A postsynaptic depolarizing pulse eliminated paired-pulse inhibition of GABAergic currents consistent with a presynaptic mechanism. Muscimol-activated currents were not altered by postsynaptic depolarization, demonstrating that the activity of GABA(A) receptors was not altered. Depolarization-induced inhibition of the GABAergic currents was not observed when a Ca2+ chelator was included in the microelectrode. Postsynaptic depolarization significantly increased the Ca2+ concentration in both the soma and dendrites. The dendritic Ca2+ levels increased faster, to a higher concentration and decayed faster than in the soma. The depolarization-induced inhibition of the evoked GABAergic current was blocked by the G-protein uncoupling agent N-ethylmaleimide, suggesting that the retrograde messenger acts on a pertussis toxin-sensitive G-protein-coupled receptor. Because the majority of SCN neurons receive GABAergic input from neighboring cells, these results describe a retrograde signaling mechanism by which SCN neurons can modulate GABAergic synaptic input.

Retrograde suppression of GABAergic currents in a subset of SCN neurons.

Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.

Mutations of presenilin genes in dilated cardiomyopathy and heart failure.

Glutamate released from retinohypothalamic tract (RHT) synapses with suprachiasmatic nucleus (SCN) neurons induces phase changes in the circadian clock presumably by using Ca2+ as a second messenger. We used electrophysiological and Ca2+ imaging techniques to simultaneously record changes in the membrane potential and intracellular calcium concentration ([Ca2+]i) in SCN neurons after stimulation of the RHT at physiologically relevant frequencies. Stimulation of the RHT sufficient to generate an EPSP did not produce detectable changes in [Ca2+]i, whereas EPSP-induced action potentials evoked an increase in [Ca2+]i, suggesting that the change in postsynaptic somatic [Ca2+]i produced by synaptically activated glutamate receptors was the result of membrane depolarization activating voltage-dependent Ca2+ channels. The magnitude of the Ca2+ response was dependent on the RHT stimulation frequency and duration, and on the SCN neuron action potential frequency. Membrane depolarization-induced changes in [Ca2+]i were larger and decayed more quickly in the dendrites than in the soma and were attenuated by nimodipine, suggesting a compartmentalization of Ca2+ signaling and a contribution of L-type Ca2+ channels. RHT stimulation at frequencies that mimicked the output of light-sensitive retinal ganglion cells (RGCs) evoked [Ca2+]i transients in SCN neurons via membrane depolarization and activation of voltage-dependent Ca2+ channels. These data suggest that for Ca2+ to induce phase advances or delays, light-induced signaling from RGCs must augment the underlying oscillatory somatic [Ca2+]i by evoking postsynaptic action potentials in SCN neurons during a period of slow spontaneous firing such as occurs during nighttime.

Calcium response to retinohypothalamic tract synaptic transmission in suprachiasmatic nucleus neurons.

