Professional Documents
Culture Documents
Institut fr Physiologie I
Geschftsfhrender Direktor: Univ.-Prof. Dr. rer. nat. H.-C. Pape
Kumulative Habilitationsschrift
zur Erlangung der Venia legendi fr das Fach Physiologie
an der Medizinischen Fakultt
der Westflischen Wilhelms-Universitt Mnster
vorgelegt von
Dr. rer. nat. Philippe Coulon
Mnster
2012
Einleitung
Inhaltsverzeichnis
Formale Grundlagen zum Habilitationsantrag ................................................................................. 2
Inhaltsverzeichnis ................................................................................................................................. 3
1.
2.
Einleitung ..................................................................................................................................... 4
1.1.
1.2.
1.3.
1.4.
Hypothesen ...................................................................................................................... 15
2.2.
Intrazellulres Calcium beeinflusst elektrophysiologische Aktivittsmuster in
thalamischen Neuronen ............................................................................................................... 22
3.
2.3.
2.4.
2.5.
Zusammenfassung ................................................................................................................... 40
Literaturverzeichnis ............................................................................................................................ 43
Anhang ................................................................................................................................................ 52
Der kumulativen Habilitationsschrift zugrundeliegende Publikationen ........................ 52
Lehrerfahrung ....................................................................................................................... 53
Eingeworbene Frdermittel und Preise ............................................................................ 56
Gesamtpublikationsliste ...................................................................................................... 56
Lebenslauf Philippe Coulon ............................................................................................. 63
Danksagungen ................................................................................................................................... 64
Einleitung
1.
Einleitung
Die Einstellung der Rhythmogenese neuronaler Netzwerke ist also entscheidend fr die Funktion des ZNS. Eines der wichtigsten Netzwerke zur
Generierung von rhythmischer Aktivitt ist das thalamokortikale System,
welches in einer vereinfachten Betrachtung aus dem Thalamus, dem
Nucleus reticularis thalami (NRT) und dem Kortex gebildet wird. Der
Thalamus ist die Schnittstelle zwischen Empfindung und Wahrnehmung.
Abhngig davon, ob der Organismus wach ist oder schlft, bestimmen dabei
die Nervenzellverbnde des Thalamus in erster Instanz, welche Informationen zur Grohirnrinde (Kortex) weitergeleitet werden. Integrative
Prozesse des Kortex werden so vom Thalamus mageblich mitbestimmt
4
Einleitung
Es erscheint verwunderlich, dass die Funktion des Schlafs nach wie vor
unklar ist. Es wird angenommen, dass sie u. a. in der Regeneration der
neuronalen Energiereserven liegt. Auerdem findet offenbar whrend des
Schlafs eine Konsolidierung von Gedchtnisinhalten statt. Obwohl Schlaf fr
Sugetiere lebensnotwendig ist und Schlafentzug von Menschen als Qual
empfunden wird, ist Schlafmangel nur sehr selten lebensbedrohend. Es
scheint, dass Schlafentzug zu einem groen Teil und ber lngere Zeitrume
kompensiert werden kann, wobei mglicherweise lokale neuronale Verbnde
in einen schlafhnlichen Zustand bergehen (Krueger et al., 2008; Coulon et
al., 2012). Eine Voraussetzung fr den Schlaf bei Sugetieren ist die
synchrone Aktivitt elektrischer Entladungen im thalamokortikalen System
(Coulon et al., 2012).
Einleitung
Auerdem
sind
hufig
Einschrnkungen
der
kognitiven
Einleitung
Ziel der Arbeiten, die hier zusammengefasst wurden ist es, gemeinsame
Mechanismen der Rhythmogenese bei Schlaf-Wach-Regulation und der
Epilepsie-Entstehung zu untersuchen und hieraus ein tieferes Verstndnis
der Steuerung rhythmischer Aktivitt im ZNS unter physiologischen und
pathophysiologischen Bedingungen zu erhalten.
In
einer
vereinfachten
Betrachtung
der
sensorischen
Informations-
verarbeitung bilden Teile des Thalamus (die sog. Relaiskerne), des NRT und
der sensorischen Areale des Kortex ein neuronales Netzwerk, das sog.
thalamokortikale System. Das Schema in Abb. 1 zeigt die Verschaltungen
sensorischer Eingnge in diesem Netzwerk an einem Beispiel. Visuelle
Signale, die ber die Sehbahn von der Netzhaut (Retina, RET) an das
Corpus geniculatum laterale pars dorsalis (CGLd) des Thalamus gesendet
werden, werden bei Wachheit an die korrespondierenden und topographisch
geordneten sensorischen Zentren des Kortex weitergeleitet. Das CGLd ist
ebenfalls topographisch geordnet und besteht aus mindestens zwei Typen
von Nervenzellen: Thalamokortikale Schaltneuronen (TC Neuronen, rote
Ellipse in Abb. 1) und lokale Interneuronen (grne Ellipse). TC Neuronen
projizieren mit ihren Axonen zum Kortex und terminieren dort mit ihren
Synapsen auf kortikalen Neuronen der Schicht 4. Gleichzeitig besitzen sie
Einleitung
Kollateralen, die zum NRT abzweigen und dort auf hemmenden Neuronen
terminieren, die ebenfalls topographisch geordnet sind (Lam et al., 2006).
Schicht 1 und 2
Schicht 6
Erregung
Hemmung
NR
T
NOT
NPB
RET
Thalamus
Cortex
BV
NPB
NOT
NPB
Abb. 1: Das thalamokortikale System. Schema der Verschaltungen der drei Hauptzentren
des thalamokortikalen Systems: Relaiskerne des Thalamus (Thalamus), Nucleus reticularis
thalami (NRT), Kortex. Nach Coulon et al. (2012), modifiziert nach Sherman & Guillery
(1996).
Einleitung
Eingnge
sowohl
aus
dem
Kortex
(von
kortikothalamischen
thalamokortikalen
NRT-Neuronen,
kortikale
Pyramidenzellen)
spielen
eine
Einleitung
ein
bistabiles Membranruhepotential
in
thalamischen
Neuronen
bedingen:
verhltnismig
langsame,
niederschwellige
und
10
Einleitung
Der NRT gilt als Zentrum fr die Modulation der sensorischen Schaltneuronen im Thalamus und scheint fr die Generierung rhythmischer Aktivitt
im thalamokortikalen System von besonderer Bedeutung zu sein. Er gilt als
Schrittmacher (engl. pacemaker) langsamer Oszillationen im Thalamus
(Bal & McCormick, 1993; Llinas & Ribary, 1993; Steriade et al., 1997;
Fuentealba & Steriade, 2005). Auerdem spielt dieser Nucleus eine
Schlsselrolle bei der Kontrolle selektiver Aufmerksamkeit (Crick, 1984;
Guillery et al., 1998).
