Gesellschaftlich Synchronisierte Zirkadian Oszillatoren Forex

Die meisten Organismen, einschließlich der Menschen, weisen tägliche physiologische und Verhaltensrhythmen auf. Fast alle Körperfunktionen zeigen signifikante tägliche Variationen wie Aktivität, Erregung, psychophysische Leistungsfähigkeit, Nahrungs - und Wasserverbrauch, Lebermetabolismus, Urinvolumen und pH - Wert, Blutdruck, Herzfrequenz, Säuresekretion im Magen - Darmtrakt und Cortisolsekretion , 55). Es ist überraschender, eine Variable zu finden, die durch die Zeit statisch ist, als eine, die einen täglichen Rhythmus zeigt. Diese zeitliche Variation spielt offensichtlich eine wichtige Rolle bei den homöostatischen Mechanismen der Bodys und hat einen signifikanten Einfluss auf die Funktion spezifischer physiologischer Systeme, einschließlich des Nervensystems. In vielen Fällen werden diese Rhythmen durch endogene Prozesse erzeugt, die als zirkadiane Oszillatoren bezeichnet werden und die vermutlich dazu dienen, biologische Prozesse mit zeitlichen Änderungen der Umgebung zu synchronisieren und die zeitliche Koordination von physiologischen Systemen zu ermöglichen. Der Begriff Circadian stammt aus lateinischen Wurzeln: circa (ungefähr) und stirbt (Tag). Ein circadianer Rhythmus ist ein endogener Rhythmus, der sich mit einer Periode von etwa 24 Stunden wiederholt. Weil diese Rhythmen aus dem Inneren eines Organismus erzeugt werden, besteht ein fundamentales Merkmal zirkadianer Oszillationen darin, dass sie persistieren, wenn sie aus Umgebungszeit-Cues isoliert werden. Da die Periodendauer dieser Rhythmen jedoch nicht genau 24 Stunden beträgt, werden die Oszillationen in Bezug auf jede 24-Stunden-Zeit driften und als Freilauf bezeichnet. Die Freilaufzeit eines Organismus ist unter genetischer Kontrolle und wird für Generationen in Abwesenheit von Umgebungszeit-Cues bestehen bleiben. Eine Manifestation davon ist, dass Mutationen isoliert werden können, die die Rhythmusperiode verändern. In Drosophila haben Forscher erfolgreich einen molekulargenetischen Ansatz zur Erforschung mehrerer Aspekte des fly circadianischen Systems (32, 80) eingesetzt. Zwei Gene wurden identifiziert: Periode und Zeitlos. Die integraler Bestandteil der Generation der zirkadianen Rhythmen in Drosophila sind. Zum Beispiel erzeugen Mutationen im Periodengenom Phänotypen, die rhythmisch oder ausdrücklich veränderte Perioden sind. Vor kurzem wurden auch zwei einzelne Genmutationen, die die Periode verändern, in Säugetieren isoliert (74, 100). Hoffentlich können in Zukunft solche Mutationen verwendet werden, um die genetische Basis der zirkadianen Säugetiersysteme zu erforschen. Um adaptiv als Zeiterfassungssysteme funktionieren zu können, müssen zirkadiane Oszillatoren im Hinblick auf Umweltstörungen präzise und stabil sein. Im allgemeinen scheinen sie diese Bedingungen zu erfüllen. Beispielsweise weist bei der Maus (Mus musculus) der zirkadiane Oszillator, der die Radlaufaktivität antreibt, eine Standardabweichung von etwa 0,6 oder knapp neun Minuten pro 24-Stunden-Zyklus (72) auf. Temperaturschwankungen müssen eine der größten Herausforderungen für diese Homöostase darstellen, da sich die Geschwindigkeit der meisten biochemischen Prozesse bei jeder Temperaturänderung von 1000 C um das 2- bis 3-fache erhöht (Q 10 23). Da selbst homöothermische Säugetiere tägliche Oszillationen bei der Körpertemperatur im Bereich von 20 ° C durchlaufen, könnte ein circadianer Oszillator mit einer Q 10 23 transiente Periodenänderungen von mehreren Stunden zeigen. Dies ist offensichtlich für ein Zeitsteuersystem nicht akzeptabel, und die Periode von zirkadianen Oszillatoren zeigt typischerweise ein Q & sub1; & sub0; von nahezu 1. Diese Temperaturkompensation der Periode scheint ein universelles Merkmal von zirkadianen Systemen zu sein, obwohl wenig über die zuständigen Mechanismen bekannt ist. Dies ist nur ein Beispiel für die Arten der homöostatischen Mechanismen, die sich entwickeln mussten, um es biologischen Oszillatoren zu ermöglichen, eine genaue Zeit zu halten. Dennoch ist die Periode eines circadianen Oszillators keine Konstante und kann sowohl durch äußere als auch innere Einflüsse wie Veränderungen der photischen Umgebung, des hormonellen Milieus und sogar des Alters eines Organismus moduliert werden. Tatsächlich sind einige dieser Änderungen wesentlich, wenn der Oszillator mit der Umgebung synchronisiert werden soll. Circadian-Oszillatoren erzeugen einen Rhythmus, der mit einer Frequenz von nahe, aber nicht gleich 24 Stunden wiederholt wird. Um adaptiv zu funktionieren, müssen diese Oszillatoren mit dem exakten 24-Stunden-Zyklus der physikalischen Welt synchronisiert werden. Schließlich gibt es einen begrenzten Nutzen darin, daß ein Zeitsteuerungssystem vorhanden ist, das nicht auf die Ortszeit zurückgesetzt werden kann. Dieser Vorgang der Synchronisation wird als Mitreißen bezeichnet. Empirisch muß die Phase (F) des Oszillators so eingestellt werden, daß die endogene Periode des Oszillators (tau, t) der 24-Stunden-Periode (T) der physikalischen Welt entspricht. Mathematisch kann dies bei t-T DF ausgedrückt werden. So für einen Primaten mit einem t von 25 Stunden, um zu einem T von 24 Stunden zu ziehen, muss der Oszillator Phase um 1 Stunde pro Tag fortgeschritten sein. Die formalen Eigenschaften von Entrainment und circadianen Oszillatoren sind im allgemeinen Gegenstand einer eleganten Analyse von Pittendrigh und Kollegen (71). Der tägliche Zyklus von Licht und Dunkel, der durch lichtinduzierte Phasenfortschritte und Verzögerungen des endogenen Rhythmus wirkt, ist das dominierende Stichwort, das von Organismen, einschließlich Menschen, verwendet wird, um ihre circadianen Oszillatoren mit der Umgebung zu synchronisieren. Dies ist wahrscheinlich, weil die Morgendämmerung und die Dämmerung die zuverlässigsten und lärmfreien Indikatoren der Zeit sind, die den Organismen in den meisten Lebensräumen zur Verfügung stehen. Aber Organismen haben die Fähigkeit, viele andere Hinweise für das Mitnehmen, einschließlich sozialer Faktoren, Temperaturzyklen, Nahrungsverfügbarkeit zu verwenden. Ein Hauptziel der zirkadianen Rhythmenforschung ist es, die Mechanismen zu verstehen, mit denen Licht und andere Signale dazu dienen, zirkadiane Oszillatoren zu synchronisieren. Kurzum, die zirkadianen Rhythmen zeigen einige grundlegende Merkmale: 1) sie sind endogen erzeugte Schwingungen, die sich mit einer Frequenz von nahezu 24 Stunden wiederholen, 2) sie weisen eine Homöostase der Periode einschließlich der Temperaturkompensation auf, 3) sie werden durch periodische Umweltsignale synchronisiert, Wobei der tägliche Hell-Dunkel-Zyklus das dominierende Stichwort ist. Offensichtlich sind nicht alle täglichen Rhythmen circadian, und das Finden eines Tagesnachtdifferenz in einem physiologischen oder Verhaltensparameter ist nicht ausreichend, um zu behaupten, dass der Parameter einen circadianen Rhythmus zeigt. Tägliche Rhythmen, die exogen angetrieben werden, verschwinden, wenn ein Organismus in konstante Bedingungen gebracht wird und die exogene Tagesvariable entfernt wird. Im Gegensatz dazu wird ein circadianer Rhythmus mit einer nahezu 24-Stunden-Periode für mindestens mehrere Zyklen unter konstanten äußeren Bedingungen fortgesetzt. Die Phase eines circadianen Rhythmus kann auch mit der Phase des Hell-Dunkel-Zyklus synchronisiert werden, der sie ausgesetzt ist. Säugetiere haben einen Satz anatomisch diskreter Zellpopulationen entwickelt, die als physiologisches System dienen, um eine zeitliche Organisation auf einer zirkadianen Zeitskala zur Verfügung zu stellen. Diese Strukturen werden allgemein als ein circadianes System bezeichnet (1). Im einfachsten Fall kann ein zirkadianes System mit drei Komponenten modelliert werden: 1) einem Oszillator oder Taktgeber, der für die Erzeugung des täglichen Rhythmus verantwortlich ist, 2) Eingabewege, durch die die Umgebung und andere Komponenten des Nervensystems Information an die Oszillator und 3) Ausgangswege, durch die der Oszillator zeitliche Information für einen weiten Bereich von physiologischen und Verhaltensregelzentren bereitstellt. Obwohl das Vorhandensein von Rückkopplungsschleifen zwischen diesen Komponenten diese Trennungen etwas willkürlich machen kann, stellen sie doch einen guten Ausgangspunkt für die Diskussion dar. Im weiteren Verlauf dieses Kapitels werden wir jede dieser Komponenten nacheinander behandeln und auch einige der medizinischen Implikationen von Menschen mit einem zirkadianen Timing-System kurz erörtern. Angesichts des Ziels dieses Buches wird sich die Diskussion auf Säugetiere und neurochemische Mechanismen konzentrieren. Angesichts Raumbeschränkungen konnten viele hervorragende und relevante Referenzen nicht berücksichtigt werden. DAS MAMMALISCHE ZIRKADISCHE OSZILLATOR: DER SUPRACHIASMATISCHE NUCLEUS (SCN) Lokalisierung eines Oszillators: Das SCN Eines der Hauptziele der neurowissenschaftlichen Forschung ist es, die anatomischen Substrate spezifischer Verhaltensregelsysteme zu identifizieren. Dies hat sich in vielen Fällen als frustrierend erwiesen. Eine der Erfolgsgeschichten in diesem Bereich war die Lokalisierung der meisten zirkadianen Timing-Funktion zu einem bilateral gepaarten Kern im Säugetier-Hypothalamus des suprachiasmatischen Kerns oder SCN (Abbildung 2). Aus diesem Grund lohnt es sich, den Nachweis zu erbringen, dass diese heterogene Zellpopulation der Ort der biologischen Uhr des Säugers ist. Erste Studien verwendeten Läsionen, um die Hypothese, dass diskrete Teile des Nervensystems als biologische Uhr in Säugetieren fungieren können. Aus diesen Studien kamen die Forscher zu dem Schluss, dass ein circadianer Oszillator irgendwo im ventralen Hypothalamus lokalisiert war. Bis Anfang der siebziger Jahre, als neue neuroanatomische Instrumente auf das Problem angewendet wurden, wurden nicht viel mehr Fortschritte erzielt. Da Läsionsstudien nahelegten, dass ein circadianer Oszillator im Hypothalamus lokalisiert ist und Lichtinformationen aus der Netzhaut diesen Oszillator erreichen müssen, war es ein logischer Ansatz, anatomische Wege zu suchen, mit denen Lichtinformationen von der Netzhaut zum Hypothalamus transportiert werden konnten. Diese Strategie führte zu dem Nachweis eines direkten Retinohypothalamus (RHT). Die Hypothalamus-Stelle, in der diese Fasern beendet waren, war die SCN-Ablation des SCN, die schnell gezeigt wurde, um circadiane Rhythmen abzuschaffen (61, 92). Natürlich gab es unter anderen Interpretationsproblemen, die bei Läsionsstudien häufig vorkamen, die Möglichkeit, dass die SCN-Läsionen den Ausdruck nur blockieren könnten, aber nicht die Erzeugung des rhythmischen Verhaltens. Nichtsdestotrotz waren diese Studien die ersten, die das SCN als einen zirkadianen Oszillator verwickeln. Wenn das SCN die Stelle eines circadianen Oszillators war, dann sollten diese Strukturen in vivo Oszillationen zeigen. Eines der ersten Beweisstücke der endogenen Rhythmizität kam von Studien, die den Zellmetabolismus mit 2-Desoxy-D-glucose (2DG, 84) untersuchten. Die Zellen des SCN zeigten prominente zirkadiane Variation in ihrer metabolischen Aktivität Glukose Verwertung spitzte während des Tages, als die nächtlichen Nager in Ruhe waren, und fiel auf ein Minimum während der Nacht, als die Tiere aktiv waren. Ein Grund für diesen metabolischen Rhythmus kann sein, dass SCN-Zellen auch einen ähnlichen Rhythmus bei spontaner neuraler Aktivität zeigen. Aber in vivo ist dieser Rhythmus nicht auf den SCN beschränkt und kann in anderen Regionen des Zentralnervensystems aufgezeichnet werden. Um die Rolle des SCN bei der Erzeugung dieser Rhythmen zu klären, haben Inouye und Kawamura (37) eine Reihe von Messerschnitten um das SCN herum gemacht, um diese Strukturen von der neuralen aber nicht hormonellen Kommunikation zu isolieren. Danach setzte sich der elektrische Rhythmus in der Insel fort, die das SCN enthielt, aber nicht in anderen Regionen des Nervensystems. So scheint das SCN tatsächlich in der Lage zu sein, circadiane Schwingungen in einem Organismus zu erzeugen. SCN-Gewebe ist auch in der Lage, zirkadiane Oszillationen in vitro zu erzeugen, wenn sie von dem Rest des Organismus isoliert werden. Erste Studien zeigten, dass der circadiane Rhythmus in der neuronalen Aktivität auch aus einer SCN-Hirnscheibenpräparation (30) aufgezeichnet werden konnte. Seit dieser Zeit haben viele Studien von dieser rhythmischen neuralen Aktivität Gebrauch gemacht, um die SCN-Funktion in vitro zu untersuchen. SCN-Gewebe sezerniert das Peptid Vasopressin auch rhythmisch (23). Derzeit werden diese in vitro-Vorbereitungen verwendet, um grundlegende Fragen zu erforschen, wie zirkadiane Schwingungen erzeugt werden. Die Ergebnisse dieser Experimente zeigen, dass der SCN ein Oszillator ist und dass, wenn der SCN zerstört oder funktionell von einem Organismus entfernt wird, viele der Verhaltensrhythmen verschwinden. Idealerweise möchte man zeigen, dass, wenn das SCN-Gewebe wieder hergestellt wird, die Rhythmen wieder auftauchen würden. Aufgrund der Fortschritte in der Hirntransplantation Techniken, ist dieses Experiment jetzt möglich. Es ist nun möglich, Rhythmen bei Tieren, die durch SCN-Läsionen arrhythmisch gemacht wurden, wiederherzustellen, indem man die SCN eines anderen Tieres verpflanzt (45, 76). Darüber hinaus ist es möglich, festzustellen, dass der Rhythmus durch das transplantierte SCN-Gewebe und nicht eine gewisse trophische Wechselwirkung mit dem Wirtsgewebe erzeugt wird. Für diese Experimente wurde ein mutierter Hamster verwendet, der einen circadianen Rhythmus mit einer wesentlich kürzeren Periode als der Normalthe der sogenannten tau-Mutante zeigt (74). Wenn SCN-Gewebe von tau-mutierten Tieren in SCN-lesionierte Wildtyp-Tiere transplantiert wurde, wurden Lokomotor-Aktivitätsrhythmen mit der kürzeren Periode des Spendergewebes wiederhergestellt. Umgekehrt zeigen die wiederhergestellten Rhythmen die normale Periode der Spendertiere (76), wenn SCN-Gewebe von Wildtyp-Hamstern in SCN-vermittelte Tau-Mutantentiere transplantiert wurde. Diese Experimente lieferten zwingenden Beweis, dass der SCN der zirkadiane Oszillator bei Nagetieren war. Ist die zirkadiane Funktion ausschließlich auf den SCN lokalisiert oder haben andere Regionen auch diese Kapazität? Einige Studien haben die Möglichkeit von anderen zirkadianen Oszillatoren erhöht. Beispielsweise können in SCN-vermittelten Tieren (57) lebensmittelverzehrbare und methamphetamininduzierte circadiane Oszillationen beobachtet werden. In vielerlei Hinsicht wäre das Vorhandensein mehrerer zirkadianer Oszillatoren nicht überraschend. Bei den meisten Wirbeltierarten wurden endogene circadiane Oszillatoren auf drei Strukturen lokalisiert: dem SCN, der Zirbeldrüse und der Netzhaut. Bis vor kurzem schienen Säugetiere eine Ausnahme von dieser Regel zu sein, wobei eine derartige Hauptkomponente der zirkadianen Funktion im SCN lokalisiert war. Die jüngste Arbeit von Tosini und Menaker (94) zeigte jedoch einen besonders deutlichen Beweis, dass auch bei Säugetieren zirkadiane Oszillatoren außerhalb des SCN-Gebietes vorhanden sind. In dieser Studie wurde eine Hamster-Retina bei Raumtemperatur gezüchtet, und die Sekretion von Melatonin wurde gemessen. Non-Säugetiere Retinas zeigen tägliche Rhythmen in vielen Variablen, einschließlich der Melatonin-Sekretion, so war es nicht unvernünftig, nach Rhythmen in Melatonin-Produktion zu suchen. Tatsächlich produzierte die Hamster-Retina Melatonin rhythmisch, mit hohen Niveaus in der Nacht und niedrigem Niveau während des Tages. Ein Licht-Dunkel-Zyklus kann diesen Rhythmus in der Kultur synchronisieren, so dass die exzidierte Retina sogar einen intakten Lichteintrittsweg hat. Diese Beobachtungen zeigten, dass ein anderer circadianer Oszillator unabhängig vom SCN existiert und die Retina als ein anderes Modellsystem einführt, um die zirkadiane Funktion bei Säugetieren zu erforschen. Circadian-Oszillationen