&#x4E0B;&#x4E18;&#x8111;&#x89C6;&#x4EA4;&#x53C9;&#x4E0A;&#x7684;&#x6838; (SCN) &#x8C03;&#x8282;&#x751F;&#x7269;&#x8282;&#x5F8B;&#x65F6;&#x95F4;&#xFF0C;&#x4ECE;&#x800C;&#x76F4;&#x63A5;&#x5F71;&#x54CD;&#x8BB8;&#x591A;&#x751F;&#x7406;&#x8FC7;&#x7A0B;&#x3002;SCN &#x51E0;&#x4E4E;&#x5B8C;&#x5168;&#x7531;&#x8BB8;&#x591A;&#x4E0E;&#x5176;&#x4ED6;&#x7684;&#x7A81;&#x89E6;&#x5B89;&#x57FA;&#x7EC6;&#x80DE;&#x7684; SCN &#x6291;&#x5236;&#x5BF9;&#x65BD;&#x52A0;&#x5F71;&#x54CD;&#x6700;&#x5927;&#xFF0C;&#x5176;&#x7A81;&#x89E6;&#x540E;&#x7684;&#x76EE;&#x6807;&#x5982;&#x679C;&#x4E0D;&#x662F;&#x6240;&#x6709;&#x5468;&#x671F;&#x9636;&#x6BB5;&#x7684;&#x663C;&#x591C;&#x8282;&#x5F8B;&#x7684; &gamma;-&#x6C28;&#x57FA;&#x4E01;&#x9178; (GABA) &#x80FD;&#x795E;&#x7ECF;&#x5143;&#x7EC4;&#x6210;&#x3002;&#x4EE5;&#x538B;&#x5012;&#x6027;&#x591A;&#x6570;&#x5B89;&#x57FA;&#x6027;&#x8D28;&#x7684; SCN&#xFF0C;&#x4EE5;&#x53CA;&#x5176;&#x5185;&#x90E8;&#x8FDE;&#x63A5;&#x5C5E;&#x6027;&#xFF0C;&#x63D0;&#x4F9B;&#x529F;&#x80FD;&#x5F3A;&#x5927;&#x7684;&#x6A21;&#x578B;&#xFF0C;&#x7814;&#x7A76;&#x5982;&#x4F55;&#x6291;&#x5236;&#x611F;&#x53D7;&#x5668;&#x4EA7;&#x751F;&#x8F93;&#x51FA;&#x4FE1;&#x53F7;&#x3002;&#x5728;&#x76EE;&#x524D;&#x7684;&#x5DE5;&#x4F5C;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A; hyperpolarizations&#xFF0C;&#x8303;&#x56F4;&#x4ECE; 5 &#x5230; 1000 ms &#x5F15;&#x8D77;&#x53CD;&#x5F39;&#x4E2D;&#x6240;&#x6709; SCN &#x795E;&#x7ECF;&#x5143;&#x6709; 63%&#x7684;&#x5CF0;&#x503C;&#x5728;&#x6210;&#x5E74;&#x5927;&#x9F20;&#x548C;&#x4ED3;&#x9F20;&#x7684; SCN &#x7684;&#x7535;&#x538B;&#x94B3;&#x8FDB;&#x884C;&#x6D4B;&#x8BD5;&#x3002;&#x5728;&#x7535;&#x6D41;&#x94B3;&#x5F55;&#x97F3; hyperpolarizations &#x5BFC;&#x81F4;&#x53CD;&#x5F39;&#x7A57;&#x5F62;&#x6210;&#x4E2D;&#x7684;&#x6240;&#x6709;&#x5355;&#x5143;&#x683C; &#xFF1B;&#x7136;&#x800C;&#xFF0C;&#x4F4E;&#x632F;&#x5E45;&#x6216;&#x77ED;&#x671F;&#x5F53;&#x524D;&#x6CE8;&#x5C04;&#x672A;&#x80FD;&#x4E00;&#x8D2F;&#x6FC0;&#x6D3B;&#x53CD;&#x5F39;&#x5CF0;&#x503C;&#x3002;&#x589E;&#x52A0;&#x4ECE; 5 &#x81F3; 1000 ms &#x8D85;&#x6781;&#x5316;&#x7684;&#x6301;&#x7EED;&#x65F6;&#x95F4;&#x662F;&#x575A;&#x51B3;&#x548C;&#x79EF;&#x6781;&#x4E0E;&#x76F8;&#x5173;&#x589E;&#x5F3A;&#x7684;&#x7A57;&#x6982;&#x7387;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6781;&#x5316;&#x7684;&#x89C4;&#x6A21;&#x5F71;&#x54CD;&#x529B;&#x5F3A;&#x8FD9;&#x4E24;&#x4E2A;&#x632F;&#x5E45;&#x7684;&#x56FE;&#x6587;&#x573A;&#x4E2D;&#xFF0C;&#x6240;&#x663E;&#x793A;&#x7684;&#x7535;&#x538B;&#x94B3;&#x5F55;&#x97F3;&#x548C;&#x5728;&#x4EFB;&#x4E00;&#x7535;&#x538B;&#x6216;&#x7535;&#x6D41;&#x94B3;&#x6A21;&#x5F0F;&#x83B7;&#x5F97;&#x7684;&#x6EDE;&#x540E;&#x65F6;&#x95F4;&#xFF0C;&#x4EE5;&#x8FBE;&#x5230;&#x5CF0;&#x503C;&#x7535;&#x6D41;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E; SCN &#x795E;&#x7ECF;&#x5143;&#x53EF;&#x80FD;&#x53CD;&#x5F39;&#x5C16;&#x5CF0;&#x7528;&#x4F5C;&#x751F;&#x4EA7;&#x8F93;&#x51FA;&#x4FE1;&#x53F7;&#x4ECE;&#x5F88;&#x5927;&#x7A0B;&#x5EA6;&#x4E0A;&#x76F8;&#x4E92;&#x5173;&#x8054;&#x7684;&#x5B89;&#x57FA;&#x795E;&#x7ECF;&#x5143;&#x7F51;&#x7EDC;&#x7684;&#x624B;&#x6BB5;&#x4E4B;&#x4E00;&#x3002;

Postinhibitory &#x53CD;&#x5F39;&#x5CF0;&#x503C;&#x88AB;&#x8C03;&#x5236;&#x800C;&#x6210;&#x7684;&#x819C;&#x8D85;&#x6781;&#x5316; SCN &#x5728;&#x5386;&#x53F2;&#x3002;