11
Einleitung
Die Neuronen des NRT bilden eine dnne Schicht aus Zellen um den
dorsalen Thalamus. Einzelne Zellverbnde innerhalb dieser Schicht sind
topographisch nach sensorischen Eingngen geordnet (Lam et al., 2006).
Alle Neuronen im NRT nutzen fr die chemisch-synaptische bertragung den
inhibitorischen Neurotransmitter -Aminobuttersure (engl. -aminobutyric
acid, GABA) (Pinault, 2004). Wie andere Neuronen des Thalamus sind auch
diese dazu in der Lage, zwei Aktivittszustnde einzunehmen. Allerdings
scheint dem NRT dabei eher die Funktion der Generierung von
Salvenaktivitt zuzukommen. Diese kann mit einer hheren Frequenz
erzeugt werden, als einzelne Aktionspotentiale (Steriade et al., 1986; Coulon
et al., 2009).
einige
NRT-Neuronen
ber
elektrische
Synapsen
miteinander
Eingnge von Neuronen des basalen Vorderhirns blockieren die Schlafspindeln und haben somit einen desynchronisierenden Effekt (Steriade et al.,
12
Einleitung
1987). Es ist allerdings nach wie vor unklar, ob der NRT diese Schlafspindeln
selbst generiert (De Gennaro & Ferrara, 2003) oder ob erst eine Interaktion
zwischen NRT und TC Neuronen dies bewerkstelligt (Bal & McCormick,
1993; Huguenard & McCormick, 2007).
Der NRT war Gegenstand zahlreicher Untersuchungen, die unter verschiedenen experimentellen Bedingungen, wie Stimulation, Lsion oder Isolation
durchgefhrt wurden, und zum Ziel hatten, die Netzwerkeigenschaften dieses
Kerns aufzuklren. Auerdem wurden einzelne Neuronen elektrophysiologisch untersucht oder deren elektrische Aktivitt wurde anhand eines
Computermodells simuliert, um die Eigenschaften der NRT-Neuronen auf
Einzelzellebene zu erfassen (siehe Pinault, 2004). Dennoch ist die genaue
Funktion dieses Kerngebiets und seiner Neuronen bislang unklar.
Einleitung
Synchrone Aktivitt rekrutiert individuelle TC Neuronen durch ein konzertiertes Wechselspiel von GABAergen inhibitorischen postsynaptischen
Potentialen, die aus dem NRT eintreffen, und zwei Typen spannungsgesteuerter Ionenkanle: T-Typ Ca2+-Kanle, durch die der Strom IT fliet
und hyperpolarisationsaktivierte Kationenkanle (HCN-Kanle), durch die der
Strom Ih fliet (McCormick & Pape, 1990; Steriade et al., 1993; McCormick &
Bal, 1994; Gerard et al., 2012).
Einleitung
Systems eingreifen (Crunelli & Leresche, 2002b). Ethosuximid, Levetiracetam, Trimethadion und Valproat konnten die Anfallshufigkeit und -dauer
verringern. Fr Ethosuximid konnte gezeigt werden, dass es u. a. niedrigschwellige Ca2+-Kanle des T-Typs hemmt (Broicher et al., 2007). Bei bis zu
40% aller Patienten, die unter Absence-Epilepsie leiden, liegt eine genetische
Ursache der Erkrankung vor, wobei Mutationen in T-Typ Ca2+-Kanlen und
GABAA-Rezeptoren nachgewiesen wurden (Wallace et al., 2001; Chen et al.,
2003; Scheffer, 2003; Khosravani et al., 2004; Khosravani & Zamponi, 2006;
Vitko
et
al.,
2007).
Auerdem
wurde
gezeigt,
dass
rhythmische,
1.4. Hypothesen
thalamischen
Neuronen;
b)
Eigenschaften
des
lokalen
15
Einleitung
Einleitung
Einwerbung
von
Frdermitteln:
Die
Rolle
elektrischer
Neuronen
des
Nucleus
Reticularis
Thalami.
Innovative
17
Eigene Arbeiten
2.
Eigene Arbeiten
Schlaf und schlafhnliche Zustnde finden sich nicht nur bei Vertebraten
sondern z. B. auch bei Nematoden (Caenorhabditis elegans, (Raizen et al.,
2008)), Fruchtfliegen (Drosophila melanogaster (Cirelli & Bushey, 2008)) und
selbst im Pflanzenreich (McClung, 2006). Beim medizinischen Blutegel kann
erhhte oder verringerte motorische Aktivitt durch Mechanismen ausgelst
werden, die mit der Schlaf-/Wachregulation bei Sugetieren vergleichbar sind
(Hashemzadeh-Gargari & Friesen, 1989). Ein Schlafzustand wurde beim
medizinischen Blutegel zwar nicht beschrieben, jedoch treten z. B. vor und
nach der Nahrungsaufnahme stille Ruhezustnde auf (quiescence resting,
(Dickinson & Lent, 1984)) die zumindest schlafhnlich sind.
Eigene Arbeiten
2+
Abb. 2: HCN-Kanle knnen im Wechselspiel mit dem T-Typ Ca -Kanal Oszillationen des
Membranpotentials verursachen. Die Rckkopplung kann dabei ber das eingestrmte Ca
2+
2+
In Neuronen erfllt dieser Strom mindestens zwei Aufgaben: 1. Rhythmogenese und 2. Stabilisierung des Membranpotentials. Diese beiden Funktionen bedingen einander, da durch eine dauerhafte Aktivierung des Ih um das
19
Eigene Arbeiten
des
Stroms
zur
Folge
hat.
Ist
der Auslser
der
Membranpotential-Vernderung z. B. ein Strom durch Ca2+-Kanle des TTyps (IT), welcher selbst spannungsaktiviert ist, so ergeben sich aus dem
Wechselspiel zwischen Aktivierung und Deaktivierung Oszillationen des
Membranpotentials: Die Aktivierung des IT depolarisiert die Membran,
wodurch der Ih deaktiviert wird. Diese Deaktivierung bewirkt aber zusammen
mit der zeitabhngigen Inaktivierung des IT eine Hyperpolarisation, die
wiederum Ih aktiviert (Pape, 1996). Dies hat eine Depolarisation zur Folge,
die wieder IT aktiviert, do dass der Zyklus von neuem beginnt.
Eigene Arbeiten
21
Eigene Arbeiten
22
Eigene Arbeiten
Zu den wichtigsten Quellen fr Zunahmen der [Ca2+]i gehren spannungsgesteuerte Ca2+-Kanle, die ber die Plasmamembran einen Einstrom von
Ca2+ aus dem Extrazellulrraum in das Cytosol ermglichen (Cain & Snutch,
2010). Dabei unterscheidet man grob zwischen hochschwelligen Ca2+Kanlen (engl. high voltage activated, HVA) und niederschwelligen Ca2+Kanlen (engl. low voltage activated, LVA).