sind ein Netzwerk oder eine intrazelluläre Eigenschaft Die SCN ist eine dichte Ansammlung von kleinen Neuronen, die dorsal zum Optikchiasma und lateral zum dritten Ventrikel liegen (Abb. 3). Wie zuvor beschrieben, zeigen diese Zellen zirkadiane Rhythmen in elektrischer Aktivität, Glucoseverwertung und Sekretion. Eine der ersten Fragen, die angegangen werden müssen, ist, ob diese Zellen den Rhythmus durch einen intrazellulären oder einen Netzwerkprozess erzeugen. Die Antwort ist noch nicht bekannt, aber mindestens drei Zeilen deuten darauf hin, dass synaptische Wechselwirkungen für die Generierung des Tagesrhythmus nicht wichtig sind. Zuerst blockierte die lokale Injektion von Tetrodotoxin (TTX) in die SCN-Region in vivo die Expression und die photonische Regulierung eines zirkadianen Rhythmus im Trinkverhalten (86). TTX blockiert spannungsempfindliche Natriumkanäle und synaptische Kommunikation im neuronalen Gewebe. Als diese Behandlungen jedoch enden, setzten sich die Rhythmen von einer Phase fort, die nahelegte, dass der SCN-Oszillator durch die experimentelle Manipulation ungestört war. Ähnliche Ergebnisse wurden mit in vitro SCN-Präparaten erhalten: Die Expression von zirkadianen Sekretionsrhythmen und spontaner neuraler Aktivität wurde durch TTX blockiert, aber der Oszillator selbst schien nicht betroffen zu sein (22, 103). Darüber hinaus erzeugen die Zellen des SCN bereits vor der Bildung der Mehrheit der Synapsen Rhythmen bei der Glukoseverwertung früh bei der Entwicklung des Kerns (84). Schließlich behalten disassoziierte SCN-Zellen in Kultur die Fähigkeit, circadiane Oszillationen (68, 90, 103) zu erzeugen. Unter diesen Bedingungen zeigten einzelne SCN-Zellen zirkadiane Schwingungen bei spontaner neuronaler Aktivität, die auseinander phasenverschoben sind, als wenn jede Zelle einen unabhängigen Oszillator (103) enthält. Die einfachste Interpretation dieser kollektiven Daten ist, dass synaptische Wechselwirkungen für die Ein - und Ausgänge des SCN-Oszillators wichtig sind, aber nicht für die Erzeugung des zirkadianen Rhythmus selbst verantwortlich sind. Allerdings muss die Lösung dieses Problems die Demonstration erwarten, dass einzelne, isolierte SCN-Zellen die Fähigkeit beibehalten, circadiane Oszillationen zu erzeugen. Das zirkadiane System der Säugetiere entwickelte sich nicht unabhängig. Vergleichende Beweise stimmen auch mit der Möglichkeit überein, dass die Erzeugung von zirkadianen Oszillationen eher ein intrazellulärer als ein Netzwerkprozess ist. Zweifellos zeigen einzelne zelluläre Organismen (z. B. die Dinoflagellat-Gonyaulax-Polyeder) und Cyanobakterien circadiane Oszillationen, so daß diese Rhythmen intrazellulär eindeutig erzeugt werden können. Weitere Unterstützung stammt aus Untersuchungen der marinen Mollusk Bulla gouldiana. Die Augen dieses Organismus enthalten einen Oszillator, der einen circadianen Rhythmus von spontanen zusammengesetzten Aktionspotentialen im Sehnerv antreibt. Eine Population von elektrisch gekoppelten Zellen, die als basale Retinalneuronen (BRNs) bekannt sind, ist verantwortlich für die Erzeugung dieses Rhythmus durch einen täglichen Zyklus in ihrem Membranpotential. Isolierte BRNs in der Kultur zeigen weiterhin einen circadianen Rhythmus in der Membranleitfähigkeit (54), was zeigt, dass einzelne Zellen in Kultur die Fähigkeit behalten, eine circadiane Oszillation zu erzeugen. Unabhängig davon, ob einzelne Zellen im SCN kompetente zirkadiane Oszillatoren sind, ist es offensichtlich wichtig zu verstehen, wie diese Zellen kommunizieren und miteinander synchronisiert bleiben. Das aktuelle Wissen über die Zell-zu-Zell-Kommunikation im SCN ist Gegenstand einer kürzlich erschienenen Übersicht (98) und wird hier nur kurz diskutiert. Intrinsische Transmitter: GABA und Peptide Der Nachweis deutet darauf hin, dass die Aminosäure G-Aminobuttersäure (GABA) der wichtigste Transmitter ist, der von SCN-Neuronen verwendet wird. GABA findet sich in mindestens der Hälfte aller präsynaptischen Terminals im SCN (18), und GABA und sein synthetisches Enzym finden sich in den meisten SCN-Zellkörpern (63). Darüber hinaus haben elektrophysiologische Studien spontane und elektrisch evozierte hemmende postsynaptische Potentiale im SCN aufgezeichnet, die GABA-vermittelt sind (43). Die Verabreichung von GABA-Agonisten verursacht Phasenverschiebungen von Verhaltensrhythmen während des Tages und verändert die photonische Regulierung des zirkadianen Systems während der Nacht (75, 95). In erwachsenem Gewebe ist GABA typischerweise ein inhibitorischer Transmitter, und die meisten SCN-Neuronen senden Projektionen zu anderen Zellen in dem SCN, so dass das typische SCN-Neuron am besten als ein inhibitorisches Interneuron betrachtet werden kann. Ultrastrukturelle Studien unterstützen die Unterteilung der Ratte SCN in drei Populationen: eine kleine rostrale Fläche mit kaudalen, dorsomedialen und ventrolateralen Unterteilungen. Viele der Zellen in den SCN-Expresspeptiden und Unterschiede in der Peptid-Expression können als Grundlage für die Segregation von SCN-Zellen dienen (58, 97). Insbesondere wird zwischen Zellen, die Vasopressin (VA) exprimieren, die in den rostralen und dorsomedialen Regionen gefunden wird, und jenen, die vasoaktives intestinales Peptid (VIP) exprimieren, die in den ventrolateralen Bereichen des SCN (4) gefunden wird, unterschieden. Retinal und andere afferenten Innervationen sind weitgehend auf die ventrolateralen Regionen beschränkt. So ist es in diesen Zellen, dass die Integration der Mehrheit der synaptischen Eingaben am ehesten stattfindet. Einige haben spekuliert, dass die VIP-immunreaktiven Zellen eine wichtige Rolle bei der Verarbeitung von fotografischen Inputs spielen können, während die VA-immunreaktiven Zellen für die Erzeugung von täglichen Rhythmen verantwortlich sein können. Jedoch können zirkadiane Rhythmen sowohl in der ventrolateralen als auch in der dorsomedialen Population von SCN-Neuronen (38) exprimiert werden, und an diesem Punkt sind die funktionellen Rollen, die von diesen verschiedenen Zelltypen gespielt werden, unklar. Darüber hinaus bleibt die Frage, ob diese anatomisch definierten Unterteilungen von Zellen auch elektrophysiologisch verschieden sind, noch zu beantworten. Zusätzlich zu VA und VIP werden eine Anzahl anderer Peptide und Wachstumsfaktoren in SCN-Neuronen exprimiert. Diese schließen gastrinfreisetzendes Peptid (GRP), das Peptid Histidin Isoleucin, Somatostatin, Substanz P, Neurotensin und Nervenwachstumsfaktor ein. In vielen Fällen scheinen diese Peptide mit Aminosäure-Transmittern co-lokalisiert zu sein und funktionieren vermutlich als Signalmoleküle. Ein Peptid, das einige neue Aufmerksamkeit erhalten hat, ist GRP, das in Neuronen in der ventrolateralen Unterteilung des SCN exprimiert wird. Die Anwendung von GRP erregte viele SCN-Neuronen in vitro und kann Phasenverschiebungen des zirkadianen Systems in vivo bewirken (69). Aber die Frage, wie diese oder andere Peptidfunktionen in lokalen SCN-Schaltungen und die Rolle von Peptiden in der zirkadianen Funktion noch nicht gelöst sind, Die Rolle von Peptiden im SCN wurde kürzlich untersucht (38). Das SCN mit seiner klar definierten zirkadianen Funktion und seinen Verhaltensausgaben ist ein ausgezeichneter Ort, um die Funktion von Peptid-Signalmolekülen zu erforschen. INPUT ZUM SCN: WIE FUNKTIONIERT DIE UMWELTUNG DAS ZIRKADISCHE OSZILLATOR Ein Ansatz zum Verständnis zirkadianer Systeme ist es, die neurochemischen Schaltkreise zu untersuchen, mit denen der SCN Informationen aus der Umgebung erhält. Neben der Behandlung von Problemen im Zusammenhang mit der sensorischen Physiologie zirkadianer Systeme könnte dieser Ansatz zur Identifikation von Komponenten des circadianen Oszillators führen. Durch systematische Verfolgung der Signaltransduktionskaskade, durch die die photonischen Informationen die SCN-Neuronen erreichen und regulieren, sollte es möglich sein, Mechanismen zu identifizieren, die circadiane Oszillationen erzeugen. Eine Strategie, die weitgehend verwendet wurde, um diese Probleme anzugehen, ist eine systemebene Analyse der Effekte von photischen, pharmakologischen und genetischen Manipulationen auf Rhythmen, die durch das zirkadiane System angetrieben werden. Eine weitere Strategie besteht darin, die Auswirkungen solcher Manipulationen auf die zelluläremolekulare Aktivität von SCN-Neuronen zu untersuchen. Beide Strategien werden erfolgreich verwendet, um die SCN-Funktion zu erforschen (siehe aktuelle Übersichten in Lit. 38, 66, 95). Retinohypothalamischer Trakt (RHT) Die Anatomie des Lichteingangswegs zum SCN wurde kürzlich untersucht (Abb. 5, Ref. 66). Bei Säugetieren werden die Wirkungen von Licht auf das SCN durch unbekannte Photorezeptoren, die sich in der Netzhaut befinden, vermittelt. Der primäre Weg für die Übertragung von fotografischen Informationen aus der Netzhaut zum Herzschrittmacher für die Mitnahme ist die RHT. Das RHT umfasst eine deutliche Untergruppe von Retinal-Ganglion-Zell-Axonen, die sich von den anderen optischen Axonen am Optikchiasma unterscheiden, um das SCN zu innervieren. Die photorelevanten Informationen, die neuronal zum SCN übertragen werden, sind sowohl notwendig als auch ausreichend für das Mitreißen. Es gibt Hinweise darauf, dass das Aminosäure-Glutamat ein Transmitter am RHTSCN-synaptischen Anschluss ist und dass dieser Transmitter eine entscheidende Rolle bei der Vermittlung der photonischen Regulation des zirkadianen Systems spielt (14). Anatomische Studien berichten, dass RHT-Terminals innervieren die SCN zeigen Glutamat-Immunreaktivität mit synaptischen Bläschen assoziiert (8, 19) identifiziert. Eine Vielzahl von Glutamat-Rezeptoren wurde lokalisiert, um die SCN sowohl in situ-Hybridisierung und Immunozytochemie (28). Es gibt elektrophysiologische Hinweise darauf, dass die exogene Applikation von GluR-Agonisten die SCN-Neuronen anregt (6, 87), und dass GluRs die im SCN (42) aufgezeichneten exzitatorischen post-synaptischen Potentiale vermitteln. Die Anwendung von GluR-Agonisten verursacht Phasenverschiebungen eines Rhythmus der neuronalen Aktivität, die von dem SCN in vitro aufgezeichnet wurden (20, 89). Schließlich blockieren GluR-Antagonisten die lichtinduzierten Phasenverschiebungen und die Fos-Induktion im SCN in vivo (2, 13). Trotz dieses starken Beweises, dass Glutamat ein Sender ist, der von der RHT freigegeben wird, gibt es viele unbeantwortete Fragen, wie die zirkadianen Oszillatoren im SCN auf diese glutamaterge Stimulation reagieren. Im einfachsten Szenario verursacht Licht die Freisetzung von Glutamat, das eine Signaltransduktionskaskade in SCN-Neuronen initiiert, was letztendlich zu einer Phasenverschiebung des zirkadianen Systems führt. Dieses Modell wird durch die Erkenntnisse gestärkt, dass GluR-Antagonisten lichtinduzierte Phasenverschiebungen in vivo verhindern. Und dass exogenes Glutamat Phasenverschiebungen in vitro verursachen kann. Problematischer ist die Feststellung, dass GluR-Agonisten, die in den SCN-Bereich injiziert wurden, keine lichtähnlichen Phasenverschiebungen verursachen (53). Natürlich gibt es viele mögliche Fallstricke bei der Interpretation dieser Art von Experiment. Zum Beispiel scheint es unwahrscheinlich, dass die Injektion von Glutamat in die SCN-Region die gleichen Zellen auf dieselbe Weise stimulieren würde wie die synaptische Aktivierung des RHT. Trotzdem stellen derzeit verfügbare Daten eine einfache Interpretation der Verhaltensexperimente mit GluR-Antagonisten in Frage. Gegenwärtig belegen Verhaltensnachweise, dass GluR-Aktivierung notwendig ist, aber nicht ausreichend ist, um lichtähnliche Phasenverschiebungen zu erzeugen. Zur Klärung der Signaltransduktionskaskaden, durch die Glutamat im SCN wirkt, und um zu verstehen, wie diese Kaskaden die Phase des zirkadianen Systems beeinflussen, ist mehr Arbeit erforderlich. Stickoxid (NO) Eine mögliche Konsequenz der NMDA GluR-Aktivierung ist die Stimulation der Stickoxidsynthase (NOS). Mehrere Beweisstücke eröffnen die Möglichkeit einer Rolle für NO im Lichteintrittsweg zum SCN. Zunächst zeigen anatomische Studien die Anwesenheit von NOS im SCN (3). Zweitens wurde die NO-Produktion im Allgemeinen mit der NMDA-induzierten cGMP-Produktion verknüpft, und die Verabreichung von cGMP erzeugt Phasenverschiebungen des zirkadianen Rhythmus der neuronalen Aktivität, die von dem SCN in vitro aufgezeichnet wurden. (73) aufweist. Schließlich verhindern NOS-Inhibitoren NMDA-induzierte Phasenverschiebungen von zirkadianen Rhythmen sowohl in vitro als auch in vivo (20). Veränderungen in der Genexpression Obwohl die Signaltransduktionsereignisse, die stromabwärts der GluR-Aktivierung im SCN auftreten, nicht gut verstanden werden, ist eine wichtige Konsequenz der photischen Stimulation die Regulation der Genexpression im SCN (44). In vielen Neuronen ist eine Konsequenz der GluR-Stimulation die Aktivierung von unmittelbaren frühen Genen, einschließlich c-fos. Die von diesen Genen codierten Proteine, einschließlich Fos, scheinen generell an der Transduktion extrazellulärer Signale an Veränderungen in der Genexpression beteiligt zu sein und in einigen Caseschenen in der unmittelbaren frühen Genexpression können als zellulärer Marker der neuronalen Aktivierung verwendet werden. Die photonische Regulation von c-fos mRNA und Fos-artiger Immunoreaktivität (Fos-LI) im SCN von Nagetieren wurde umfassend demonstriert. Diese Studien haben gezeigt, dass die photochemische Regulation von Fos in SCN-Neuronen mit lichtinduzierten Phasenverschiebungen des zirkadianen Systems korreliert. Beispielsweise zeigt die Induktion der c-fos-mRNA durch Licht im Hamster-SCN dieselbe Phasenabhängigkeit und Intensitätsschwelle wie die Phasenverschiebung. Die funktionelle Bedeutung der lichtinduzierten Fos-Expression ist noch unklar. Licht kann das zirkadiane System von Mäusen, denen das c-fos-Gen fehlt, noch entziehen, obwohl eine Reduktion in der Größe von lichtinduzierten Phasenverschiebungen beobachtet wurde (35). In einer anderen Studie verringerte die intraventrikuläre Verabreichung von c-fos und jun-B Antisense-Oligonukleotiden die Expression dieser Transkriptionsfaktoren und inhibierte lichtinduzierte Phasenverschiebungen (104). Diese Ergebnisse deuten darauf hin, dass, während c-fos-Aktivierung kann dazu beitragen, die normale Entrainment-Prozess, ist es nicht unbedingt erforderlich für die photologische Regulation oder Generation von circadianen Rhythmen. Die Fos-Induktion wurde auch weithin als zellulärer Marker für lichtempfindliche Zellen verwendet, um Fragen zu beantworten, wie experimentelle Manipulationen die fotografische Eingabe in das SCN ändern. In einer Reihe von Studien wurde berichtet, dass sowohl NMDA - als auch AMPAKA-GluR-Antagonisten die Lichteindung der Fos-Expression im SCN inhibieren (2, 25). Mindestens eine Studie hat auch festgestellt, dass die intraventrikuläre Injektion von NMDA induziert Fos-Expression in der SCN (25). Die Wirkungen von AMPAKA GluR-Agonisten wurden nicht untersucht. Diese pharmakologischen Studien deuten generell auf eine Rolle sowohl für NMDA als auch für AMPAKA GluRs hin, die die photonische Regulation von SCN-Neuronen in vivo vermitteln. Die Möglichkeit von Peptid-Co-Transmittern in RHT-Terminals wird durch die Anwesenheit von Dichte-Vesikeln unter den glutamathaltigen synaptischen Vesikeln (8) spezifisch vorgeschlagen. Zwei mögliche Kandidatenübermittler sind N-Acetylaspartylglutamat (NAAG) und Substanz P (SP). Immunzytochemische Lokalisation von NAAG zu vielen retinalen Ganglienzellen und dem SCN wurde berichtet (58). Die Sehnerven-Transektion verminderte die NAAG-Immunreaktivität im SCNa-Befund, was mit dem Vorschlag zusammenhängt, dass NAAG in terminalen Feldern des RHT enthalten ist. Obwohl die funktionelle Rolle von NAAG unklar ist, kann es sowohl direkt GluRs aktivieren und Glutamat durch extrazelluläre Hydrolyse bilden. Eine physiologische