The suprachiasmatic nucleus (SCN) of the hypothalamus regulates biological circadian time thereby directly impacting numerous physiological processes. The SCN is composed almost exclusively of gamma-aminobutyric acid (GABA)ergic neurons, many of which synapse with other GABAergic cells in the SCN to exert an inhibitory influence on their postsynaptic targets for most, if not all, phases of the circadian cycle. The overwhelmingly GABAergic nature of the SCN, along with its internal connectivity properties, provide a strong model to examine how inhibitory neurotransmission generates output signals. In the present work we show that hyperpolarizations that range from 5 to 1000 ms elicit rebound spikes in 63% of all SCN neurons tested in voltage-clamp in the SCN of adult rats and hamsters. In current-clamp recordings, hyperpolarizations led to rebound spike formation in all cells; however, low-amplitude or short-duration current injections failed to consistently activate rebound spikes. Increasing the duration of hyperpolarization from 5 to 1000 ms is strongly and positively correlated with enhanced spike probability. Additionally, the magnitude of hyperpolarization exerts a strong influence on both the amplitude of the spike, as revealed by voltage-clamp recordings, and the latency to peak current obtained in either voltage- or current-clamp mode. Our results suggest that SCN neurons may use rebound spikes as one means of producing output signals from a largely interconnected network of GABAergic neurons.

Postinhibitory rebound spikes are modulated by the history of membrane hyperpolarization in the SCN.

Le noyau suprachiasmatique (NSC) de l&#x26;#39;hypothalamus r&#xE9;gule biologique rythme circadien ce qui influe directement sur de nombreux processus physiologiques. Le SCN est compos&#xE9;e presque exclusivement de acide gamma-aminobutyrique (GABA) neurones ergiques, dont beaucoup font synapse avec les autres cellules GABAergiques dans le SCN pour exercer une influence inhibitrice sur leurs cibles post-synaptiques pour la plupart, sinon tous, les phases du cycle circadien. La nature extr&#xEA;mement GABAergique de la SCN, ainsi que ses propri&#xE9;t&#xE9;s de connectivit&#xE9; interne, fournir un mod&#xE8;le fort pour examiner comment la neurotransmission inhibitrice g&#xE9;n&#xE8;re des signaux de sortie. Dans le pr&#xE9;sent travail, nous montrons que hyperpolarisations qui vont de 5 &#xE0; 1000 ms pointes rebond suscitent dans 63% de tous les neurones du SCN test&#xE9;s dans voltage-clamp dans le SCN de rats adultes et les hamsters. En courant-clamp, hyperpolarisations conduit &#xE0; la formation de rebondir pic dans toutes les cellules, mais de faible amplitude ou de courte dur&#xE9;e injections de courant n&#x26;#39;a pas constante activer pointes de rebond. L&#x26;#39;augmentation de la dur&#xE9;e de l&#x26;#39;hyperpolarisation de 5 &#xE0; 1000 ms est fortement et positivement corr&#xE9;l&#xE9;e avec des pointes probabilit&#xE9; accrue. En outre, l&#x26;#39;ampleur de l&#x26;#39;hyperpolarisation exerce une forte influence &#xE0; la fois sur l&#x26;#39;amplitude de la pointe, tel que r&#xE9;v&#xE9;l&#xE9; par la tension de serrage des enregistrements, et la latence de pic de courant obtenu en mode de tension ou de courant-clamp. Nos r&#xE9;sultats sugg&#xE8;rent que les neurones du SCN peut utiliser des pointes de rebond comme un moyen de produire des signaux de sortie &#xE0; partir d&#x26;#39;un r&#xE9;seau largement interconnect&#xE9;s de neurones GABAergiques.

Pointes de rebond Postinhibitory sont modul&#xE9;s par l&#x26;#39;histoire de l&#x26;#39;hyperpolarisation de la membrane dans le SCN