Eine
weitere
wichtige
Quelle
fr
Erhhungen
der
[Ca2+]i
ist
das
23
Eigene Arbeiten
2+
2+
2+
2+
Typs vermutet (Budde et al., 2000). Modifiziert nach Coulon et al. (2012).
24
Eigene Arbeiten
Mechanismus
wurde
die
Aktivierung
von
Ca 2+-
Eigene Arbeiten
Bislang konnte ein CICR im NRT nicht gezeigt werden (Richter et al., 2005;
Cueni et al., 2008). Mit Hilfe der whole-cell patch-clamp Technik und der 2Photonen-Laserscanning-Fluoreszenzmikroskopie mit Ca2+-Indikatorfarbstoffen konnte gezeigt werden, dass eine vergleichbare Freisetzung im NRT
durch rhythmisch-oszillatorische Aktivitt erzielt wird. Der dafr erforderliche
Ca2+-Einstrom findet ber T-Typ Ca2+-Kanle statt. Die Freisetzung von Ca2+
findet, hnlich wie im dorsalen Thalamus, ber Ryanodin-Rezeptoren statt
(Abb. 4). Dieser CICR ber Ryanodin-Rezeptoren erfolgt im NRT hauptschlich im Bereich des Zellsomas (Coulon et al., 2009). Das dabei
freigesetzte Ca2+ konnte durch einen hier ebenfalls noch ungeklrten Mechanismus oszillatorisch ausgelste LTS frequenzabhngig unterdrcken und so
die Fhigkeit der NRT-Neuronen, einer vorgegebenen Frequenz zu folgen,
behindern (Coulon et al., 2009). In der Folge bedeutet dies einen Verlust der
Fhigkeit zur
Generierung von
Salvenaktivitt
und
eine
Dmpfung
Anhand dieser Erkenntnisse wird klar, dass sowohl L-Typ-, als auch T-Typ
Ca2+-Kanle zelltypspezifisch einen CICR auslsen knnen. Obwohl dies auf
gnzlich unterschiedliche Weise geschieht und auf gegenstzliche zellulre
26
Eigene Arbeiten
Dabei wird eine Aktivierung von Ca -abhngigen K -Kanlen vermutet, die zu den
Membranpotential-Oszillationen phasenverschoben ist (Coulon et al., 2009). Modifiziert nach
Coulon et al. (2012).
27
Eigene Arbeiten
Eigene Arbeiten
beschriebenen Ryanodin-Rezeptoren dazu dienen, stereotype Entladungsmuster zu detektieren und ber die dauerhafte Aktivierung Ca2+-aktivierter
Ionenkanle zu unterbinden (Coulon et al., 2012).
bei
Bedarf
zu
unterbinden.
Im
Rahmen
der
hier
Eigene Arbeiten
Ba2+
ersetzt
oder
durch
den
Ca2+-Chelator
1,2-Bis(2-
30
Eigene Arbeiten
Eigene Arbeiten
der Antworten
wurde
in
den
supragranulren
Schichten
gemessen, von wo aus die Amplitude mit zunehmender Entfernung von der
Pia mater encephali abnahm. Entwicklungsabhngig nahm die Erregung der
infragranulren Schichten zunehmend ab, so dass diese in jngeren Tieren
32
Eigene Arbeiten
strker aktiviert waren als bei ausgewachsenen Tieren. Zwischen einem Alter
von 31 und 64 Tagen postnatal (p31 bis p64) nahm die Aktivitt in den
infragranulren Schichten kontinuierlich ab und war ab einem Alter von ca.
65 Tagen (p65) kaum mehr messbar. Eine Stimulation mit hoher Frequenz
hatte vergleichsweise geringe Effekte im auditorischen Kortex zur Folge, im
Gegensatz zum somatosensorischen Kortex, wo durch Stimulation des VB
starke Aktivierung erzielt werden konnte (Laaris et al., 2000; Beierlein et al.,
2002; Llinas et al., 2002). Auch die Latenzen waren in supragranulren und
granulren Schichten krzer als in den infragranulren. Die Ergebnisse zur
Amplitude und Latenz stimmen gut mit den anatomisch-morphologischen
Beschreibungen der Terminalien thalamokortikaler Synapsen aus dem
ventralen Teil des Nucleus geniculatum mediale berein (Linden & Schreiner,
2003; Winer & Lee, 2007). Whrend die geringe Amplitude ein Artefakt der
geringeren Neuronendichte sein knnte, deuten die langen Latenzen in den
infragranulren Schichten auf eine intrakortikale Aktivierung hin. Die laterale
Ausbreitung in den supragranulren und granulren Schichten war zudem
nicht von der infragranulren Aktivitt abhngig, so dass davon ausgegangen
werden kann, dass diese Ausbreitung ber die hheren Schichten luft.
Infragranulre Schichten allein zeigten keine laterale Ausbreitung. Wenn die
ausgelste Aktivitt sich ber die Grenzen des A1 ausbreitete, dann waren
die krzesten Latenzen ausschlielich in den supragranulren Schichten des
A1 zu finden. Im AuV und TeA waren die krzesten Latenzen in den
granulren Schichten zu finden, whrend die grten Amplituden ebenfalls in
33
Eigene Arbeiten
34
Eigene Arbeiten
Konzentrationen
volatiler
Ansthetika
moduliert
werden
35
Eigene Arbeiten
und
IKL)
aktiviert.
Gleichzeitig
wurde
dort
gezeigt,
dass
die
36
Eigene Arbeiten
37
Eigene Arbeiten
Eigene Arbeiten
39
Zusammenfassung
3.
Zusammenfassung
Die in der vorliegenden Arbeit referierten Publikationen liefern neue Erkenntnisse bezglich der Rhythmogenese des thalamokortikalen Systems. Dabei
wurden
sowohl
elektrophysiologische
Ableitungen
von
Ionenstrmen
untersucht, als auch Vernderungen intrazellulrer Ca2+-Ionenkonzentrationen, die durch diese Ionenstrme bedingt waren.
Zusammenfassung
erfolgreich
bezglich
der
rumlichen
und
zeitlichen
Zusammenfassung
Die hier beschriebenen Arbeiten zeigen Mechanismen auf, die zu Vernderungen der Aktivittsmuster von Neuronen im thalamokortikalen System
beitragen. Hierdurch wurde es ermglicht, in einem umfangreichen bersichtsartikel das neue Prinzip der Koexistenz zentraler und dezentraler
Schlafmechanismen zu beschreiben.
[COULON P, BUDDE T, PAPE HC (2012): The sleep relay - the role of the
thalamus in central and de-central sleep regulation. Pflgers Arch. 463(1),
53-71.]