Studie ergab, dass die iontophoretische Anwendung von NAAG die Zündgeschwindigkeit und die potenzierten Glutamat-induzierten Reaktionen in SCN-Neuronen in der Kultur erhöhte (6). Es gibt auch einige Hinweise darauf, dass SP eine Rolle als Retina-Co-Transmitter spielen kann. Anatomische Evidenz für die Anwesenheit von SP im RHT wurde für eine Anzahl von Spezies, einschließlich Menschen, gefunden (64). Die Anwendung von SP in vitro erhöhte die 2DG-Aufnahme, erregte eine Population von SCN-Neuronen, induzierte Fos-Expression und verursachte Phasenverschiebungen des circadianen Rhythmus der elektrischen Aktivität (88). In neuerer Zeit wurde festgestellt, dass ein SP-Antagonist die lichtinduzierte Fos-Expression in vivo blockiert (1). Es ist wichtig, die Auswirkungen dieses Inhibitors auf lichtinduzierte Phasenverschiebungen des zirkadianen Systems zu untersuchen und zu sehen, ob SP die Glutamatfreisetzung aus dem RHT ändert. Bisher stimmen die Ergebnisse mit der Möglichkeit überein, dass dieses Peptid ein Co-Transmitter an der RHTSCN-synaptischen Verbindung ist. Geniculohypothalamus-Trakt (GHT) Die Netzhautganglienzellen, die das SCN innervieren, projizieren ebenfalls zu einer Unterteilung des lateralen Geniculat des intergeniculären Faltblatts (IGL, Abb. 6, Ref. 66). Die IGL wiederum hat eine Population von Neuronen, die zum SCN durch einen geniculohypothalamic Trakt (GHT) projizieren. Die Projektion Neuronen, aus denen sich die GHT scheinen Neuropeptid Y (NPY) und GABA enthalten, und diese Sender können co-lokalisiert werden. Beide Moleküle üben Effekte auf das circadiane System aus. Dieser Weg scheint sowohl bei der Verarbeitung von fotografischen Informationen als auch bei der Vermittlung der Effekte einiger nicht-photischer Mitreiz-Stimuli beteiligt zu sein. Es ist bekannt, dass Zellen im IGL lichtempfindlich sind (33), und der GHT-Signalweg kann zur Verarbeitung von fotografischer Eingabe beitragen. In vielen sensorischen Neuronen wirkt NPY präsynaptisch, um die Freisetzung von Transmittern zu modulieren und kann eine ähnliche Rolle bei der Regulierung der RHT-Eingabe an den SCN spielen. Ebenso können GABAB-Rezeptoren die lichtinduzierten Phasenverschiebungen (75) und die Freisetzung von Glutamat durch das RHT (40) modulieren. IGL-Läsionen sind mit relativ subtilen Veränderungen in der Reaktion des zirkadianen Systems auf Licht assoziiert, einschließlich Änderungen in der Größe von lichtinduzierten Phasenverschiebungen, Periodenänderungen und langsamerer Anpassung an neue Hell-Dunkel-Zyklen (66). Allerdings sind viele Merkmale der zirkadianen Systeme Reaktion auf Licht nicht betroffen in IGL-läsionierten Tieren, was darauf hindeutet, dass dieser Weg ist nicht wesentlich für den Großteil der photischen Regulierung des zirkadianen Systems. Aber dieser Weg scheint eine wichtige Rolle bei der Vermittlung der Wirkungen anderer nicht-photischer Reize zu haben. A number of experimental treatments produce phase shifts during the day but not during the night, including activity induced by novel stimuli (67) and benzodiazepines (95). These phase shifts are dependent upon an intact IGL lesions abolish both benzodiazepine (41) and activity-induced (67) phase shifts. The use of antibodies to reduce NPY binding also reduced activity-induced phase shifts (4). A similar pattern of phase shifts during the day was generated by NPY administration in vitro and in vivo through a mechanism which may be dependent on GABAergic transmission (36). Recent studies in cultured SCN cells suggest that NPY can act presynaptically to inhibit GABA-mediated synaptic transmission through inhibition of calcium currents (9). Thus, the GHT plays a critical role in mediating the effects of some non-photic stimuli on the circadian system. Other Neurotransmitter Systems The SCN receives a dense serotonergic projection from the midbrain raphe nuclei that terminates predominantly in the retinorecipient region of the nucleus (62). It is well established that 5-HT receptor agonists cause phase shifts of the SCN circadian oscillator when administered at times in the circadian cycle during which light does not cause phase shifts both in vitro (52) and in vivo (26). In addition, evidence suggests that this projection modulates photic input to the SCN. Neurotoxic destruction of the serotonergic input to the SCN alters the relationship between the light-dark cycle and locomotor activity, and increases in 5-HT levels alter the effects of light on the circadian system (65). Finally, 5-HT and 5-HT agonists inhibit optic nerve-induced field potentials in the SCN brain slice preparation, light-induced Fos expression and phase shifts of the circadian rhythm of wheel-running activity (78). Interestingly, 5-HT antagonists have been reported to enhance light-induced increases in the firing rates of SCN neurons (105) and light-induced phase shifts (77). These results raise the possibility that 5-HT may be involved in a tonic inhibition of the light-input pathway to the SCN. In addition, these studies are all consistent with the hypothesis that the serotonergic innervation of the SCN serves to modulate light-regulated glutaminergic input. Understanding this pathway is likely to be important in understanding the links between disruptions in circadian function and affective disorders (see later discussion). It has long been suggested that ACh plays a role in the light-input pathway. Fibers immunoreactive for choline acetyltransferase innervate the SCN (99), apparently from the cholinergic regions of the basal forebrain and brain stem (5). Furthermore, electrophysiological studies indicate that some SCN neurons are excited by cholinergic agents. In addition, administration of the ACh receptor agonist carbachol caused large phase shifts in SCN neuronal activity rhythms this response is mediated by muscarinic receptors perhaps of the M1 subtype (50). The intraventricular administration of carbachol caused phases shifts in vivo which can be blocked by GluR antagonists (12). This result raises the possibility that some of the behavioral effects of carbachol may be due to stimulation of glutamate release. So, while ACh does not appear to be a transmitter directly in the light input pathway, it may act to modulate photic information reaching the SCN. The SCN receives a prominent histaminergic innervation from the tuberomammillary nucleus. Based on anatomical considerations, the histaminergic transmitter system may represent a regulatory center capable of altering arousal throughout the nervous system. Depending on the subtype of receptor activated, HA can have excitatory (H1) or inhibitory (H2) actions on SCN neurons (49). Administration of HA can cause phase shifts of the in vitro neural activity rhythm and the in vivo locomotor activity rhythm (15). These results suggest that HA may be involved in modulation of light input to the circadian system but, at present, the functional significance of this regulation is unknown. The mammalian pineal gland secretes melatonin rhythmically under the neural control of the SCN (see discussion below). The SCN is also a target of this hormonal output, as the SCN has a high density of melatonin receptors (102). One consequence of activation of these receptors is the inhibition of neural activity, perhaps through the activation of potassium currents in SCN cells (40). Melatonin also caused phase shifts of the circadian rhythm of neuronal activity of SCN neurons in vitro (51). Behaviorally, administration of melatonin caused phase shifts of the locomotor activity rhythm during the day and may modulate the effects of light during the night (7). OUTPUT FROM THE SCN: HOW DOES THE OSCILLATOR IN THE SCN REGULATE OTHER PHYSIOLOGICAL AND BEHAVIORAL SYSTEMS Most of an organisms physiological and behavioral parameters show a daily rhythm. In many cases, these rhythms are driven from a circadian oscillator located in the SCN. Physiological or behavioral parameters which exhibit daily rhythmicity due to the activity of cells in the SCN are known as outputs of the circadian system. Metaphorically, these outputs are sometimes referred to as hands of the clock to distinguish them from the mechanisms responsible for the generation of the rhythms. It is not clear if this intellectual distinction will hold up experimentally. In general, outputs of the circadian system are rhythmic but not temperature compensated. Theoretically, if an output is held constant, this should not alter other rhythms driven from the SCN. A major problem in circadian rhythms research is to understand the mechanisms by which the circadian oscillator located in the SCN regulates such a wide range of physiological outputs. There is evidence for two types of signals originating from the SCN and conveying phase information: hormonal and neural outputs. Evidence for hormonal or some other diffusible signals comes from transplantation experiments (76). These studies have shown that rhythmicity can be restored to SCN-lesioned animals following implantation into the third ventricle of tissue containing the SCN. As previously discussed, these experiments provided compelling evidence that the SCN is a circadian oscillator in mammals. These studies also provide an important tool to look at the mechanisms by which SCN output alters other physiological systems. Several pieces of evidence suggest that a hormonaldiffusible factor produced by the SCN is an important output signal for the circadian system (91). First, some behavioral rhythms recover within four days after transplantation of the SCN, before much axonal outgrowth from the transplant is noted (45). In addition, some successful transplants can be placed in locations distant from the SCNhypothalamus (45). Third, transplantation of disassociated SCN cells can restore rhythmicity (90). The interpretational problem common to all of these studies is the difficulty in ruling out all neural outgrowth. Resolution of this problem may come from studies which place SCN grafts into a polymer capsule or into the anterior chamber of the eye such conditions allow humoral communication with the brain but prevent neurite outgrowth (76). Of course, interpretation of negative results from these type of experiments would be extremely difficult. One of the best studied examples of hormonal output from the SCN is the rhythmic secretion of the peptide VA. Peripherally, this peptide is secreted by the pituitary and functions as an antidiuretic hormone. Centrally, VA also acts as a signaling molecule and is highly expressed in a population of cells in the SCN. These cells drive a prominent daily rhythm in the levels of VA in the cerebrospinal fluid in mammals. If the SCN are lesioned, then VA levels are dramatically reduced and no rhythmicity can be detected (81). Furthermore, SCN cells express a circadian rhythm in VA mRNA in vivo (96) and SCN cells in vitro secrete VA rhythmically (23). Finally, if embryonic SCN tissue is transplanted from a normal rat into a VA-deficient Brattleboro rat, the rhythm in VA is restored (24). Since host animals can not make VA, the source must be donor tissue containing the SCN. The function of this rhythm in VA is currently unknown and is an interesting area for future work. Anatomical knowledge of the output pathways of the SCN has been reviewed by Watts (101). In short, axonal projections from SCN neurons terminate within the SCN, other hypothalamic regions, and a few regions outside the hypothalamus. The largest projection from the SCN goes to the subparaventricular region of the hypothalamus, a region with widespread connections throughout the limbic system. Most SCN neurons contain GABA ( 18. 63, 99 ), suggesting that the output is generally inhibitory. There is evidence that these neuronal pathways are functional and are involved in the communication of signals from the SCN to other parts of the nervous system. First, the local injection of TTX into the SCN region blocks the expression of a rhythm in drinking activity (86). Second, knife cuts around the SCN which cut efferent fibers block the expression of several rhythms (37, 92). Third, some transplants appear to be healthy, are in an appropriate position, and have cells expressing normal peptides, yet they do not restore rhythmicity. These data are all consistent with the idea that neural connections play an important role in driving overt rhythms. The interpretation problem common to all of these studies is that the experimental treatments might interrupt hormonal as well as neural output from the SCN. Among the rhythms under neural control of the SCN is the circadian synthesis and secretion of the pineal hormone melatonin. In mammals, the pineal gland is not directly light sensitive but is photically regulated through a complicated neuronal pathway involving the SCN. Melatonin is the hormone secreted by the pineal gland, and the SCN drives a daily rhythm in its secretion. Control relies on a multisynaptic pathway via the sympathetic nervous system to maintain and entrain the rhythmic synthesis and secretion of this hormone ( Fig. 7 ref. 60). The neural pathway from the SCN to the pineal passes first to the paraventricular nuclei (PVN). Ablation of either the SCN or PVN results in loss of the rhythm in pineal melatonin levels. Most SCN neurons contain GABA, an inhibitory neurotransmitter, and it is most likely that the excitation of SCN neurons inhibits neurons in the PVN. The PVN neurons project to the spinal cord and make synaptic connections with preganglionic cell bodies which, in turn, innervate the superior cervical ganglia of the sympathetic nervous system. Stimulation of the PVN increases sympathetic outflow through activation of cholinergic preganglionic sympathetic neurons. Sympathetic neurons release norepinephrine which drives a rhythm in pineal melatonin by increasing N-acetyltransferase activity. Disruption of the pathway from the SCN to the pineal gland at any level (destruction of the SCN itself, knife cuts of SCN afferents, or pharmacologic blockade of the sympathetic innervation) interrupts the circadian pattern in the synthesis and secretion of the hormone (60). SCN transplants do not appear to restore the melatonin-mediated photoperiodic response in hamsters (45). Clearly, this output of the circadian system is under neural control. There is evidence for both neuronal and hormonal outputs from the SCN. Signals could vary with the specific physiological system being regulated. For example, rhythmic secretion of melatonin could be under neural control while locomotor activity is under hormonal control. Alternatively, the signals could be redundant, with a specific physiological system receiving both neural and hormonal signals from the SCN. The issue of how cells in the SCN regulate other physiological systems is clearly an important area and requires further study. MEDICAL IMPLICATIONS OF THE CIRCADIAN SYSTEM Although the experimental studies described in this review were mostly performed in rodents, the general principles developed by this research are likely to apply to humans. Humans, like other organisms, exhibit daily rhythms in many physiological and behavioral parameters (11, 46, 55). Because of experimental difficulties in isolating humans from environmental influences, in many cases, it is not yet clear whether these rhythms are really circadian or instead are diurnal, i. e. driven by external cues. Nevertheless, it is clear that humans have an endogenous circadian timing system including the SCN and RHT. There is every reason to think that the SCN functions as a circadian oscillator in humans. First, humans suffering from dementia have problems with the timing of their sleepwake cycle this is correlated with loss of neurons in the SCN (56). Furthermore, patients with tumors or other types of damage to the hypothalamic area, including the SCN, exhibit