Gefundenes Kern (SCN) im Hypothalamus regelt biologischen circadiane Zeit damit direkt beeinflussen zahlreiche physiologische Prozesse. Die SCN besteht fast ausschlie&szlig;lich von Gamma - Aminobutters&auml;ure (GABA) ergic Neuronen, viele von denen Synapse mit anderen GABAergic in der SCN auf inhibitorisches postsynaptisches Zielvorgaben f&uuml;r die meisten, beeinflussen wenn nicht in allen Phasen des zirkadianen Zyklus Zellen. Die &uuml;berwiegend GABAergic Natur des SCN, zusammen mit seinen Eigenschaften interne Konnektivit&auml;t bieten ein starkes Modell zu untersuchen, wie inhibitorischen Neurotransmission erzeugt Ausgangssignale. In der vorliegenden Arbeit, die wir zeigen, dass Hyperpolarizations, die im Bereich von 5 bis 1000 ms Zugstufe entlocken Spitzen in 63 % aller SCN-Neuronen im Spannung-Klammer in der SCN von Erwachsenen Ratten und Hamster getestet. Klemm-aktuelle Aufnahmen f&uuml;hrte Hyperpolarizations Spitze Bildung in allen Zellen Rebound; jedoch nicht niedriger Amplitude oder kurzer Dauer Aktuelle Injektionen, Zugstufe Spitzen konsequent zu aktivieren. Erh&ouml;ht die Dauer von 5 bis 1000 ms mit einem Gatter ist mit verst&auml;rkten Spitze Wahrscheinlichkeit stark und positiv korreliert. Dar&uuml;ber hinaus das Ausma&szlig; der Hyperpolarisation &uuml;bt einen starken Einfluss auf beide die Amplitude der Spike, wie durch Spannung Klemm-Aufnahmen aufgedeckt, und die Wartezeit bis aktuelle Spitzenwert erreicht entweder Spannung oder Strom-Klemm-Modus. Unsere Ergebnisse zufolge SCN Neuronen Zugstufe Spitzen als ein Mittel zur Herstellung Ausgangssignale von einem weitgehend miteinander verbundenen GABAergic Neuronen verwenden k&ouml;nnen.

Postinhibitory Erholung Spitzen werden durch die Geschichte der Membran mit einem Gatter in der SCN moduliert.

Il nucleo soprachiasmatico (SCN) dell&#39;ipotalamo regola biologica tempo circadiano che incidono quindi direttamente numerosi processi fisiologici. Il SCN &egrave; composto quasi esclusivamente da neuroni ergic acido gamma - aminobutirrico (GABA), che molti dei quali sinapsi con altri GABAergico cells in SCN di esercitare un&#39;influenza inibitoria sui loro bersagli postsinaptici per la maggior parte, se non in tutte le fasi del ciclo circadiano. La stragrande maggioranza GABAergico natura di SCN, insieme alle sue propriet&agrave; connettivit&agrave; interna, forniscono un modello forte di esaminare come inibitorio neurotrasmissione genera segnali in uscita. Nel presente lavoro che ci mostrano che hyperpolarizations che vanno da 5 a 1000 ms suscitare rimbalzo picchi nel 63% dei neuroni ANK tutti testati in tensione-morsetto in SCN di ratti adulti e criceti. In corrente-morsetto registrazioni, hyperpolarizations portato a rimbalzo spike formazione in tutte le cellule; Tuttavia, iniezioni di corrente bassa ampiezza o di breve durata non &egrave; riuscito a attivare costantemente picchi di rimbalzo. Aumentando la durata di hyperpolarization da 5 a 1000 ms &egrave; fortemente e positivamente correlato con la probabilit&agrave; maggiore spike. Inoltre, la grandezza di hyperpolarization esercita una forte influenza su entrambi l&#39;ampiezza del picco, come rivelato da registrazioni morsetto di tensione, e la latenza a picco corrente ottenuta in entrambe le modalit&agrave; morsetto di tensione o di corrente. I nostri risultati suggeriscono che i neuroni SCN possono utilizzare picchi di rimbalzo come uno dei mezzi di produzione di segnali in uscita da una rete interconnessa in gran parte dei neuroni GABAergico.

Rimbalzo postinhibitory picchi sono modulati dalla storia di hyperpolarization membrana in SCN.