42
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Literaturverzeichnis
Literaturverzeichnis
51
Anhang
Anhang
Der kumulativen Habilitationsschrift zugrundeliegende Publikationen
Anhang
Lehrerfahrung
Der Antragsteller war vom 09.01.2001 bis 31.03.2005 in der curricularen
Lehre des Fachbereichs Biologie an der Mathematisch-Naturwissenschaftlichen Fakultt der Heinrich-Heine-Universitt Dsseldorf ttig. Im
Rahmen dieser Ttigkeit wurden folgende, auf einem Arbeitszeugnis
besttigte und dokumentierte Lehrveranstaltungen inhaltlich, fachlich und
didaktisch gestaltet und durchgefhrt:
bungen in Neurophysiologie und Stoffwechselphysiologie fr Studierende
im Grundstudium: Computersimulationen. Mit dem Programm Neurosimul
(H. Kettenmann) wurden komplexe neurophysiologische Zusammenhnge
erklrt und demonstriert. Die Ttigkeit umfasste eine umfangreiche
Vorbesprechung ber 1,5 h. Die Veranstaltung wurde durchschnittlich von ca.
180 Studenten besucht, der Umfang betrug 4 SWS und die Veranstaltung
fand jeweils im Wintersemester statt. Der Antragsteller war zusammen mit
AOR Dr. Eberhard von Berg alleinig fr die inhaltliche Gestaltung verantwortlich.
bungen
in
Neurophysiologie
fr
Studierende
im
Hauptstudium:
Extrazellulre Ableitungen am Regenwurm Lumbricus spec.. Am Frischprparat des Riesennervenfasersystems von Lumbricus spec. wurde das
Prinzip der extrazellulren Ableitung demonstriert und vermittelt. Das
Praktikumsmodul wurde dabei vom Antragsteller vollstndig selbstttig
gestaltet, weiterentwickelt und alleinig durchgefhrt. Diese bung hatte einen
Umfang von 4 SWS und fand im Sommersemester statt.
bungen in Neurobiologie fr Studierende im Hauptstudium. In diesem
sechswchigen Blockpraktikum fr fortgeschrittene Studierende der Biologie,
das als Einzelkurs auch fr Examenskandidaten durchgefhrt wurde, wurden
die Studierenden vom Antragsteller in verschiedenen neurobiologischen
53
Anhang
Umfang
von
SWS.
Der
Ablauf
des
Einzelkurses
fr
Themen
eigenverantwortlich
behandelt:
Atmung,
Niere
Anhang
Fr
den
Sonderforschungsbereich
TRR
58
Furcht,
Angst,
Angst-
erkrankungen wurden seit 2009 ca. vier Mal pro Jahr (nach Vereinbarung)
Laborfhrungen und Kurzvorlesungen fr Oberstufenschler durchgefhrt.
Die Veranstaltung wurde von durchschnittlich ca. 20 Teilnehmern besucht.
Innerhalb des Instituts fr Physiologie wurden fr Doktoranden die Vorlesungen Basic principles of electrophysiology und Calcium Imaging
(insgesamt 1 SWS) abgehalten.
Fr das Otto Creutzfeld Center for Cognitive and Behavioral Neuroscience
wird am 08.11.2012 die Vorlesung Sleep and Epilepsy: From Spindles to
Spikes im Rahmen der Ringvorlesung OCC Lecture Series WS 2012/13
(Nr.: 132906) gehalten.
55
Anhang
Gesamtpublikationsliste
Artikel in peer-review-Journalen
1. DIERKES PW, COULON P, NEUMANN S, SCHLUE WR (2002):
Potentiometric measurement of cell volume changes and intracellular
ion concentrations under voltage-clamp conditions in invertebrate
nerve cells. Anal Bioanal Chem, 373, 762-766. IF 2011: 3.778 Klasse 1
2. COULON P, W STEN HJ, HOCHSTRATE P, DIERKES PW (2008): Swellingactivated chloride channels in leech Retzius neurons. J Exp Biol, 211,
630-641. IF 2011: 2.996 - Klasse 2
3. BUDDE T, COULON P*, PAWLOWSKI M, MEUTH P, MEUTH SG, PAPE HC
(2008): Reciprocal modulation of Ih and ITASK in thalamocortical relay
neurons by halothane. Pflgers Arch, 456(6), 1061-73. *(T.B. and P.C.
are equally contributing first authors). IF 2011: 4.463 - Klasse 1
4. BROICHER T, WETTSCHURECK N, MUNSCH T, COULON P, MEUTH SG,
KANYSHKOVA T, SEIDENBECHER T, OFFERMANNS S, PAPE HC, BUDDE T
(2008): Muscarinic ACh receptor-mediated control of thalamic activity
via Gq/G11-family G-proteins. Pflgers Arch, 456(6), 1049-60. IF 2011:
4.463 - Klasse 1
5. DNGI M, HIRNET D, COULON P, PAPE HC, DEITMER JW, LOHR C (2009):
GABA uptake-dependent Ca2+ signaling in olfactory bulb astrocytes: a
56
Anhang
Anhang
Anhang
Anhang
Anhang
Anhang
62
Anhang
Besetzte Positionen
09/2011 heute
01/2006 09/2011
07/2005 01/2006
12/2001 03/2005
09/2000 11/2001
Ausbildung
01/2005 heute
12/2001 01/2005
10/94 12/2001
11/87 6/94
07/81 11/87
Post-Doc
Promotion zum Dr. rer. nat. (magna cum laude)
Studium
der
Biologie
(Heinrich-Heine-Universitt
Dsseldorf), Abschluss: Dipl.-Biol. (sehr gut)
Erzbischfliches St. Suitbertus Gymnasium in DsseldorfKaiserswerth, Abitur (1,8)
Schulen in Nrnberg, Hamburg und London
63
Danksagungen
Danksagungen
Prof. Dr. Hans-Christian Pape gilt besonderer Dank fr seine Untersttzung und die
hervorragende Infrastruktur des Instituts. Prof. Dr. Thomas Budde bin ich fr seine
Ideen und die stetige, tatkrftige und freundschaftliche Untersttzung zu groem
Dank verpflichtet. Prof. em. Dr. Erwin-Josef Speckmann gilt besonderer Dank fr
seine Ermunterungen und seine guten Ratschlge. David Herr, Christina Neyer,
Manuela Cerina und Denise Kohmann gilt mein besonderer Dank dafr, dass sie
sich dafr entschieden haben, mit mir zusammen zu arbeiten. Dr. Susan Sangha,
Dr. Ludmila Sosulina, Dr. Tilman Broicher, Dr. Christian Kluge, Dr. Jrg Lesting, Dr.
Kay Jngling, PD Dr. Christian Stock und Dr. Michael Dngi danke ich sehr fr die
zahllosen
fachlichen
und
freundschaftlichen
Gesprche.