disruptions in their daily rhythms (10, 85). Since the human SCN expresses many of the same neurochemical markers described in rodents (59), it seems likely that many of the neural pathways described in rodents will also be relevant for humans. In recent years, it has become clear that light is an important environmental signal for the synchronization of the human circadian system (16). This has led several groups to investigate the use of light for therapeutic manipulations of the human circadian system (93). With these observations also comes the recognition that, within the last 100 years, dramatic changes have occurred in the temporal environment to which humans are exposed. With the widespread use of artificial lights and airplanes, many people experience rapid changes in their light-dark cycle. These changes can disrupt our endogenous timing system. Let us next briefly consider a few aspects of the human circadian system: desynchronization caused by jet travel or shift work circadian variation in the effects of pharmacological agents the possible use of melatonin to manipulate the human circadian system and, finally, the possible link between the circadian system and affective disorders. While there are many other interesting aspects of the human circadian system, these topics at least provide an introduction as to how the circadian system impacts human biology. Desynchronization of the Circadian System In our modern world, increasing numbers of people move rapidly across time zones or work during the night. The result is a group of symptoms collectively known as jet lag. While there is a lot of variation in individual symptoms, many people experience disruption of sleep, gastrointestinal disturbances, decreased vigilance and attention span, and lack of energy. While most people have no difficulties tolerating an occasional case of jet lag, repeated shifts create greater problems. One recent report even suggested that jet lag of players may be a factor affecting the outcome of baseball games (79). More seriously, consider people whose jobs require constant changes of schedule (e. g. health care professionals, pilots, and other shift workers). While it is difficult to link shift work directly to demonstrable physical illnesses, commonly reported health consequences include stomach diseases, sleep disturbances, and fatigue (55, 93). Besides physical problems, these workers are unlikely to be performing optimally. Humans undergo daily oscillations in many cognitive and motor functions. Human performance is normally at a minimum between 3 and 5 AM (11, 55). Persons working during these hours are likely to be sleepy, inefficient, and accident-prone. Many factors contribute to jet lag, including fatigue and stress, which may be independent of the circadian system. But other symptoms are undoubtedly a direct result of the desynchronization of the circadian system. In general, circadian systems can be thought of as serving at least two different kinds of functions. One is to ensure that an organism is synchronized to the physical world. Another, less appreciated, function is to ensure that the various physiological systems inside the organism remain synchronized. Both of these functions are compromised under conditions of rapid travel between time zones or changes in the scheduling of work. For example, consider a person traveling from North America to Europe, which involves a shift of at least 8 time zones and may require up to a week for the body to resynchronize. In the meantime, the travelers performance minimum (3:00 to 5:00 AM, old time) will now be occurring at 11:00-13:00. In order to maximize synchronization to a new time zone or schedule, the best strategy appears to be to maximize exposure to entraining signalsespecially light and social cues. So, the best advice to speed adjustment to a new schedule is to be immediately active in the new daytime and to sleep during the new night, eat meals at local times, and spend the day out in well lit environs. But even after exposure to all of these new environmental signals, it will still take a few days to readjust, so it may just be best to simply recognize this and to allow some time for adjustment after shifting to a new schedule or time zone. Circadian Variation in Drug Effects Most physiological and behavioral parameters exhibit daily rhythms, so it is not surprising that drug effects both desired and undesired (i. e. toxicity) vary with the time of day. In one early observation of this phenomenon, the mortality of mice after an injection of E. coli endotoxin was 80 during the middle of their inactivitysleep time but below 20 in the middle of their activity time (31). This is not an isolated or unusual result, and the effects of many drugs are now known to vary depending on the time of day (46). This daily variation is not due to some mysterious process but rather can be mostly explained by two observations. First, temporal variation has been documented in the rates of absorption, metabolism, and excretion. These factors will all impact the concentration of drug that actually reaches the intended target. Second, many tissues and cells show temporal variations in their response to the drugs which do reach them. These observations form the basis of the hope that clinical outcomes can be improved by scheduling drug treatments for certain times of day. Daily variation in the time of symptom onset may be common in a number of diseases and medical emergencies. One extensively studied example is asthma, in which the majority of patients experience symptoms mainly at night (70). These patients have a daily rhythm of bronchial constriction with the greatest constriction seen between midnight and 8:00 A. M. Accordingly, this is the time when most respiratory failures occur. Understanding this type of rhythm can, at the very least, lead to improved monitoring at certain times of day as a preventive measure. In addition, drugs used for treatment of asthma are apparently more effective when given before bed than during other times of day (46). So, in this case, the normal pharmacologic goal of keeping drug concentrations constant through time appears to be less effective than allowing drug concentrations to fluctuate. Even in diseases in which the symptoms are not so obviously temporally patterned, there is still evidence for diurnal variation in drug effects. For example, most of the drugs used in chemotherapy are toxic to both host and malignant cells, so there is an unusually narrow window between therapeutic and toxic effects. There are good reasons to think that the timing of drug administration may be an important therapeutic variable. Data suggest that both the toxicity and therapeutic benefits of anticancer drugs vary with the daily cycle (27). If these rhythmic variations are out of phase with each other, then time of treatment may represent a variable which can be exploited to maximize the benefitcost ratio for the use of anticancer drugs. In one early study, mice were injected with leukemia cells and treated with DNA synthesis inhibitors (cyclophosphamide and 1-B-D-arabinofuranosylcytosine) at different times of day (83). Without treatment, most of the mice died during the 75-day trial. The number of animals that survived the tumor inoculation varied depending on the timing of cyclophosphamide administration. Treatment in the beginning of the inactivitysleep period led to a 40 survival rate the same treatment given at the beginning of the animals active period led to over 90 survival. Of course, there are many possible reasons that might account for these data, and the question of whether such scheduling strategies can actually improve clinical outcome for humans is one that still needs to be answered. But, it seems likely that varying drug doses over the course of the day could lead to much more effective treatment strategies. In humans, as in other mammals, melatonin is secreted by the pineal gland during the night but not during the day. This rhythm is due to both circadian regulation and acute light-induced suppression of melatonin secretion. Both mechanisms ensure that the secretion of melatonin fairly accurately follows the night. Thus, melatonin is referred to as a dark hormone. In many temperate-zone mammals, this nightly dark signal is intimately involved in the control of seasonal changes in physiology and behavior. Although there is little direct evidence that melatonin mediates seasonal changes in humans, the onset of melatonin secretion at night does appear to be a good marker for the phase of the human circadian system (47). The human SCN contains melatonin binding sites, and administration of melatonin (0.5 mg), by itself, caused phase shifts of the human circadian system: phase delays during late night and phase advances during the morning (47). In addition, melatonin can act as a sleep-inducing agent. In one study, a group of healthy adults were administered melatonin (1-10 mg) in the middle of the day. Melatonin induced sleep, lowered body temperature, and caused feelings of sleepiness and fatigue (21). On the basis of these results, it was proposed that melatonin might be used to help workers or travelers adjust to new schedules. There is some evidence that melatonin may be therapeutically useful in this regard. Several studies have reported that timed melatonin administration can help with re-adjusting the circadian system after jet-lag or shift work (17, 47 ). Moreover, in some insomnia patients, administration of melatonin improved the sleep quality or the phasing of sleep (29). Overall, our understanding of melatonins effects on human biology is still in its infancy. Much more work will have to be done before the possible therapeutic value of melatonin can be determined. Several observations suggest a link between disruptions in circadian function and affective disorders (34). Certainly, many patients suffering from depression exhibit disruptions in the timing of sleepwake cycles, cortisol levels, melatonin secretion and body temperature. Similarly, many treatments that have antidepressant effects (e. g. timed photic stimulation, sleep-deprivation, serotonin reuptake inhibitors) also alter circadian rhythms. Dysfunction in serotonergic pathways has been suggested to play a role in affective disorders, which are frequently treated with agents that alter serotonergic neurotransmission. The serotonergic projection from the raphe to the SCN (see previous discussion) may very well be the anatomical substrate by which affective disorders alter the human circadian system. Of course, it has been extremely difficult to establish more then a correlative link. Perhaps the best evidence comes from studies of seasonal affective disorder (SAD). SAD is characterized by recurrent cycles of fall-winter depression and spring-summer remission (82). The seasonal nature of the symptoms immediately suggests a role for the circadian system which, in many mammals, plays a central role in mediating seasonal changes in behavior and physiology. Furthermore, SAD has been successfully treated by timed exposure to bright light (48, 93). Overall, the exact role of the circadian system in affective disorders is still open to debate and remains an important area for future research. The finding that humans and other organisms have endogenous circadian timing systems raises a number of issues. What are the mechanisms by which light and other environmental stimuli synchronize these systems What are the mechanisms by which cells generate these oscillations How are these cells and cell populations integrated to form a coherent timing system How are these oscillators coupled to the various outputs they control What are the consequences for human biology, as well as the natural history of other organisms In this review, I have briefly described some of the current work in each of these areas. Some of the knowledge gained from addressing these issues may very well be unique to this physiological system. For example, the biochemical processes involved in the generation and temperature compensation of rhythms with a 24-hour time base are likely to be novel. Other questions involving sensory input to the circadian system, coupling between oscillators, and output from the circadian system are all basic issues of communication central to neuroscience research. In closing, I would like to argue that some features of the circadian system make it an excellent model system to address many core issues in the neurosciences. Many of the behavioral and physiological outputs of the circadian system are precise, quantifiable, and functionally important. This allows the productive use of both neuropharmacologic and genetic approaches. Anatomically discrete and well defined pathways control these behaviors. Finally, SCN neurons are amenable to detailed cellular and molecular analysis by all of the tools of modern neuroscience. For these reasons, I believe that the circadian system will be one of the first mammalian behavioral control systems to be understood at a variety of levelsfrom behavioral to molecular. I would like to thank Drs. E. Nansen and N. Wayne for reading an early draft of this chapter. Synchronized Circadian Rhythms: Natures Cancer Fighter Timing is everything. Time is of the essence. Perfect timing. Humanitys captivation by temporal reality goes much deeper than the unifying framework of schedules, calendars, rituals and histories it goes straight to our DNA. We live in a universe of cycles, natures clockwork, shaping and directing us since the literal dawn of time. All life on earth is governed by a multitude of oscillating rhythms a perpetual ebb and flow of energy and movement. The rhythms of day and night, the seasons and celestial motions, the rhythms of nature and our societies, all of these cyclical movements affect us at every layer of our being. We have evolved in synchronicity with our rhythmic environment, anticipating and adapting to our surroundings for optimization of our internal and external resources. Our bodies follow innate biological clocks governing cellular, metabolic and developmental processes which fluctuate minute to minute, hour to hour, day to day, following 24-hour cycles, 2-day cycles, weekly cycles, monthly lunar cycles, seasonal cycles annual cycles, 7,8,12, and 60 year cycles, life cycles, and more. The Harmony of Traditional Chinese Medicine Traditional Chinese Medicine (TCM) charts two hour daily energy cycles for each organ weekly, monthly, yearly, and 7 and 8 year female and male developmental cycles 12 and 60 years astrological cycles, along with other calendric patterns as they relate to our health and development. Based on this ebb and flow of energetic activity, the most appropriate medical treatments can be administered at specific optimal times in order to achieve maximum therapeutic effects. Much of TCM approach focuses on promoting health on all levels by synchronizing a patient with the rhythms of time, nature and the seasons. From a psycho-spiritual perspective, becoming familiar and in tune with these cycles serves as preparation for a harmonious transition of our most important cycle, the cycle of birth, life and death. Chronotherapy: Fine tuning your health journey Western medicine has also long theorized about our internal biological clocks, known today as circadian oscillators or circadian clocks, and the physiological, metabolic and behavioral processes they rule over which are known as circadian rhythms. Modern analysis of our circadian rhythms has given birth to a new therapeutic modality known as chronotherapy, which, whether influenced by it or not, corresponds to much of TCMs daily internal energy cycles. Emphasizing the most beneficial times of day to take certain medications such as statins and chemotherapy drugs, receive