&#x8996;&#x5E8A;&#x4E0B;&#x90E8;&#x306E;&#x8996;&#x4EA4;&#x53C9;&#x4E0A;&#x6838;&#xFF08;SCN&#xFF09;&#x306F;&#x3001;&#x76F4;&#x63A5;&#x591A;&#x6570;&#x306E;&#x751F;&#x7406;&#x5B66;&#x7684;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x751F;&#x7269;&#x5B66;&#x7684;&#x306A;&#x6982;&#x65E5;&#x6642;&#x9593;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002; SCN&#x306F;&#x3001;&#x307B;&#x307C;&#x72EC;&#x5360;&#x7684;&#x306B;&#x3B3;-&#x30A2;&#x30DF;&#x30CE;&#x916A;&#x9178;&#xFF08;GABA&#xFF09;&#x306F;&#x3001;&#x3059;&#x3079;&#x3066;&#x306E;ERGIC&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3001;SCN&#x5185;&#x306E;&#x4ED6;&#x306E;GABA&#x4F5C;&#x52D5;&#x6027;&#x7D30;&#x80DE;&#x3068;&#x305D;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x591A;&#x304F;&#x306F;&#x3001;&#x307B;&#x3068;&#x3093;&#x3069;&#x5F7C;&#x3089;&#x306E;&#x5F8C;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306E;&#x963B;&#x5BB3;&#x5F71;&#x97FF;&#x3092;&#x767A;&#x63EE;&#x3059;&#x308B;&#x3001;&#x305D;&#x3046;&#x3067;&#x306A;&#x3044;&#x5834;&#x5408;&#x306F;&#x3001;&#x6982;&#x65E5;&#x30B5;&#x30A4;&#x30AF;&#x30EB;&#x306E;&#x30D5;&#x30A7;&#x30FC;&#x30BA;&#x3067;&#x69CB;&#x6210;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; SCN&#x306E;&#x5727;&#x5012;&#x7684;&#x306A;GABA&#x4F5C;&#x52D5;&#x6027;&#x306E;&#x6027;&#x8CEA;&#x306F;&#x3001;&#x305D;&#x306E;&#x5185;&#x90E8;&#x306E;&#x63A5;&#x7D9A;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x3068;&#x5171;&#x306B;&#x3001;&#x6291;&#x5236;&#x795E;&#x7D4C;&#x4F1D;&#x9054;&#x306F;&#x3001;&#x51FA;&#x529B;&#x4FE1;&#x53F7;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x5F37;&#x529B;&#x306A;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;5&#x304B;&#x3089;1000&#x30DF;&#x30EA;&#x79D2;&#x306E;&#x7BC4;&#x56F2;&#x306F;&#x3001;&#x6210;&#x4F53;&#x30E9;&#x30C3;&#x30C8;&#x3068;&#x30CF;&#x30E0;&#x30B9;&#x30BF;&#x30FC;&#x306E;SCN&#x306E;&#x96FB;&#x5727;&#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x3067;&#x30C6;&#x30B9;&#x30C8;&#x3057;&#x305F;&#x3059;&#x3079;&#x3066;&#x306E;SCN&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;63&#xFF05;&#x306E;&#x56DE;&#x5FA9;&#x306E;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x3092;hyperpolarizations&#x3092;&#x5F15;&#x304D;&#x51FA;&#x3059;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x73FE;&#x5728;&#x306E;&#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x8A18;&#x9332;&#x3067;&#x306F;&#x3001;hyperpolarizations&#x306F;&#x3059;&#x3079;&#x3066;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x5F62;&#x6210;&#x3092;&#x56DE;&#x5FA9;&#x306B;&#x5C0E;&#x3044;&#x305F;&#x304C;&#x3001;&#x4F4E;&#x632F;&#x5E45;&#x307E;&#x305F;&#x306F;&#x77ED;&#x6642;&#x9593;&#x306E;&#x96FB;&#x6D41;&#x6CE8;&#x5165;&#x306F;&#x3001;&#x4E00;&#x8CAB;&#x3057;&#x3066;&#x30EA;&#x30D0;&#x30A6;&#x30F3;&#x30C9;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x5316;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002; 5&#x304B;&#x3089;1000&#x306B;&#x904E;&#x5206;&#x6975;&#x306E;&#x6301;&#x7D9A;&#x6642;&#x9593;&#x3092;&#x5897;&#x3084;&#x3059;&#x3068;&#x3001;MS&#x304C;&#x5F37;&#x304F;&#x3001;&#x7A4D;&#x6975;&#x7684;&#x306B;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x306E;&#x78BA;&#x7387;&#x3068;&#x76F8;&#x95A2;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x904E;&#x5206;&#x6975;&#x306E;&#x5927;&#x304D;&#x3055;&#x306F;&#x3001;&#x96FB;&#x5727;&#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x8A18;&#x9332;&#x306B;&#x3088;&#x3063;&#x3066;&#x660E;&#x3089;&#x304B;&#x306B;&#x3055;&#x308C;&#x305F;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x306E;&#x632F;&#x5E45;&#x3001;&#x304A;&#x3088;&#x3073;&#x96FB;&#x5727;&#x307E;&#x305F;&#x306F;&#x96FB;&#x6D41;&#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x30E2;&#x30FC;&#x30C9;&#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x3067;&#x5F97;&#x3089;&#x308C;&#x305F;&#x96FB;&#x6D41;&#x3092;&#x30D4;&#x30FC;&#x30AF;&#x306B;&#x5F85;&#x3061;&#x6642;&#x9593;&#x306E;&#x4E21;&#x65B9;&#x306B;&#x5F37;&#x3044;&#x5F71;&#x97FF;&#x3092;&#x53CA;&#x307C;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x6211;&#x3005;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;SCN&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306F;GABA&#x4F5C;&#x52D5;&#x6027;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x5927;&#x90E8;&#x5206;&#x306F;&#x76F8;&#x4E92;&#x63A5;&#x7D9A;&#x3055;&#x308C;&#x305F;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x304B;&#x3089;&#x306E;&#x51FA;&#x529B;&#x4FE1;&#x53F7;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x624B;&#x6BB5;&#x306E;&#x4E00;&#x3064;&#x3068;&#x3057;&#x3066;&#x3001;&#x30EA;&#x30D0;&#x30A6;&#x30F3;&#x30C9;&#x306E;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;&#x3002;