Birgit
Herrenpoth,
64
227
The Journal of Experimental Biology 215, 227-238
2012. Published by The Company of Biologists Ltd
doi:10.1242/jeb.062836
RESEARCH ARTICLE
Functional properties and cell type specific distribution of Ih channels in leech
neurons
Ednan Gerard1, Peter Hochstrate1, Paul-Wilhelm Dierkes2 and Philippe Coulon3,*
1
Institut fr Neurobiologie, Heinrich-Heine-Universitt, 40225 Dsseldorf, Germany, 2Abteilung fr Didaktik der Biowissenschaften,
Johann Wolfgang Goethe-Universitt, Sophienstrae 1-3, 60487 Frankfurt/Main, Germany and 3Institut fr Physiologie I,
Westflische Wilhelms-Universitt, Robert-Koch-Str. 27a, 48149 Mnster, Germany
*Author for correspondence (coulon@uni-muenster.de)
SUMMARY
The hyperpolarisation-activated cation current (Ih) has been described in many vertebrate and invertebrate species and cell types.
In neurons, Ih is involved in rhythmogenesis, membrane potential stabilisation and many other functions. In this work, we
investigate the distribution and functional properties of Ih in identified leech neurons of intact segmental ganglia. We found Ih in
the mechanosensory touch (T), pressure (P) and noxious (N) neurons, as well as in Retzius neurons. The current displayed its
largest amplitude in P neurons and we investigated its biophysical and pharmacological properties in these cells. Ih was halfmaximally activated at 65mV and fully activated at 100mV. The current mutually depended on both Na+ and K+ with a
permeability ratio pNa/pK of ~0.21. The reversal potential was approximately 35mV. The time course of activation could be
approximated by a single time constant of ~370ms at 60mV, but required two time constants at 80mV of ~80 and ~560ms. The
current was half-maximally blocked by 0.3mmoll1Cs+ but was insensitive to the bradycardic agent ZD7288. The physiological
function of this channel could be a subtle alteration of the firing behaviour of mechanosensory neurons as well as a stabilisation
of the resting membrane potential.
Key words: leech, P neuron, Ih channel, caesium pharmacology, ZD7288.
INTRODUCTION
228
1999; Coulon et al., 2008). Marked voltage sags have been described
in pressure (P) neurons in situ (Jansen and Nicholls, 1973; Klees
et al., 2005), but the underlying mechanism has not been
investigated. Ih was also found in salivary glands of the giant
Amazon leech, Haementeria ghilianii (Wuttke and Berry, 1992).
The aims of this study were to: (1) determine which neurons show
Ih or the characteristic voltage sag; (2) characterise key properties
such as activation kinetics, voltage dependence, effects of
impermeable cations, pharmacological profile and ion fluxes that
constitute the current; and (3) elucidate the possible functional role
this current plays in leech P neurons by assessing the excitability
and the effects on the time course of AP firing.
We found that Ih preferentially appears in leech mechanosensory
neurons and is largest in P neurons. We concentrated on P neurons
for the biophysical and pharmacological characterisation of the
current properties and for a discussion of the currents functional
role.
lateral packet there are two pairs of P neurons (P1, P2), which can
be distinguished by their electrophysiological characteristics.
Depolarising current injections evoke large-amplitude action
potentials and hyperpolarising current injections evoke characteristic
voltage sags (see Fig.1). The two anterior-lateral packets contain
N, T and AP neurons. N and AP neurons are similar in size to P
neurons, but N neurons generate very large action potentials with
a prominent afterhyperpolarisation immediately after microelectrode
impalement. AP neurons show spontaneous action potentials with
low amplitude (~10mV) at a frequency of ~5Hz and a characteristic
pagoda shape (Nicholls, 1987). T neurons are smaller in size and
do not show spontaneous action potentials. AE neurons are situated
adjacent to neuron 251 and generate spontaneous low-amplitude
(~7mV) action potentials at ~10Hz. Leydig neurons are adjacent
to P2 neurons, smaller in size and fire spontaneously at less than
4Hz (Arbas and Calabrese, 1990).
The standard leech saline (SLS) used for bath perfusion had the
following composition (in mmoll1): 85 NaCl, 4 KCl, 2 CaCl2, 1
MgCl2 and 10 HEPES. The pH was adjusted to 7.40 with 1moll1
NaOH, which increased the Na+ concentration by 4mmoll1. The
osmolality of the SLS was 190mosmolkg1 H2O (Osmomat 030,
Gonotec, Berlin, Germany). Na+-free solution was prepared by
substitution of Na+ with N-methyl-D-glucammonium (NMDG+,
prepared from N-methyl-D-glucamine and HCl; Sigma-Aldrich,
Munich, Germany). Because of differences in osmotic activity,
105mmoll1 NMDG-Cl was used to substitute 89mmoll1 NaCl.
In solutions containing 89mmoll1 Li+, Na+ was omitted and the
pH was adjusted using LiOH. Finally, in solutions with varying K+
concentrations, K+ was either removed, or added to the solution
without osmotic compensation. Monovalent and polyvalent cations
were added as chloride salts without osmotic compensation.
In most preparations, the activation curve of Ih can be shifted
towards more positive potentials by elevations in the intracellular
cAMP concentration (Pape, 1996), thus increasing active Ih under
resting conditions. In order to determine whether leech Ih channels
are also sensitive to cAMP, we used the membrane-permeable cAMP
analogue dibutyryl-cAMP (dbcAMP). Similarly, we applied 3isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich) to inhibit
phosphodiesterases and, thus, increase the intracellular cAMP
concentration (Bobker and Williams, 1989). To directly block
Ih, we used Cs+ (CsCl, Sigma-Aldrich) or ZD7288
(4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium
chloride; Tocris, Bristol, UK). For experiments in the presence of
ZD7288, the bath perfusion was stopped after adding ZD7288 to
the SLS.
K + e
o
zFErev
RT
zFErev
RT
K +
i
Na+ Na+
i
o
(1)
where [K+]o and [Na+]o are the extracellular and [K+]i and [Na+]i
the intracellular concentrations of K+ and Na+, respectively, z is the
number of elementary charges transferred, R is the universal gas
constant, T is the absolute temperature and Erev is the reversal
(2)
where EK and ENa are the equilibrium potentials for K+ and Na+,
respectively. At a given Em, Ih can be calculated by (Aidley, 1989):
Ih gK(Em EK) + gNa(Em ENa).
(3)
Table1. Size of the voltage sag in touch (T), pressure (P), noxious
(N) and Retzius neurons
Cell type
Em@ICC (mV)
ICC (nA)
T
P
N
Retzius
1.81.0
24.74.7
9.04.2
2.11.9
10021
1056
10717
10111
2
3
2
6
4
8
8
9
The sag amplitude was measured using injection currents (ICC) that
hyperpolarised the cells to approximately 100mV (Em@ICC, where Em is
membrane potential). Voltage sags were absent in Leydig, annulus
erector (AE) and anterior pagoda (AP) neurons (see Fig.1).