specific medical treatments such as radiation therapy, and perform various functions. modern chronotherapy can be likened to a Western equivalent of TCMs ancient chronobiology protocols. Still in its early stages, Chronotherapy is gaining a reputation in conventional oncology, as new understandings in environmental influences on gene expression unravel some of the mysteries of cellular cycles. In general, circadian rhythms relate to the 24 hour solar cycle and can be found in plants, animals, and even fungi and bacteria. Body temperature, blood pressure, muscular strength, levels of circulating hormones, neurotransmitters, and numerous other metabolic compounds and physiological processes follow the tides of circadian rhythm. Diseases have their own rhythms, too. For example, some breast cancers have been found to grow faster during the day than at night. Circadian rhythms and your health Accumulating evidence demonstrates that disruption of circadian rhythms is linked to the development of cancer, and in particular breast cancer. The proper synchronization of biological rhythms is crucial for healthy cell cycles, DNA damage responses, and tumor suppression, among a multitude of other functions, many of which are still not completely understood. However, scientists have discovered specific genes that control our circadian clocks, and several molecular components of the clock machinery have been found to interact closely with regulators of the cell cycle. Thus, disruptions in circadian clock function can contribute to abnormal cellular metabolism and the proliferation of cancerous cells. A disrupted circadian clock has been called a Group 2A carcinogen by scientists, placing it in the same carcinogenic class as lead compounds and diesel exhaust fumes. Recently, there have been studies linking shift work working during the night hours to breast and other hormone-related cancers. Along with frequent jet lag and other factors such as alcohol excess, shift work is a major disruptor of circadian rhythms, mostly due to the excessive exposure to bright light during normal sleeping hours. Of all external cues affecting our circadian rhythms, light is the single most powerful influence. Prolonged exposure to bright light at night disrupts melatonin production, a serious affront to our health. Melatonin is perhaps the most important endogenous component in the regulation of our circadian clocks and circadian rhythmic processes. Its production is highest during the dark hours of the night when we can sleep in total darkness. Melatonin helps reset your circadian clock As a supplement, melatonin plays in important role in cancer treatment and prevention, especially with breast cancer. In addition to having powerful immune modulating action, Melatonin has been shown to reduce estrogen receptor cells on ER tumors, exhibit cytotoxic (tumor killing) activity, promote antioxidant activity protecting DNA from oxidative stress, protect against side effects of chemotherapy and radiation, and assist healing after surgery. Melatonin is considered a master hormone, influencing and regulating other hormones along with a multitude of biological processes, and it also exhibits neurotransmitter activity, making it a very powerful molecule. People with diminished levels of melatonin are at a much higher risk for developing cancer, and in particular, hormone related cancers. Supplementing with melatonin should be done with guidance and supervision from your health care provider. Testing melatonin levels prior to supplementation will provide an accurate base line for appropriate treatment. Cancer patients can take up to 20 mg before bed time, but for most people, the dose is 0.5-3 mg nightly. Learn about other cancer-fighting supplements by downloading a complimentary wellness guide here. Other tips to rebalance your rhythms Along with melatonin supplementation, there are a number of other ways to reset your circadian clocks. Following extremely consistent sleeping, eating and exercise habits at the appropriate times of day can greatly synchronize your innate rhythms, benefitting your health far beyond many other methods. Lastly, but perhaps most importantly, Traditional Chinese Medicine recommends very specific practices and therapies developed over millennia, which help to harmonize people with the ebb and flow of the rhythms of nature, the seasons, and the movements of celestial bodies. In ancient times, these natural cycles would serve to effectively set and regulate our internal rhythm keepers. Ideally, we should not require elaborate scientific mechanisms to synchronize ourselves with nature. It is only now, engulfed in a world that in many ways functions counter-intuitively to our innate biology, that we have learned what it really means to be out of sync. If we want to survive and thrive today, we need to take persistent measures to harmonize our internal and external symphonies. Dr. Isaac Eliaz, M. D. MS. L. Ac. has been a pioneer in preventive medicine since the early 1980s, and serves as a respected researcher, clinical practitioner, author and lecturer. Dr. Eliaz integrates his background in Western medicine with extensive knowledge of traditional Chinese, Tibetan, Ayervedic, Homeopathic and complementary medical systems. Dr. Eliaz is also the Director of the Amitabha Medical Clinic amp Healing Center (amitabhaclinic ) in Sebastopol, Calif. where he and his team of integrative practitioners treat cancer and other chronically ill patients using a wide range of cutting edge and traditional therapies drawn from diverse medical systems. For more information about his work, visit DrEliaz. org. hot on elephant Elephant8217s Valentine8217s Day Gift Guide. 25 shares Share A letter to the Anger that refuses to Leave Me. 1,795 share Share Jupiter Retrograde in Libra: The Fairytale does Exist, so Fight for It. 7,396 shares Share The Local Business I cannot Support. 1,745 share Share Twin Flame Forecast for February: Its Time to Reunite. 1,868 share Share Moon in Taurus: Cosmic Couple Mars 038 Venus causing Explosions. 1,600 share Share February 2017 Forecast: The Month for Deep, Unshakeable Love. 1,590 share Share The Year of the Rooster: Finding our Best Romantic Match. 777 shares Share You are Everything I Never knew I Always Wanted. 1,101 share Share Dear Body, I am Sorry. 442 shares ShareMost organisms, including humans, exhibit daily physiological and behavioral rhythms. Nearly all body functions show significant daily variations including activity, arousal, psychophysical performance, consumption of food and water, hepatic metabolism, urine volume and pH, blood pressure, heart rate, acid secretion in the gastrointestinal tract, and cortisol secretion (11, 46, 55). It is more surprising to find a variable that is static through time than one that shows a daily rhythm. This temporal variation obviously plays an important role in the bodys homeostatic mechanisms and has a significant impact on the function of specific physiological systems, including the nervous system. In many cases, these rhythms are generated by endogenous processes referred to as circadian oscillators which presumably function to synchronize biological processes with temporal changes in the environmental and to allow the temporal coordination of physiological systems. The term circadian comes from Latin roots: circa (about) and dies (day). A circadian rhythm is an endogenous rhythm that repeats with a period of about 24 hours. Because these rhythms are generated from inside an organism, a fundamental feature of circadian oscillations is that they will persist when isolated from environmental time cues. However, because the period of these rhythms is not equal to exactly 24 hours, the oscillations will drift with respect to any 24-hour-based time and are referred to as free-running. The free-running period of an organism is under genetic control and will persist for generations in the absence of environmental time cues. One manifestation of this is that mutations can be isolated which alter the rhythm period. In Drosophila, investigators have successfully used a molecular genetic approach to explore several aspects of the fly circadian system (32, 80). Two genes have been identified: period and timeless . which are integral to the generation of circadian rhythms in Drosophila . For example, mutations in the period gene produce phenotypes which are arrhythmic or express altered periods. Recently, two single gene mutations that alter period have also been isolated in mammals (74, 100). Hopefully, in the future, such mutations can be used to explore the genetic basis of mammalian circadian systems. In order to function adaptively as time-keeping systems, circadian oscillators need to be precise and stable in the face of environmental perturbations. In general, they appear to satisfy these conditions. For example, in the mouse ( Mus musculus ), the circadian oscillator that drives wheel-running activity exhibits a standard deviation of about 0.6, or just under nine minutes per 24-hour cycle (72). Changes in temperature must be one of the major challenges to this homeostasis of period, as the rates of most biochemical processes change 2- to 3-fold for every 1000C change in temperature (Q 10 23). Since even homeothermic mammals undergo daily oscillations in body temperature in the range of 20C, a circadian oscillator with a Q 10 23 could exhibit transient period changes of several hours. This is obviously unacceptable for a timing system, and the period of circadian oscillators typically shows a Q 10 of close to 1. This temperature compensation of period appears to be a universal feature of circadian systems, although little is known about the mechanisms responsible. This is just one example of the types of homeostatic mechanisms which had to evolve in order to enable biological oscillators to keep accurate time. Nevertheless, the period of a circadian oscillator is not a constant and can be modulated by both external and internal influences, such as changes in photic environment, hormonal milieu, and even the age of an organism. In fact, some of these changes are essential if the oscillator is to be synchronized to the environment. Circadian oscillators generate a rhythm that repeats with a frequency of close to but not equal to 24 hours. In order to function adaptively, these oscillators must be synchronized to the exact 24-hour cycle of the physical world. After all, there is limited utility in having a timing system which cannot be reset to local time. This process of synchronization is referred to as entrainment. Empirically, the phase ( F ) of the oscillator must be adjusted so that the endogenous period of the oscillator (tau, t ) equals the 24-hour period (T) of the physical world. Mathematically, this can be expressed at t - T DF. So for a primate with a t of 25 hours to entrain to a T of 24 hours, the oscillator needs to be phase advanced by 1 hour per day. The formal properties of entrainment and circadian oscillators in general have been the subject of elegant analysis by Pittendrigh and colleagues (71). The daily cycle of light and dark, acting through light-induced phase advances and delays of the endogenous rhythm, is the dominant cue used by organisms, including humans, to synchronize their circadian oscillators to the environment. This is probably because dawn and dusk are the most reliable and noise-free indicators of time available to organisms in most habitats. But organisms have the ability to use many other cues for entrainment, including social factors, temperature cycles, food availability. A major goal of circadian rhythms research is to understand the mechanisms by which light and other cues act to synchronize circadian oscillators. In short, circadian rhythms show several fundamental features: 1) they are endogenously generated oscillations that repeat with a frequency of close to 24 hours, 2) they exhibit homeostasis of period, including temperature compensation, 3) they are synchronized by periodic environmental signals, with the daily light-dark cycle being the dominant cue used. Obviously, not all daily rhythms are circadian, and finding a daynight difference in a physiological or behavioral parameter is not sufficient to claim that the parameter shows a circadian rhythm. Daily rhythms that are driven exogenously will disappear when an organism is placed in constant conditions and the exogenous daily variable is removed. In contrast, a circadian rhythm will continue with a near 24-hour period for at least several cycles in constant external conditions. The phase of a circadian rhythm can also be synchronized to the phase of the light-dark cycle to which it is exposed. Mammals have evolved a set of anatomically discrete cell populations that function as a physiological system to provide temporal organization on a circadian time scale. These structures are commonly referred to as a circadian system ( Fig. 1 ). In the simplest case, a circadian system can be modeled as having three components: 1) an oscillator or clock responsible for the generation of the daily rhythm, 2) input pathways by which the environment and other components of the nervous system provide information to the oscillator, and 3) output pathways by which the oscillator provides temporal information to a wide range of physiological and behavioral control centers. Although the presence of feedback loops between these components may make these separations somewhat arbitrary, they nevertheless represent a good starting point for discussion. In the rest of this chapter, we will consider each of these components in turn, as well as to briefly discuss some of the medical implications of humans having a circadian timing system. Given the aim of this book, the discussion will focus on mammals and neurochemical mechanisms. Given space restrictions, many excellent and relevant references could not be included. THE MAMMALIAN CIRCADIAN OSCILLATOR: THE SUPRACHIASMATIC NUCLEUS (SCN) Localization of an Oscillator: The SCN One of the major goals of neuroscience research is to identify the anatomical substrates of specific behavioral control systems. This has proven frustratingly difficult in many cases. One of the success stories in this area has been the localization of most of circadian timing function to a bilaterally paired nucleus in the mammalian hypothalamusthe suprachiasmatic nucleus or SCN ( Figure 2 ). For this reason, it is worth describing in some detail the evidence that this heterogenous population of cells is the locus of the mammalian biological clock. Initial studies utilized lesions to address the hypothesis that discrete parts of the nervous system can function as a biological clock in mammals. From these studies, researchers concluded that a circadian oscillator was located somewhere in the ventral hypothalamus. Not much more progress was made until the early 1970s, when new neuroanatomical tools were applied to the problem. Since lesion studies suggested that a circadian oscillator is located in the hypothalamus and light information from the retina must reach this oscillator, a logical approach was to look for anatomical pathways by which light information could be carried from the retina to the hypothalamus. This strategy lead to the demonstration of a direct retinohypothalamic tract (RHT). The hypothalamic site in which these fibers terminated was the SCN ablation of the SCN was quickly shown to abolish circadian rhythms (61, 92). Of course, among other interpretational problems common to lesion studies, there was the possibility that the SCN lesions could just be blocking the expression but not the generation of the rhythmic behavior. Nevertheless, these studies were the first to implicate the SCN as a circadian oscillator. If the SCN was the site of a circadian oscillator, then