Postinhibitory&#x30EA;&#x30D0;&#x30A6;&#x30F3;&#x30C9;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x306F;SCN&#x3067;&#x819C;&#x904E;&#x5206;&#x6975;&#x306E;&#x6B74;&#x53F2;&#x306B;&#x3088;&#x3063;&#x3066;&#x5909;&#x8ABF;&#x3055;&#x308C;

&#xC2DC;&#xC0C1; &#xD558; &#xBD80;&#xC758; suprachiasmatic &#xD575; (SCN) &#xD568;&#xC73C;&#xB85C;&#xC368; &#xC9C1;&#xC811; &#xC218;&#xB9CE;&#xC740; &#xC0DD;&#xB9AC; &#xC801; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC0DD;&#xBB3C; &#xD559;&#xC801; circadian &#xC2DC;&#xAC04; &#xC870;&#xC808;. SCN &#xAC10;&#xB9C8;-aminobutyric &#xC0B0; (GABA) ergic &#xC2E0;&#xACBD;, &#xB9CE;&#xC740; &#xB2E4;&#xB978; &#xC5B4;&#xB5A4; &#xB0C5; GABAergic &#xACBD;&#xC6B0; circadian &#xC8FC;&#xAE30;&#xC758; &#xD558;&#xC9C0; &#xBAA8;&#xB4E0; &#xB2E8;&#xACC4; &#xB300;&#xBD80;&#xBD84;&#xC744; &#xC704;&#xD574;, &#xADF8;&#xB4E4;&#xC758; postsynaptic &#xBAA9;&#xD45C;&#xC5D0; &#xC5B5;&#xC81C; &#xC601;&#xD5A5;&#xC744; SCN&#xC5D0; &#xC140; &#xAC70;&#xC758; &#xB3C5;&#xC810;&#xC801;&#xC73C;&#xB85C; &#xAD6C;&#xC131; &#xB418;&#xC5B4;&#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC555;&#xB3C4;&#xC801;&#xC73C;&#xB85C; GABAergic &#xC790;&#xC5F0;&#xC758; &#xB0B4;&#xBD80; &#xC5F0;&#xACB0; &#xC18D;&#xC131;&#xACFC; &#xD568;&#xAED8; SCN&#xC758; &#xCD9C;&#xB825; &#xC2E0;&#xD638;&#xB97C; &#xC0DD;&#xC131; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xC744; &#xAE08;&#xC9C0; neurotransmission&#xB97C; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xAC15;&#xB825;&#xD55C; &#xBAA8;&#xB378; &#xC81C;&#xACF5;. &#xC6B0;&#xB9AC;&#xB294; 1000 &#xBC00;&#xB9AC;&#xCD08; &#xBC94;&#xC704;&#xB294; 5&#xC5D0;&#xC11C; hyperpolarizations &#xB9AC;&#xBC14;&#xC6B4;&#xB4DC; &#xC720;&#xB3C4; &#xD45C;&#xC2DC; &#xD604;&#xC7AC; &#xC791;&#xC5C5; &#xBAA8;&#xB4E0; SCN &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; 63%&#xC5D0;&#xC11C; &#xC2A4;&#xD30C;&#xC774;&#xD06C; &#xC131;&#xC778; &#xC950;, &#xD584;&#xC2A4;&#xD130;&#xC758; SCN&#xC5D0; &#xC804;&#xC555; &#xD074;&#xB7A8;&#xD504;&#xC5D0; &#xD14C;&#xC2A4;&#xD2B8;. &#xC804;&#xB958; &#xD074;&#xB7A8;&#xD504; &#xB179;&#xC74C;, hyperpolarizations; &#xBAA8;&#xB4E0; &#xC140;&#xC5D0;&#xC11C; &#xC2A4;&#xD30C;&#xC774;&#xD06C; &#xD615;&#xC131; &#xBC18; &#xB4F1;&#xC744; &#xC8FC;&#xB3C4; &#xADF8;&#xB7EC;&#xB098;, &#xB0AE;&#xC740; &#xC9C4;&#xD3ED; &#xB610;&#xB294; &#xC9E7;&#xC740; &#xAE30;&#xAC04; &#xD604;&#xC7AC; &#xC8FC;&#xC0AC; &#xB9AC;&#xBC14;&#xC6B4;&#xB4DC; &#xC2A4;&#xD30C;&#xC774;&#xD06C;&#xB97C; &#xC9C0;&#xC18D;&#xC801;&#xC73C;&#xB85C; &#xD65C;&#xC131;&#xD654; &#xD558;&#xC9C0; &#xBABB;&#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. 5&#xC5D0;&#xC11C; 1000 ms&#xC5D0; hyperpolarization&#xC758; &#xAE30;&#xAC04;&#xC744; &#xB298;&#xB9AC;&#xBA74;&#xC740; &#xAC15;&#xB825; &#xD558; &#xACE0; &#xAE0D;&#xC815;&#xC801;&#xC73C;&#xB85C; &#xC11C;&#xB85C; &#xD5A5;&#xC0C1; &#xB41C; &#xC2A4;&#xD30C;&#xC774;&#xD06C; &#xD655;&#xB960;. &#xB610;&#xD55C;, &#xC804;&#xC555; &#xD074;&#xB7A8;&#xD504; &#xB179;&#xC74C; &#xD558; &#xC5EC; &#xACF5;&#xAC1C; hyperpolarization &#xC9C4;&#xB3C4; &#xC2A4;&#xD30C;&#xC774;&#xD06C;, &#xC9C4;&#xD3ED;&#xC5D0; &#xAC15;&#xD55C; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xACE0; &#xD604;&#xC7AC; &#xC808;&#xC815;&#xC5D0; &#xB300;&#xAE30; &#xC804;&#xC555; &#xB610;&#xB294; &#xC804;&#xB958; &#xD074;&#xB7A8;&#xD504; &#xBAA8;&#xB4DC;&#xC5D0;&#xC11C; &#xC5BB;&#xC740;. &#xACB0;&#xACFC; SCN &#xC2E0;&#xACBD; GABAergic &#xC2E0;&#xACBD;&#xC758; &#xC8FC;&#xB85C; &#xC0C1;&#xD638; &#xC5F0;&#xACB0; &#xB41C; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC5D0;&#xC11C; &#xCD9C;&#xB825; &#xC2E0;&#xD638;&#xB97C; &#xC0DD;&#xC0B0; &#xD558;&#xB294; &#xD558;&#xB098;&#xC758; &#xC218;&#xB2E8;&#xC73C;&#xB85C; &#xB9AC;&#xBC14;&#xC6B4;&#xB4DC; &#xC2A4;&#xD30C;&#xC774;&#xD06C;&#xB97C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