20
Retzius
40
6 nA
3 nA
4.5 nA
P
4 nA
60
80
Em (mV)
100
120
500 ms
20
Leydig
2 nA
40
60
AE
5 nA
5 nA
AP
229
80
100
120
230
50 mV
80 mV
4
3
Tail current
Instantaneous current, Ii
2
Steady-state current, ISS
Hyperpolarisation-activated current, Ih
1 nA
200 ms
B
50 mV
Em (mV)
85
80
75
70
65
60
55
50
45
0.0
80 mV
1.0
Ii
1.5
2.0
1 nA
ISS
200 ms
0.5
2.5
3.0
Fig.3. Voltage dependence of Ii and ISS. (A)Membrane currents upon shifting Em from 50 to 80mV (in 2mV steps) for 2s, recorded at intervals of 30s.
With increasing hyperpolarisation, the activation of Ih became progressively faster, while the decay of the tail current appeared to follow an invariant time
course. (B)Relationship between membrane current and Em, either at the beginning of the hyperpolarising step (Ii, open circles) or at its end, after activation
of the Ih channels (ISS, closed circles). Data points are means s.d. of 23 experiments. The voltage dependence of Ii was well fitted by a straight line (red
line), the slope of which corresponded to an input resistance of 23M. In contrast, ISS increased over-proportionally because of the increasing activation of
the Ih channels. The difference between the two currentvoltage relationships (ISSIi) gives the amplitude of Ih activated by the respective voltage step.
231
Em (mV)
60 mV
=368 ms
90
80
70
60
50
40
30
0
0.1
0.3
0.4
80 mV
0.5
0.2
0.6
=158 ms
1=78 ms
2=557 ms
0.7
0.8
1 nA
0.9
200 ms
0
0.2 nA
0
Fig.4. Kinetics of Ih activation and deactivation. (A)At moderate
hyperpolarisations (e.g. 60mV), the activation of Ih was well fitted by a
single exponential function. The upper trace shows the membrane current
upon a hyperpolarisation from 50 to 60mV (black trace), together with
the approximation of Ih by a single exponential function with t368ms and
an amplitude of 0.38nA (red curve). The lower trace shows the difference
between recorded trace and calculated curve at an increased
magnification, allowing an assessment of fit quality. See B for scale bars.
(B)At stronger hyperpolarisations, the approximation of Ih by a single
exponential function became progressively worse, but we obtained a
satisfying fit using a sum of two exponential functions. The black trace
shows the membrane current upon a hyperpolarisation to 80mV, together
with the approximation by a single exponential function (t158ms; red
curve) or the sum of two exponential functions (t178ms, t2557ms; green
curve). The fast component contributed 0.93nA to the total current and
the slow component contributed 0.33nA. The lower two traces show the
difference between the recorded trace and the curves calculated with one
exponential function (red) or the sum of two exponential functions (green).
The decline of the tail currents in A and B could be reasonably fitted by a
single exponential function with a time constant of approximately 200ms.
232
50 mV
90
2s
80
70
Em (mV)
60
50
40
30
0.5
80 mV
80 mV
100 mV
1.0
1.5
2s
Ih (nA)
0.5
2.0
2.5
3.0
Ih
3.5
1 nA
50 ms
1s
Fig.6. Reversal potential of Ih. (A)Experimental protocol. To determine the instantaneous membrane current (Ii) at a given test potential (here, 80mV), Em
was shifted correspondingly for 50ms (upper trace). During this period, the membrane current increased slightly, probably because of the onset of Ih
channel activation (lower trace). Ii was estimated by visual extrapolation of the recorded trace to the beginning of the voltage jump (dark grey circle). After a
2s pause, Em was shifted to 100mV for 3s to achieve full activation of the Ih channels. Subsequently, Em was shifted back to the test potential, in order to
determine the maximum Ih at this potential. For this, the previously recorded Ii was subtracted from the current recorded immediately after the potential jump
from 100mV to the test potential (light grey circle). This protocol was applied every 10s at different test potentials to determine the membrane currents
mediated by the fully activated Ih channels at 30 to 90mV in 10mV increments. (B)Currentvoltage relationships. The data points represent the
membrane currents mediated by the fully activated Ih channels at different Em, as recorded from seven P neurons. In two cells, a reversal of Ih was not
observed; in one cell Ih could only be measured between 90 and 60mV. An error-weighted linear fit of the averaged data gave an Erev of Ih of 35mV
(N7, red line).
2 mmol l1 Cs+
3 min
5mmoll1 Cs+, the block was virtually complete (Fig.7). The onset
of the block was fast and reversible within a few minutes. The time
course of Ih activation was unaffected. The extracellular application
of Ba2+, Co2+ or Ni2+ (5mmoll1 each), or of the K+ channel blocker
tetraethylammonium (TEA, 20mmoll1), for up to 10min had no
effect on Ih (data not shown).
The bradycardic agent ZD7288 selectively blocks Ih channels in
a variety of vertebrate and invertebrate preparations. The halfmaximal effect is mostly exerted at concentrations of 10 to
1.0
3 min
Relative Ih
0.8
0.5 nA
0.6
0.4
0.2
500 ms
0
0
1
Cs+ (mmol l1)
10
Fig.7. Ih is blocked by Cs+. (A)The presence of 2mmoll1 Cs+ reversibly suppressed Ih. Traces show the membrane current upon shifting Em from 50 to
80mV for 1s. Wash-in and wash-out time of Cs+ was 3min, as indicated. (B)Ih in solutions containing various Cs+ concentrations were normalised to the
mean current recorded before and after Cs+ application. The approximation of the data by using the Hill formalism (red line) gave a half-maximal block of Ih
at a Cs+ concentration of 0.3mmoll1. The Hill coefficient was 0.98. Data points are means s.d. of four to six experiments.
233
B
Em(mV)
110 100
90
80
70
60
50
0
1
110 mV
Ii
Iss, Na+-free
4
Iss
50 mV
5
0.5 nA
500 ms
Fig.8. Ih in Na+-free solution. (A)Membrane currents upon shifting Em from 50 to 110mV (in 5mV steps) for 2s, recorded at intervals of 30s. The
recordings were started 3min after replacing extracellular Na+ by NMDG+. In Na+-free solution, Ih was virtually abolished. The holding current for clamping
the cells to the reference potential was slightly reduced (0.20.16nA, N7, cf. Fig.2). (B)Relationship between the membrane current and Em, either at the
beginning of the hyperpolarising step (Ii; open circles) or at its end, after activation of the Ih channels (ISS, Na+-free; closed circles). Data points are means
s.d. of five experiments. The voltage dependence of Ii was fitted by a straight line, the slope of which indicated an input resistance of 27M (see Fig.3). For
comparison, the ISS recorded in SLS is also given (black squares; means s.d., N7).