these structures should show oscillations in vivo . One of the first pieces of evidence of endogenous rhythmicity came from studies looking at cellular metabolism with 2-deoxy-D-glucose (2DG, 84). The cells of the SCN showed prominent circadian variation in their metabolic activity glucose utilization peaked during the day, when nocturnal rodents were at rest, and fell to a minimum during the night, when the animals were active. One reason for this metabolic rhythm may be that SCN cells also show a similar rhythm in spontaneous neural activity. But in vivo this rhythm is not restricted to the SCN and can be recorded in other regions of the central nervous system. In order to clarify the role of the SCN in the generation of these rhythms, Inouye and Kawamura (37) made a series of knife cuts around the SCN in order to isolate these structures from neural but not hormonal communication. When this was done, the rhythm in electrical activity continued inside the island containing the SCN but not in other regions of the nervous system. So, the SCN does indeed appear to be capable of generating circadian oscillations in an organism. SCN tissue is also capable of generating circadian oscillations in vitro when isolated from the rest of the organism. Initial studies showed that the circadian rhythm in neural activity could also be recorded from an SCN brain slice preparation (30). Since that time, many studies have made use of this rhythmic neural activity to explore SCN function in vitro . SCN tissue also secretes the peptide vasopressin rhythmically (23). Currently, these in vitro preparations are being utilized to explore basic questions about how circadian oscillations are generated. The results of these experiments show that the SCN is an oscillator and that, if the SCN are destroyed or functionally removed from an organism, many of the behavioral rhythms disappear. Ideally, one would want to show that if SCN tissue is restored, the rhythms would reappear. Due to the advances in brain transplantation techniques, this experiment is now feasible. It is now possible to restore rhythms in animals made arrhythmic due to SCN lesions by transplanting the SCN of another animal (45, 76). Furthermore, it is possible to establish that the rhythm is being generated by the transplanted SCN tissue and not some trophic interaction with the host tissue. For these experiments, a mutant hamster was used which shows a circadian rhythm with a period much shorter than normalthe so-called tau mutant (74). When SCN tissue from tau mutant animals was transplanted into SCN-lesioned wild-type animals, locomotor activity rhythms having the shorter period of the donor tissue were restored with. Conversely, if SCN tissue from wild-type hamsters was transplanted into SCN-lesioned tau mutant animals, the restored rhythms exhibited the normal period of the donor animals (76). These experiments provided compelling evidence that the SCN was the circadian oscillator in rodents. Is circadian function localized exclusively to the SCN, or do other regions also have this capacity A few studies raised the possibility of other circadian oscillators. For example, food-entrainable and methamphetamine-induced circadian oscillations can be observed in SCN-lesioned animals (57). In many ways, the presence of multiple circadian oscillators would not be surprising. In most vertebrate species, endogenous circadian oscillators have been localized to three structures: the SCN, the pineal gland and the retina. Until recently, mammals seemed to be an exception to this rule, having such a major component of circadian function localized in the SCN. But recent work by Tosini and Menaker (94) provided a particularly clear demonstration that circadian oscillators exist outside of the SCN region even in mammals. In this study, a hamsters retina was cultured at room temperature and the secretion of melatonin was measured. Non-mammalian retinas exhibit daily rhythms in many variables, including melatonin secretion, so it was not unreasonable to look for rhythms in melatonin production. Indeed, the hamster retina produced melatonin rhythmically, with high levels at night and low levels during the day. A light-dark cycle can synchronize this rhythm in culture, so the excised retina even has an intact light-input pathway. These observations proved that another circadian oscillator exists independently of the SCN and introduces the retina as another model system in which to explore circadian function in mammals. Are Circadian Oscillations a Network or Intracellular Property The SCN is a dense accumulation of small neurons lying dorsal to the optic chiasm and lateral to the third ventricle ( Fig. 3 ). As previously described, these cells exhibit circadian rhythms in electrical activity, glucose utilization, and secretion. One of the first questions which needs to be addressed is whether these cells generate the rhythm through an intracellular or a network process. The answer is not yet known, but at least three lines of evidence suggest that synaptic interactions are not important for the generation of the daily rhythm. First, local injection of tetrodotoxin (TTX) into the SCN region in vivo blocked expression and photic regulation of a circadian rhythm in drinking behavior (86). TTX blocks voltage-sensitive sodium channels and synaptic communication in neural tissue. However, when these treatments ended, the rhythms continued from a phase which suggested that the SCN oscillator was undisturbed by the experimental manipulation. Similar results were obtained with in vitro SCN preparations: the expression of circadian rhythms of secretion and spontaneous neural activity were blocked by TTX, but the oscillator itself appeared to be unaffected (22, 103). In addition, the cells of the SCN generate rhythms in glucose utilization early in the development of the nucleus (84), prior to the formation of the majority of synapses. Finally, disassociated SCN cells in culture retain the ability to generate circadian oscillations (68, 90, 103). Under these condition, single SCN cells showed circadian oscillations in spontaneous neural activity which drift out of phase with one another as if each cell contains an independent oscillator (103). The simplest interpretation of this collective data is that synaptic interactions are important for the inputs to and outputs from the SCN oscillator but are not responsible for the generation of the circadian rhythm itself. However, resolution of this issue will have to await the demonstration that single, isolated SCN cells retain the ability to generate circadian oscillations. The mammalian circadian system did not evolve independently. Comparative evidence is also consistent with the possibility that generation of circadian oscillations is an intracellular rather than a network process. Certainly, single celled organisms (e. g. the dinoflagellate Gonyaulax polyhedra ) and Cyanobacteria exhibit circadian oscillations, so clearly these rhythms can be generated intracellularly. Further support comes from studies of the marine mollusk Bulla gouldiana . The eyes of this organism contain an oscillator that drives a circadian rhythm of spontaneous compound action potentials in the optic nerve. A population of electrically coupled cells known as basal retinal neurons (BRNs) is responsible for the generation of this rhythm through a daily cycle in their membrane potential. Isolated BRNs in culture continue to show a circadian rhythm in membrane conductance (54), thus demonstrating that single cells in culture retain the ability to generate a circadian oscillation. Regardless of whether individual cells in the SCN are competent circadian oscillators, it is obviously important to understand how these cells communicate and remain synchronized with each other. Current knowledge about cell-to-cell communications in the SCN is the subject of a recent review (98) and will only be briefly discussed here. Intrinsic Transmitters: GABA and Peptides Evidence suggests that the amino acid g - aminobutyric acid (GABA) is the major transmitter used by SCN neurons. GABA is found in at least half of all presynaptic terminals in the SCN (18), and GABA and its synthetic enzyme are found in most SCN cell bodies (63). Furthermore, electrophysiological studies have recorded spontaneous and electrically evoked inhibitory postsynaptic potentials in the SCN that are GABA-mediated (43). Administration of GABA agonists cause phase shifts of behavioral rhythms during the day and alter photic regulation of the circadian system during the night (75, 95). In adult tissue, GABA is typically an inhibitory transmitter, and most SCN neurons send projections to other cells in the SCN thus, the typical SCN neuron may best be thought of as an inhibitory interneuron. Ultrastructural studies support the subdivision of the rat SCN into three populations: a small rostral area, with caudal, dorsomedial, and ventrolateral subdivisions. Many of the cells in the SCN express peptides, and differences in peptide expression can serve as a basis to segregate SCN cells (58, 97). In particular, a distinction is commonly made between cells expressing vasopressin (VA), found in the rostral and dorsomedial regions, and those expressing vasoactive intestinal peptide (VIP), found in the ventrolateral regions of the SCN ( Fig. 4 ). Retinal and other afferent innervations are largely confined to the ventrolateral regions. So, it is in these cells that integration of the majority of synaptic inputs is most likely taking place. Some have speculated that the VIP-immunoreactive cells may play a critical role in processing photic input, while the VA-immunoreactive cells may be responsible for the generation of daily rhythms. However, circadian rhythms can be expressed in both the ventrolateral and dorsomedial populations of SCN neurons (38) and, at this point, the functional roles played by these different cell types are unclear. Moreover, the question of whether these anatomically defined subdivisions of cells are also electrophysiologically distinct remains to be answered. In addition to VA and VIP, a number of other peptides and growth factors are expressed in SCN neurons. These include gastrin releasing peptide (GRP), the peptide histidine isoleucine, somatostatin, substance P, neurotensin and nerve growth factor. In many cases, these peptides appear to be co-localized with amino acid transmitters and presumably function as signaling molecules. One peptide that has received some recent attention is GRP, which is expressed in neurons in the ventrolateral subdivision of the SCN. Application of GRP excited many SCN neurons in vitro and can cause phase shifts of the circadian system in vivo (69). But questions of how this or any other peptide functions in local SCN circuits and the role of peptides in circadian function have not yet been resolved. The role of peptides in the SCN has been recently reviewed (38). The SCN, with its clearly defined circadian function and behavioral outputs, is an excellent location to begin exploring the function of peptide signaling molecules. INPUT TO THE SCN: HOW DOES THE ENVIRONMENT REGULATE THE CIRCADIAN OSCILLATOR One approach to understanding circadian systems is to examine the neurochemical circuitry by which the SCN receives information from the environment. Besides addressing issues related to the sensory physiology of circadian systems, this approach could lead to the identification of components of the circadian oscillator. By systematically following the signal transduction cascade by which photic information reaches and regulates SCN neurons, it should be possible to identify mechanisms that generate circadian oscillations. One strategy that has been widely used to address these issues is a systems-level analysis of the effects of photic, pharmacologic, and genetic manipulations on rhythms driven by the circadian system. Another strategy is to examine the effects of such manipulations on the cellularmolecular activities of SCN neurons. Both strategies are being successfully used to explore SCN function (See recent reviews in refs. 38, 66, 95). Retinohypothalamic Tract (RHT) The anatomy of the light input pathway to the SCN has been recently reviewed ( Fig. 5 ref. 66). Briefly, in mammals, the effects of light on the SCN are mediated by unknown photoreceptors located in the retina. The primary pathway for transmission of photic information from the retina to the pacemaker for entrainment is the RHT. The RHT comprises a distinct subset of retinal ganglion cell axons that separate from the other optic axons at the optic chiasm to innervate the SCN. The photic information transmitted neurally to the SCN appears to be both necessary and sufficient for entrainment. There is evidence that the amino acid glutamate is a transmitter at the RHTSCN synaptic connection, and that this transmitter plays a critical role in mediating photic regulation of the circadian system (14). Anatomical studies report that identified RHT terminals innervating the SCN show glutamate immunoreactivity associated with synaptic vesicles (8, 19). A variety of glutamate receptors have been localized to the SCN by both in situ hybridization and immunocytochemistry (28). There is electrophysiological evidence that exogenous application of GluR agonists excite SCN neurons (6, 87), and that GluRs mediate the excitatory post-synaptic potentials recorded in the SCN (42). Application of GluR agonists causes phase shifts of a rhythm of neural activity recorded from the SCN in vitro (20, 89). Finally, GluR antagonists block light-induced phase shifts and Fos-induction in the SCN in vivo (2, 13). Despite this strong evidence that glutamate is a transmitter released by the RHT, there are many unanswered questions as to how the circadian oscillators in the SCN respond to this glutamatergic stimulation. In the simplest scenario, light causes the release of glutamate, which initiates a signal-transduction cascade in SCN neurons, ultimately resulting in a phase shift of the circadian system. This model is strengthened by the findings that GluR antagonists prevent light-induced phase shifts in vivo . and that exogenous glutamate can cause phase shifts in vitro . More problematic is the finding that GluR agonists injected into the SCN region do not cause light-like phase shifts (53). Of course there are many possible pitfalls in the interpretation of this type of experiment. For example, It seems unlikely that the injection of glutamate into the SCN region would stimulate the same cells in the same manner as synaptic activation of the RHT. Nevertheless, currently available data clearly challenge any simple interpretation of the behavioral experiments with GluR antagonists. At present, behavioral evidence suggests that GluR activation is necessary but not sufficient to generate light-like phase shifts. More work is clearly needed to delineate the signal-transduction cascades by which glutamate acts in the SCN and to understand how these cascades influence the phase of the circadian system. Nitric Oxide (NO) One possible consequence of NMDA GluR activation is the stimulation of nitric oxide synthase (NOS). Several pieces of evidence raise the possibility of a role for NO in the light-input pathway to the SCN. First, anatomical studies show the presence of NOS in the SCN (3). Second, NO production has generally been linked to NMDA-induced cGMP production, and administration of cGMP produces phase shifts of the circadian rhythm of neuronal activity recorded from the SCN in vitro . (73). Finally, NOS inhibitors prevent NMDA-induced phase shifts of circadian rhythms both in vitro and in vivo (20). Changes in Gene Expression Although the signal transduction events occurring downstream from GluR activation in the SCN are not well understood, one