Postinhibitory &#xB9AC;&#xBC14;&#xC6B4;&#xB4DC; &#xC2A4;&#xD30C;&#xC774;&#xD06C;&#xB294; SCN&#xC5D0; &#xB9C9; hyperpolarization &#xC5ED;&#xC0AC;&#xB85C; &#xBCC0;&#xC870; &#xB429;&#xB2C8;&#xB2E4;.

El n&#xFA;cleo supraquiasm&#xE1;tico (NSQ) del hipot&#xE1;lamo regula el reloj biol&#xF3;gico circadiano lo que impacta directamente numerosos procesos fisiol&#xF3;gicos. El SCN est&#xE1; compuesto casi exclusivamente de &#xE1;cido gamma-aminobut&#xED;rico (GABA), las neuronas ERGIC, muchas de las cuales hacen sinapsis con otras c&#xE9;lulas GABA&#xE9;rgicas en el NSQ de ejercer una influencia inhibidora en sus objetivos postsin&#xE1;pticos para la mayor&#xED;a, si no todas, las fases del ciclo circadiano. El car&#xE1;cter eminentemente GABA&#xE9;rgicas de la SCN, junto con sus propiedades de conectividad interna, proporcionar un modelo fuerte para examinar la forma en la neurotransmisi&#xF3;n inhibitoria genera se&#xF1;ales de salida. En el presente trabajo muestran que hyperpolarizations que van desde 5 a 1000 ms provocan picos de rebote en el 63% de todas las neuronas del SCN probados en fijaci&#xF3;n de voltaje en el SNC de ratas adultas y h&#xE1;msters. En las grabaciones actuales-clamp, hyperpolarizations llev&#xF3; a repuntar la formaci&#xF3;n de aumento en todas las c&#xE9;lulas, sin embargo, las inyecciones de corriente de baja amplitud o de corta duraci&#xF3;n no se activaba constantemente picos de rebote. El aumento de la duraci&#xF3;n de la hiperpolarizaci&#xF3;n 5 a 1000 ms fuerte y positivamente correlacionada con una mayor probabilidad de alza. Adicionalmente, la magnitud de hiperpolarizaci&#xF3;n ejerce una fuerte influencia en tanto la amplitud de la espiga, como se revela por abrazadera grabaciones de voltaje, y la latencia al pico de corriente obtenida en cualquiera de los modos de voltaje o corriente de pinza. Nuestros resultados sugieren que las neuronas del SCN puede utilizar picos de rebote como un medio de producir se&#xF1;ales de salida de una red interconectada en gran parte de las neuronas GABA&#xE9;rgicas.