B
[K+]o (mmol l1)
50 mV
K+-free
48
16
80 mV
12
1
Ih (nA)
K+-free
1
2
4
7
40
8
1 nA
5
3
8
200 ms
16 mmol l1 K+
Fig.9. Effect of extracellular K+ on Ih. (A)Superimposed membrane currents upon shifting Em from 50 to 80mV for 2s, recorded in the same P neuron
5min after adjusting the K+ concentration in the bath as indicated. For better comparison, the traces were slightly shifted vertically to bring the instantaneous
currents recorded immediately after shifting Em to congruence. (B)Amplitudes of Ih at 80mV at different K+ concentrations in the bath solution. Data are
means s.d. of five to 40 experiments (N-values are shown to the right of the error bars).
A
80
75
70
Em (mV)
65 60
B
55
50
0
80
75
70
Em (mV)
65
60
55
0.5
ISS
0.2
1.5
0.3
2.0
0.4
2.5
3.0
80
75
70
65
60
55
0.1
1.0
50
0
0.5
Ii
0.5
D
80
75
70
65
1.0
Ii
50
60
55
50
0
234
0.5
1.5
2.0
1.0
2.5
ISS
3.0
3.5
1.5
Fig.10. Effect of cyclic nucleotides on Ih. (A,C)Ii (open symbols) and ISS (closed symbols) in the presence (circles) and absence (squares) of dibutyryl-cAMP
(dbcAMP, 1mmoll1; A) or the phosphodiesterase blocker IBMX (1mmoll1; C). For clarity, error bars are only shown exemplarily. Although Ih was
decreased by ~50% in the presence of dbcAMP, the instantaneous current was unchanged. In IBMX, the amplitude of Ih remained unchanged but an overall
decrease in the holding current was observed. (B,D)Relationship between tail currents at 50mV and the preceding membrane hyperpolarisation, measured
in the presence of dbcAMP (B) or IBMX (D). The recordings were started 10min after adding dbcAMP or IBMX to the bath solution; single data points were
measured at 10s intervals. The continuous lines indicate approximations of the data by the Boltzmann equation, according to which the Ih channels were
half-maximally activated at 66mV (dbcAMP) or 67mV (IBMX). Note that in the presence of IBMX, the maximum tail current was similar to that in SLS,
whereas it was reduced by more than 50% in the presence of dbcAMP (cf. Fig.5).
235
20
0
Em (mV)
20
3 nA
40
60
+5 nA
80
100
Cs+
120
140
100 ms
10 ms
100 ms
10 ms
10 ms
Fig.11. Effect of Ih channel activation on membrane excitability and action potential waveform. (A)Membrane excitability. In this P neuron, action potentials
could be reliably elicited by injecting a depolarising current of +5nA for 5ms, provided that the Ih channels had been activated by a preceding
hyperpolarisation (3nA for 0.3s; black trace). However, if the Ih channels were blocked by 1mmoll1 Cs+, action potentials were never generated in
response to this stimulus (red trace). Each trace represents the superimposition of five single recordings to demonstrate the reproducibility of the waveforms.
The effect of Cs+ was fully reversible within a few minutes. Identical results were obtained from five cells in total. (B)Action potential waveform. After
activation of the Ih channels by a hyperpolarising current (3nA for 0.3s), the afterhyperpolarisation following an action potential was shortened, and
subsequently reached a transient maximum (red trace, arrow). This was never observed without preceding Ih channel activation (black trace). Amplitude and
time course of the action potentials were not affected by Ih channel activation. As in A, each trace represents the superimposition of five single recordings.
Traces were horizontally shifted to bring action potentials into congruence. In this P neuron, the injection of +2nA for 5ms was sufficient to reliably elicit an
action potential, even without Ih channel activation. (C)Effect of Cs+ on action potentials. Superimposed traces of five consecutively elicited action potentials
under control conditions (black) and in the presence of 1mmoll1 Cs+ (red). Traces were horizontally shifted to bring action potentials into congruence. Cs+
reduced the peak amplitudes and the afterhyperpolarisation. Moreover, the latency from the beginning of the current injection (arrows mark the stimulus
artefact, +2nA for 10ms) to the peak of the action potential was also reduced. The effect of Cs+ was fully reversible. Similar results were obtained for five
cells in total.
For leech HN cells, and briefly for Retzius and P neurons, Ih has
been described before (Angstadt, 1999; Angstadt and Calabrese,
1989; Arbas and Calabrese, 1987b; Coulon et al., 2008; Klees et
al., 2005). However, a detailed description of the distribution and
biophysical properties of leech Ih across cell types was lacking. The
findings presented here can be summarised as follows. First, voltage
sags and the underlying Ih were observed in T, P, N and Retzius
neurons. Second, the current was largest in P neurons. Third, the
biophysical properties of the current largely matched those described
in other preparations: (a) half-maximum activation occurred at
65mV, complete activation at 100mV; (b) the activation could
be fitted by a single time constant of ~370ms at 60mV but required
two time constants of ~80 and ~560ms at 80mV; (c) Erev was
determined to be 3517mV; and (d) the current mutually depended
on both extracellular Na+ and K+, with a pNa/pK of 0.21. Fourth,
half-maximal block of Ih was achieved at a Cs+ concentration of
0.3mmoll1. The current was insensitive to ZD7288. Fifth, after
elevation of the intracellular cAMP concentration, Ih in leech P
neurons showed a blockade, rather than a voltage shift of the
activation curve. Sixth, the channels caused bursts of APs when a
neuron was relieved from hyperpolarisation and modulated the form
of afterhyperpolarisations. And finally, the function of Ih seems to
include a subtle alteration of firing behaviour in leech
mechanosensory neurons as well as a stabilising effect on Em.
Activation kinetics
Ih (nA)
ICs (nA)
%ICs of ISS
AP
Leydig
N
P
T
Retzius
0.10.2
+0.40.2
0.60.4
1.90.4
0.20.3
0.40.4
0.80.6
0.20.8
0.91.0
2.81.4
0.40.3
1.41.7
159
0.227
3929
4724
169
1019
5
5
8
7
4
8
Ih was measured 500ms after a voltage step from 50mV to the holding
potential that gave the largest Ih (see Fig.2). When the current required for
the voltage step in the presence of Cs+ was deducted from the current
measured under control conditions with active Ih (ICs), the resulting
values were always larger. The contribution of the current blocked by Cs+
to the overall current required for the voltage step (%ICs of ISS) reveals
that N and P neurons have the largest relative Ih and will thus be
influenced most by Ih activation. Because AE neurons are rather small and
difficult to access using the two electrode current- and voltage-clamp
techniques, AE neurons were not analysed (but see Fig.1).