important consequence of photic stimulation is the regulation of gene expression in the SCN (44). In many neurons, one consequence of GluR stimulation is activation of immediate-early genes, including c-fos. The proteins coded for by these genes, including Fos, appear to be generally involved in the transduction of extracellular signals to changes in gene expression andin some caseschanges in immediate-early gene expression can be used as a cellular marker of neuronal activation. Photic regulation of c-fos mRNA and Fos-like immunoreactivity (Fos-LI) in the SCN of rodents has been extensively demonstrated. These studies have shown that photic regulation of Fos in SCN neurons is correlated with light-induced phase shifts of the circadian system. For example, induction of c-fos mRNA by light in the hamster SCN shows the same phase dependence and intensity threshold as does phase shifting. The functional significance of light-induced Fos expression is still unclear. Light can still entrain the circadian system of mice lacking the c-fos gene, although a reduction was seen in the magnitude of light-induced phase shifts (35). In another study, the intraventricular administration of c-fos and jun-B antisense oligonucleotides decreased expression of these transcription factors and inhibited light-induced phase shifts (104). These results suggest that, while c-fos activation may contribute to the normal entrainment process, it is not absolutely required for photic regulation or generation of circadian rhythms. Fos induction has also been widely used as a cellular marker for light-responsive cells to address questions about how experimental manipulations alter photic input to the SCN. A number of studies have reported that both NMDA and AMPAKA GluR antagonists inhibit light induction of Fos expression in the SCN (2, 25). At least one study has also found that the intraventricular injection of NMDA induces Fos expression in the SCN (25). The effects of AMPAKA GluR agonists have not been examined. These pharmacological studies generally suggest a role for both NMDA and AMPAKA GluRs in mediating photic regulation of SCN neurons in vivo . The possibility of peptide co-transmitters within RHT terminals is specifically suggested by the presence of dense-core vesicles among the glutamate-containing synaptic vesicles (8). Two likely candidate co-transmitters are N-acetylaspartylglutamate (NAAG) and substance P (SP). Immunocytochemical localization of NAAG to many retinal ganglion cells and the SCN has been reported (58). Optic nerve transection decreased NAAG immunoreactivity in the SCNa finding consistent with the suggestion that NAAG is contained in terminal fields of the RHT. Although the functional role of NAAG is unclear, it can both directly activate GluRs and form glutamate by extracellular hydrolysis. One physiological study found that the iontophoretic application of NAAG increased the firing rate and potentiated glutamate-induced responses in SCN neurons in culture (6). There is also some evidence that SP may play a role as a retinal co-transmitter. Anatomical evidence for the presence of SP in the RHT has been found for a number of species, including humans (64). Application of SP in vitro increased 2DG uptake, excited a population of SCN neurons, induced Fos expression, and caused phase shifts of the circadian rhythm of electrical activity (88). More recently, an SP antagonist has been found to block light-induced Fos expression in vivo (1). It will be important to examine the effects of this inhibitor on light-induced phase shifts of the circadian system and to see if SP alters glutamate release from the RHT. To date, the results are all consistent with the possibility that this peptide is a co-transmitter at the RHTSCN synaptic connection. Geniculohypothalamic Tract (GHT) The retinal ganglion cells which innervate the SCN also project to a subdivision of the lateral geniculatethe intergeniculate leaflet (IGL Fig. 6 ref. 66). The IGL, in turn, has a population of neurons that project to the SCN through a geniculohypothalamic tract (GHT). The projection neurons that make up the GHT appear to contain neuropeptide Y (NPY) and GABA, and these transmitters may be co-localized. Both of these molecules exert effects on the circadian system. This pathway appears to be involved both in the processing of photic information and the mediation of the effects of some non-photic entraining stimuli. Cells in the IGL are known to be light-sensitive (33), and the GHT pathway may contribute to the processing of photic input. In many sensory neurons, NPY acts presynaptically to modulate transmitter release and may play a similar role in the regulation of RHT input to the SCN. Likewise, GABAB receptors may modulate light-induced phase shifts (75) and the release of glutamate by the RHT (40). IGL lesions are associated with relatively subtle changes in the circadian systems response to light, including changes in the magnitude of light-induced phase shifts, period changes, and slower adjustment to new light-dark cycles (66). However, many features of the circadian systems response to light are unaffected in IGL-lesioned animals, suggesting that this pathway is not essential for the major portion of photic regulation of the circadian system. But this pathway does appear to have an important role in mediating the effects of other non-photic stimuli. A number of experimental treatments produce phase shifts during the day but not during the night, including activity induced by novel stimuli (67) and benzodiazepines (95). These phase shifts are dependent upon an intact IGL lesions abolish both benzodiazepine (41) and activity-induced (67) phase shifts. The use of antibodies to reduce NPY binding also reduced activity-induced phase shifts (4). A similar pattern of phase shifts during the day was generated by NPY administration in vitro and in vivo through a mechanism which may be dependent on GABAergic transmission (36). Recent studies in cultured SCN cells suggest that NPY can act presynaptically to inhibit GABA-mediated synaptic transmission through inhibition of calcium currents (9). Thus, the GHT plays a critical role in mediating the effects of some non-photic stimuli on the circadian system. Other Neurotransmitter Systems The SCN receives a dense serotonergic projection from the midbrain raphe nuclei that terminates predominantly in the retinorecipient region of the nucleus (62). It is well established that 5-HT receptor agonists cause phase shifts of the SCN circadian oscillator when administered at times in the circadian cycle during which light does not cause phase shifts both in vitro (52) and in vivo (26). In addition, evidence suggests that this projection modulates photic input to the SCN. Neurotoxic destruction of the serotonergic input to the SCN alters the relationship between the light-dark cycle and locomotor activity, and increases in 5-HT levels alter the effects of light on the circadian system (65). Finally, 5-HT and 5-HT agonists inhibit optic nerve-induced field potentials in the SCN brain slice preparation, light-induced Fos expression and phase shifts of the circadian rhythm of wheel-running activity (78). Interestingly, 5-HT antagonists have been reported to enhance light-induced increases in the firing rates of SCN neurons (105) and light-induced phase shifts (77). These results raise the possibility that 5-HT may be involved in a tonic inhibition of the light-input pathway to the SCN. In addition, these studies are all consistent with the hypothesis that the serotonergic innervation of the SCN serves to modulate light-regulated glutaminergic input. Understanding this pathway is likely to be important in understanding the links between disruptions in circadian function and affective disorders (see later discussion). It has long been suggested that ACh plays a role in the light-input pathway. Fibers immunoreactive for choline acetyltransferase innervate the SCN (99), apparently from the cholinergic regions of the basal forebrain and brain stem (5). Furthermore, electrophysiological studies indicate that some SCN neurons are excited by cholinergic agents. In addition, administration of the ACh receptor agonist carbachol caused large phase shifts in SCN neuronal activity rhythms this response is mediated by muscarinic receptors perhaps of the M1 subtype (50). The intraventricular administration of carbachol caused phases shifts in vivo which can be blocked by GluR antagonists (12). This result raises the possibility that some of the behavioral effects of carbachol may be due to stimulation of glutamate release. So, while ACh does not appear to be a transmitter directly in the light input pathway, it may act to modulate photic information reaching the SCN. The SCN receives a prominent histaminergic innervation from the tuberomammillary nucleus. Based on anatomical considerations, the histaminergic transmitter system may represent a regulatory center capable of altering arousal throughout the nervous system. Depending on the subtype of receptor activated, HA can have excitatory (H1) or inhibitory (H2) actions on SCN neurons (49). Administration of HA can cause phase shifts of the in vitro neural activity rhythm and the in vivo locomotor activity rhythm (15). These results suggest that HA may be involved in modulation of light input to the circadian system but, at present, the functional significance of this regulation is unknown. The mammalian pineal gland secretes melatonin rhythmically under the neural control of the SCN (see discussion below). The SCN is also a target of this hormonal output, as the SCN has a high density of melatonin receptors (102). One consequence of activation of these receptors is the inhibition of neural activity, perhaps through the activation of potassium currents in SCN cells (40). Melatonin also caused phase shifts of the circadian rhythm of neuronal activity of SCN neurons in vitro (51). Behaviorally, administration of melatonin caused phase shifts of the locomotor activity rhythm during the day and may modulate the effects of light during the night (7). OUTPUT FROM THE SCN: HOW DOES THE OSCILLATOR IN THE SCN REGULATE OTHER PHYSIOLOGICAL AND BEHAVIORAL SYSTEMS Most of an organisms physiological and behavioral parameters show a daily rhythm. In many cases, these rhythms are driven from a circadian oscillator located in the SCN. Physiological or behavioral parameters which exhibit daily rhythmicity due to the activity of cells in the SCN are known as outputs of the circadian system. Metaphorically, these outputs are sometimes referred to as hands of the clock to distinguish them from the mechanisms responsible for the generation of the rhythms. It is not clear if this intellectual distinction will hold up experimentally. In general, outputs of the circadian system are rhythmic but not temperature compensated. Theoretically, if an output is held constant, this should not alter other rhythms driven from the SCN. A major problem in circadian rhythms research is to understand the mechanisms by which the circadian oscillator located in the SCN regulates such a wide range of physiological outputs. There is evidence for two types of signals originating from the SCN and conveying phase information: hormonal and neural outputs. Evidence for hormonal or some other diffusible signals comes from transplantation experiments (76). These studies have shown that rhythmicity can be restored to SCN-lesioned animals following implantation into the third ventricle of tissue containing the SCN. As previously discussed, these experiments provided compelling evidence that the SCN is a circadian oscillator in mammals. These studies also provide an important tool to look at the mechanisms by which SCN output alters other physiological systems. Several pieces of evidence suggest that a hormonaldiffusible factor produced by the SCN is an important output signal for the circadian system (91). First, some behavioral rhythms recover within four days after transplantation of the SCN, before much axonal outgrowth from the transplant is noted (45). In addition, some successful transplants can be placed in locations distant from the SCNhypothalamus (45). Third, transplantation of disassociated SCN cells can restore rhythmicity (90). The interpretational problem common to all of these studies is the difficulty in ruling out all neural outgrowth. Resolution of this problem may come from studies which place SCN grafts into a polymer capsule or into the anterior chamber of the eye such conditions allow humoral communication with the brain but prevent neurite outgrowth (76). Of course, interpretation of negative results from these type of experiments would be extremely difficult. One of the best studied examples of hormonal output from the SCN is the rhythmic secretion of the peptide VA. Peripherally, this peptide is secreted by the pituitary and functions as an antidiuretic hormone. Centrally, VA also acts as a signaling molecule and is highly expressed in a population of cells in the SCN. These cells drive a prominent daily rhythm in the levels of VA in the cerebrospinal fluid in mammals. If the SCN are lesioned, then VA levels are dramatically reduced and no rhythmicity can be detected (81). Furthermore, SCN cells express a circadian rhythm in VA mRNA in vivo (96) and SCN cells in vitro secrete VA rhythmically (23). Finally, if embryonic SCN tissue is transplanted from a normal rat into a VA-deficient Brattleboro rat, the rhythm in VA is restored (24). Since host animals can not make VA, the source must be donor tissue containing the SCN. The function of this rhythm in VA is currently unknown and is an interesting area for future work. Anatomical knowledge of the output pathways of the SCN has been reviewed by Watts (101). In short, axonal projections from SCN neurons terminate within the SCN, other hypothalamic regions, and a few regions outside the hypothalamus. The largest projection from the SCN goes to the subparaventricular region of the hypothalamus, a region with widespread connections throughout the limbic system. Most SCN neurons contain GABA ( 18. 