Spikes Postinhibitory rebote son modulados por la Historia de la hiperpolarizaci&#xF3;n de la membrana en el SCN

Intercellular communication between gamma-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca(2+) imaging techniques to record changes in the intracellular calcium concentration ([Ca(2+)](i)) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca(2+) responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA(A) and GABA(B) receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca(2+)](i). Application of the GABA(A) receptor antagonist gabazine induced changes in baseline [Ca(2+)](i) in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca(2+) response. GABA application induced Ca(2+) responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca(2+)](i). These results suggest that physiological GABA induces opposing effects on [Ca(2+)](i) based on the chloride equilibrium potential, and may play an important role in neuronal Ca(2+) balance, synchronization and modulation of light input signaling in the SCN network.

GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network.

Neuroactive peptides and the intracellular calcium concentration ([Ca(2+) ](i) ) play important roles in light-induced modulation of gene expression in the suprachiasmatic nucleus (SCN) neurons that ultimately control behavioral rhythms. Vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) are expressed rhythmically within populations of SCN neurons. Pituitary adenylate cyclase-activating peptide (PACAP) is released from retinohypothalamic tract (RHT) terminals synapsing on SCN neurons. Nociceptin/orphanin FQ (OFQ) receptors are functionally expressed in the SCN. We examined the role of several neuropeptides on Ca(2+) signaling, simultaneously imaging multiple neurons within the SCN neural network. VIP reduced the [Ca(2+) ](i) in populations of SCN neurons during the day, but had little effect at night. Stimulation of the RHT at frequencies that simulate light input signaling evoked transient [Ca(2+) ](i) elevations that were not altered by VIP. AVP elevated the [Ca(2+) ](i) during both the day and night, PACAP produced variable responses, and OFQ induced a reduction in the [Ca(2+) ](i) similar to VIP. During the day, VIP lowered the [Ca(2+) ](i) to near nighttime levels, while AVP elevated [Ca(2+) ](i) during both the day and night, suggesting that the VIP effects on [Ca(2+) ](i) were dependent, and the AVP effects independent of the action potential firing activity state of the neuron. We hypothesize that VIP and AVP regulate, at least in part, Ca(2+) homeostasis in SCN neurons and may be a major point of regulation for SCN neuronal synchronization.

Neuropeptide-mediated calcium signaling in the suprachiasmatic nucleus network.

Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities.

Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase.

Hyperexcited states, including depolarization block and depolarized low amplitude membrane oscillations (DLAMOs), have been observed in neurons of the suprachiasmatic nuclei (SCN), the site of the central mammalian circadian (~24-hour) clock. The causes and consequences of this hyperexcitation have not yet been determined. Here, we explore how individual ionic currents contribute to these hyperexcited states, and how hyperexcitation can then influence molecular circadian timekeeping within SCN neurons. We developed a mathematical model of the electrical activity of SCN neurons, and experimentally verified its prediction that DLAMOs depend on post-synaptic L-type calcium current. The model predicts that hyperexcited states cause high intracellular calcium concentrations, which could trigger transcription of clock genes. The model also predicts that circadian control of certain ionic currents can induce hyperexcited states. Putting it all together into an integrative model, we show how membrane potential and calcium concentration provide a fast feedback that can enhance rhythmicity of the intracellular circadian clock. This work puts forward a novel role for electrical activity in circadian timekeeping, and suggests that hyperexcited states provide a general mechanism for linking membrane electrical dynamics to transcription activation in the nucleus.

Article	Year
Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels. Journal of neurophysiology\| PubMed ID: 16709723	2006
Retrograde suppression of GABAergic currents in a subset of SCN neurons. The European journal of neuroscience\| PubMed ID: 16820011	2006
Mutations of presenilin genes in dilated cardiomyopathy and heart failure. American journal of human genetics\| PubMed ID: 17186461	2006
Calcium response to retinohypothalamic tract synaptic transmission in suprachiasmatic nucleus neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 17959816	2007
Postinhibitory rebound spikes are modulated by the history of membrane hyperpolarization in the SCN. The European journal of neuroscience\| PubMed ID: 18783377	2008
GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network. The European journal of neuroscience\| PubMed ID: 19821838	2009
Neuropeptide-mediated calcium signaling in the suprachiasmatic nucleus network. The European journal of neuroscience\| PubMed ID: 21039959	2010
Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase. Journal of neurophysiology\| PubMed ID: 21389307	2011
Causes and consequences of hyperexcitation in central clock neurons. PLoS computational biology\| PubMed ID: 23990770	2013

Robert P. Irwin

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