63, 99 ), suggesting that the output is generally inhibitory. There is evidence that these neuronal pathways are functional and are involved in the communication of signals from the SCN to other parts of the nervous system. First, the local injection of TTX into the SCN region blocks the expression of a rhythm in drinking activity (86). Second, knife cuts around the SCN which cut efferent fibers block the expression of several rhythms (37, 92). Third, some transplants appear to be healthy, are in an appropriate position, and have cells expressing normal peptides, yet they do not restore rhythmicity. These data are all consistent with the idea that neural connections play an important role in driving overt rhythms. The interpretation problem common to all of these studies is that the experimental treatments might interrupt hormonal as well as neural output from the SCN. Among the rhythms under neural control of the SCN is the circadian synthesis and secretion of the pineal hormone melatonin. In mammals, the pineal gland is not directly light sensitive but is photically regulated through a complicated neuronal pathway involving the SCN. Melatonin is the hormone secreted by the pineal gland, and the SCN drives a daily rhythm in its secretion. Control relies on a multisynaptic pathway via the sympathetic nervous system to maintain and entrain the rhythmic synthesis and secretion of this hormone ( Fig. 7 ref. 60). The neural pathway from the SCN to the pineal passes first to the paraventricular nuclei (PVN). Ablation of either the SCN or PVN results in loss of the rhythm in pineal melatonin levels. Most SCN neurons contain GABA, an inhibitory neurotransmitter, and it is most likely that the excitation of SCN neurons inhibits neurons in the PVN. The PVN neurons project to the spinal cord and make synaptic connections with preganglionic cell bodies which, in turn, innervate the superior cervical ganglia of the sympathetic nervous system. Stimulation of the PVN increases sympathetic outflow through activation of cholinergic preganglionic sympathetic neurons. Sympathetic neurons release norepinephrine which drives a rhythm in pineal melatonin by increasing N-acetyltransferase activity. Disruption of the pathway from the SCN to the pineal gland at any level (destruction of the SCN itself, knife cuts of SCN afferents, or pharmacologic blockade of the sympathetic innervation) interrupts the circadian pattern in the synthesis and secretion of the hormone (60). SCN transplants do not appear to restore the melatonin-mediated photoperiodic response in hamsters (45). Clearly, this output of the circadian system is under neural control. There is evidence for both neuronal and hormonal outputs from the SCN. Signals could vary with the specific physiological system being regulated. For example, rhythmic secretion of melatonin could be under neural control while locomotor activity is under hormonal control. Alternatively, the signals could be redundant, with a specific physiological system receiving both neural and hormonal signals from the SCN. The issue of how cells in the SCN regulate other physiological systems is clearly an important area and requires further study. MEDICAL IMPLICATIONS OF THE CIRCADIAN SYSTEM Although the experimental studies described in this review were mostly performed in rodents, the general principles developed by this research are likely to apply to humans. Humans, like other organisms, exhibit daily rhythms in many physiological and behavioral parameters (11, 46, 55). Because of experimental difficulties in isolating humans from environmental influences, in many cases, it is not yet clear whether these rhythms are really circadian or instead are diurnal, i. e. driven by external cues. Nevertheless, it is clear that humans have an endogenous circadian timing system including the SCN and RHT. There is every reason to think that the SCN functions as a circadian oscillator in humans. First, humans suffering from dementia have problems with the timing of their sleepwake cycle this is correlated with loss of neurons in the SCN (56). Furthermore, patients with tumors or other types of damage to the hypothalamic area, including the SCN, exhibit disruptions in their daily rhythms (10, 85). Since the human SCN expresses many of the same neurochemical markers described in rodents (59), it seems likely that many of the neural pathways described in rodents will also be relevant for humans. In recent years, it has become clear that light is an important environmental signal for the synchronization of the human circadian system (16). This has led several groups to investigate the use of light for therapeutic manipulations of the human circadian system (93). With these observations also comes the recognition that, within the last 100 years, dramatic changes have occurred in the temporal environment to which humans are exposed. With the widespread use of artificial lights and airplanes, many people experience rapid changes in their light-dark cycle. These changes can disrupt our endogenous timing system. Let us next briefly consider a few aspects of the human circadian system: desynchronization caused by jet travel or shift work circadian variation in the effects of pharmacological agents the possible use of melatonin to manipulate the human circadian system and, finally, the possible link between the circadian system and affective disorders. While there are many other interesting aspects of the human circadian system, these topics at least provide an introduction as to how the circadian system impacts human biology. Desynchronization of the Circadian System In our modern world, increasing numbers of people move rapidly across time zones or work during the night. The result is a group of symptoms collectively known as jet lag. While there is a lot of variation in individual symptoms, many people experience disruption of sleep, gastrointestinal disturbances, decreased vigilance and attention span, and lack of energy. While most people have no difficulties tolerating an occasional case of jet lag, repeated shifts create greater problems. One recent report even suggested that jet lag of players may be a factor affecting the outcome of baseball games (79). More seriously, consider people whose jobs require constant changes of schedule (e. g. health care professionals, pilots, and other shift workers). While it is difficult to link shift work directly to demonstrable physical illnesses, commonly reported health consequences include stomach diseases, sleep disturbances, and fatigue (55, 93). Besides physical problems, these workers are unlikely to be performing optimally. Humans undergo daily oscillations in many cognitive and motor functions. Human performance is normally at a minimum between 3 and 5 AM (11, 55). Persons working during these hours are likely to be sleepy, inefficient, and accident-prone. Many factors contribute to jet lag, including fatigue and stress, which may be independent of the circadian system. But other symptoms are undoubtedly a direct result of the desynchronization of the circadian system. In general, circadian systems can be thought of as serving at least two different kinds of functions. One is to ensure that an organism is synchronized to the physical world. Another, less appreciated, function is to ensure that the various physiological systems inside the organism remain synchronized. Both of these functions are compromised under conditions of rapid travel between time zones or changes in the scheduling of work. For example, consider a person traveling from North America to Europe, which involves a shift of at least 8 time zones and may require up to a week for the body to resynchronize. In the meantime, the travelers performance minimum (3:00 to 5:00 AM, old time) will now be occurring at 11:00-13:00. In order to maximize synchronization to a new time zone or schedule, the best strategy appears to be to maximize exposure to entraining signalsespecially light and social cues. So, the best advice to speed adjustment to a new schedule is to be immediately active in the new daytime and to sleep during the new night, eat meals at local times, and spend the day out in well lit environs. But even after exposure to all of these new environmental signals, it will still take a few days to readjust, so it may just be best to simply recognize this and to allow some time for adjustment after shifting to a new schedule or time zone. Circadian Variation in Drug Effects Most physiological and behavioral parameters exhibit daily rhythms, so it is not surprising that drug effects both desired and undesired (i. e. toxicity) vary with the time of day. In one early observation of this phenomenon, the mortality of mice after an injection of E. coli endotoxin was 80 during the middle of their inactivitysleep time but below 20 in the middle of their activity time (31). This is not an isolated or unusual result, and the effects of many drugs are now known to vary depending on the time of day (46). This daily variation is not due to some mysterious process but rather can be mostly explained by two observations. First, temporal variation has been documented in the rates of absorption, metabolism, and excretion. These factors will all impact the concentration of drug that actually reaches the intended target. Second, many tissues and cells show temporal variations in their response to the drugs which do reach them. These observations form the basis of the hope that clinical outcomes can be improved by scheduling drug treatments for certain times of day. Daily variation in the time of symptom onset may be common in a number of diseases and medical emergencies. One extensively studied example is asthma, in which the majority of patients experience symptoms mainly at night (70). These patients have a daily rhythm of bronchial constriction with the greatest constriction seen between midnight and 8:00 A. M. Accordingly, this is the time when most respiratory failures occur. Understanding this type of rhythm can, at the very least, lead to improved monitoring at certain times of day as a preventive measure. In addition, drugs used for treatment of asthma are apparently more effective when given before bed than during other times of day (46). So, in this case, the normal pharmacologic goal of keeping drug concentrations constant through time appears to be less effective than allowing drug concentrations to fluctuate. Even in diseases in which the symptoms are not so obviously temporally patterned, there is still evidence for diurnal variation in drug effects. For example, most of the drugs used in chemotherapy are toxic to both host and malignant cells, so there is an unusually narrow window between therapeutic and toxic effects. There are good reasons to think that the timing of drug administration may be an important therapeutic variable. Data suggest that both the toxicity and therapeutic benefits of anticancer drugs vary with the daily cycle (27). If these rhythmic variations are out of phase with each other, then time of treatment may represent a variable which can be exploited to maximize the benefitcost ratio for the use of anticancer drugs. In one early study, mice were injected with leukemia cells and treated with DNA synthesis inhibitors (cyclophosphamide and 1-B-D-arabinofuranosylcytosine) at different times of day (83). Without treatment, most of the mice died during the 75-day trial. The number of animals that survived the tumor inoculation varied depending on the timing of cyclophosphamide administration. Treatment in the beginning of the inactivitysleep period led to a 40 survival rate the same treatment given at the beginning of the animals active period led to over 90 survival. Of course, there are many possible reasons that might account for these data, and the question of whether such scheduling strategies can actually improve clinical outcome for humans is one that still needs to be answered. But, it seems likely that varying drug doses over the course of the day could lead to much more effective treatment strategies. In humans, as in other mammals, melatonin is secreted by the pineal gland during the night but not during the day. This rhythm is due to both circadian regulation and acute light-induced suppression of melatonin secretion. Both mechanisms ensure that the secretion of melatonin fairly accurately follows the night. Thus, melatonin is referred to as a dark hormone. In many temperate-zone mammals, this nightly dark signal is intimately involved in the control of seasonal changes in physiology and behavior. Although there is little direct evidence that melatonin mediates seasonal changes in humans, the onset of melatonin secretion at night does appear to be a good marker for the phase of the human circadian system (47). The human SCN contains melatonin binding sites, and administration of melatonin (0.5 mg), by itself, caused phase shifts of the human circadian system: phase delays during late night and phase advances during the morning (47). In addition, melatonin can act as a sleep-inducing agent. In one study, a group of healthy adults were administered melatonin (1-10 mg) in the middle of the day. Melatonin induced sleep, lowered body temperature, and caused feelings of sleepiness and fatigue (21). On the basis of these results, it was proposed that melatonin might be used to help workers or travelers adjust to new schedules. There is some evidence that melatonin may be therapeutically useful in this regard. Several studies have reported that timed melatonin administration can help with re-adjusting the circadian system after jet-lag or shift work (17, 47 ). Moreover, in some insomnia patients, administration of melatonin improved the sleep quality or the phasing of sleep (29). Overall, our understanding of melatonins effects on human biology is still in its infancy. Much more work will have to be done before the possible therapeutic value of melatonin can be determined. Several observations suggest a link between disruptions in circadian function and affective disorders (34). Certainly, many patients suffering from depression exhibit disruptions in the timing of sleepwake cycles, cortisol levels, melatonin secretion and body temperature. Similarly, many treatments that have antidepressant effects (e. g. timed photic stimulation, sleep-deprivation, serotonin reuptake inhibitors) also alter circadian rhythms. Dysfunction in serotonergic pathways has been suggested to play a role in affective disorders, which are frequently treated with agents that alter serotonergic neurotransmission. The serotonergic projection from the raphe to the SCN (see previous discussion) may very well be the anatomical substrate by which affective disorders alter the human circadian system. Of course, it has been extremely difficult to establish more then a correlative link. Perhaps the best evidence comes from studies of seasonal affective disorder (SAD). SAD is characterized by recurrent cycles of fall-winter depression and spring-summer remission (82). The seasonal nature of the symptoms immediately suggests a role for the circadian system which, in many mammals, plays a central role in mediating seasonal changes in behavior and physiology. Furthermore, SAD has been successfully treated by timed exposure to bright light (48, 93). Overall, the exact role of the circadian system in affective disorders is still open to debate and remains an important area for future research. The finding that humans and other organisms have endogenous circadian timing systems raises a number of issues. What are the mechanisms by which light and other environmental stimuli synchronize these systems What are the mechanisms by which cells generate these oscillations How are these cells and cell populations integrated to form a coherent timing system How are these oscillators coupled to the various outputs they control What are the consequences for human biology, as well as the natural history of other organisms In this review, I have briefly described some of the current work in each of these areas. Some of the knowledge gained from addressing these issues may very well be unique to this physiological system. For example, the biochemical processes involved in the generation and temperature compensation of rhythms with a 24-hour time base are likely to be novel. Other questions involving sensory input to the circadian system, coupling between oscillators, and output from the circadian system are all basic issues of communication central to neuroscience research. In closing, I would like to argue that some features of the circadian system make it an excellent model system to address many core issues in the neurosciences. Many of the behavioral and physiological outputs of the circadian system are precise, quantifiable, and functionally important. This allows the productive use of both neuropharmacologic and genetic approaches. Anatomically discrete and well defined pathways control these behaviors. Finally, SCN neurons are amenable to detailed cellular and molecular analysis by all of the tools of modern neuroscience. For these reasons, I believe that the circadian system will be one of the first mammalian behavioral control systems to be understood at a variety of levelsfrom behavioral to molecular. I would like to thank Drs. E. Nansen and N. Wayne for reading an early draft of this chapter. Gain up to 92 every 60 seconds Ag forex marketing in 616. 15721576. MD Consult L. 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