236
Ion selectivity
Multi-ion pores
cAMP
EK
Em
ENa
Erev
gNa/gK
HN
IBMX
Ih
Ii
ISS
N
P
pNa/pK
R
SLS
T
T
z
ZD7288
237
ACKNOWLEDGEMENTS
The authors thank Claudia Roderigo and Simone Durry for excellent technical
assistance and Prof. Dr Thomas Budde and Dr Susan Sangha for helpful
comments on the manuscript.
FUNDING
This research received no specific grant from any funding agency in the public,
commercial, or not-for-profit sectors.
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Cell Calcium
journal homepage: www.elsevier.com/locate/ceca
Institut fr Physiologie I, Westflische Wilhelms-Universitt Mnster, Robert-Koch-Str. 27a, 48149 Mnster, Germany
Institut fr Experimentelle Epilepsieforschung, Westflische-Wilhelms-Universitt Mnster, Robert-Koch-Str. 27a, 48149 Mnster, Germany
a r t i c l e
i n f o
Article history:
Received 15 June 2009
Received in revised form 1 September 2009
Accepted 27 September 2009
Available online 14 November 2009
Keywords:
Thalamic reticular nucleus
Ryanodine receptors
SERCA
CICR
Calcium
Two-photon microscopy
a b s t r a c t
The nucleus reticularis thalami (NRT) is a layer of inhibitory neurons that surrounds the dorsal thalamus. It
appears to be the pacemaker of certain forms of slow oscillations in the thalamus and was proposed to be
a key determinant of the internal attentional searchlight as well as the origin of hypersynchronous activity
during absence seizures. Neurons of the NRT exhibit a transient depolarization termed low threshold spike
(LTS) following sustained hyperpolarization. This is caused by the activation of low-voltage-activated
Ca2+ channels (LVACC). Although the role of these channels in thalamocortical oscillations was studied
in great detail, little is known about the downstream intracellular Ca2+ signalling pathways and their
feedback onto the oscillations. A signalling triad consisting of the sarco(endo)plasmic reticulum calcium
ATPase (SERCA), Ca2+ activated K+ channels (SK2), and LVACC is active in dendrites of NRT neurons and
shapes rhythmic oscillations. The aim of our study was to nd out (i) if and how Ca2+ -induced Ca2+
release (CICR) via ryanodine receptors (RyR) can be evoked in NRT neurons and (ii) how the released
Ca2+ affects burst activity. Combining electrophysiological, immunohistochemical, and two-photon Ca2+
imaging techniques, we show that CICR in NRT neurons takes place by a cell-type specic coupling of
LVACC and RyR. CICR could be evoked by the application of caffeine, by activation of LVACC, or by repetitive
LTS generation. During the latter, CICR contributed 30% to the resulting build-up of [Ca2+ ]i . CICR was
abolished by cyclopiazonic acid, a specic blocker for SERCA, or by high concentrations of ryanodine
(50 M). Unlike other thalamic nuclei, in the NRT the activation of high-voltage-activated Ca2+ channels
failed to evoke CICR. While action potentials contributed little to the build-up of [Ca2+ ]i upon repetitive LTS
generation, the Ca2+ released via RyR signicantly reduced the number of action potentials during an LTS
and reduced the neurons low threshold activity, thus potentially reducing hypersynchronicity. This effect
persisted in the presence of the SK2 channel blocker apamin. We conclude that the activation of LVACC
specically causes CICR via RyR in neurons of the NRT, thereby adding a Ca2+ -dependent intracellular
route to the mechanisms determining rhythmic oscillatory bursting in this nucleus.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
The nucleus reticularis thalami (NRT) was proposed to be a key
determinant of the internal attentional searchlight as well as the
origin of hypersynchronous activity during absence seizures [1,2].
Endogenous NRT oscillations were shown to be regulated by Ca2+
entry through LVACC and the subsequent interplay between SERCA
activity and Ca2+ activated K+ channels (SK2) [3]. This intracellular
Ca2+ signalling complex was proposed to play a role in the amplication of low-frequency thalamic oscillations into large scale EEG
waves and it was suggested that physiologically controlled SERCA
activity could represent a method of regulating intrinsic oscillatory
Corresponding author. Tel.: +49 0251 83 58112; fax: +49 0251 83 55551.
E-mail address: coulon@uni-muenster.de (P. Coulon).
0143-4160/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2009.09.005
strength. Our specic hypothesis was that Ca2+ released from intracellular stores directly modulates such oscillatory activity. Intrinsic
oscillations can facilitate CICR, since during such activity considerable amounts of Ca2+ enter the cell. However, whether CICR occurs
in the NRT under these conditions was not known.
The NRT is the central modulatory site for the thalamocortical (TC) relay, and it is involved in generating the major brain
rhythms observed during sleep, wakefulness, and dreaming [46].
Oscillations in this nucleus arise as cyclic interactions between
the GABAergic NRT neurons and TC neurons of other thalamic
nuclei [7]. Burst ring in NRT neurons evokes rhythmic inhibitory
post-synaptic potentials (IPSPs) in TC neurons and an associated deinactivation of the low-voltage-activated (LVA) Ca2+ current that is
then activated upon repolarization and triggers a burst of fast action
potentials. This burst activity is transferred via excitatory synaptic
connections back to NRT neurons.
334
335
Fig. 1. Basic properties of NRT neurons. (A) The NRT was visible as a thin band between the internal capsule and the external medullary lamina or ventral posterolateral
thalamic nucleus. The arrows indicate two patch pipettes, one for the application of drugs (right) and another for whole cell patch clamp recordings. (B) NRT neuron in a
three-dimensional reconstruction after establishing the whole cell patch clamp conguration and equilibration of the pipette solution with the cytosol. The patch electrode
was removed. (C) Superposition of three current-clamp traces from an NRT neuron (upper traces) obtained by varying current injections (lower traces). By applying a DC
offset (not shown), cells were set to 60 mV. During the injection of depolarizing currents the cells showed a series of APs (tonic ring), and after cessation of hyperpolarizing
currents, the neurons produced one or several LTS (burst ring). The LTS is shown to the right at an expanded timescale. It consisted of a long-lasting depolarization crowned
by several APs. APs are truncated. (D) The frequency (fAP ), the number of action potentials (nAP ), and the width of the LTS (widthLTS ) of 180 NRT neurons were plotted in a
three-dimensional graph. The black squares represent the three-dimensional value set. The grey symbols show the projections onto the plane spanning between each pair of
axes: fAP nAP (circles), fAP widthLTS (triangles), widthLTS nAP (squares). Neurons of the NRT showed variations in the values shown but could not be subdivided into different
cell types.
336
2.3. Immunohistochemistry
SpragueDawley rats (postnatal days 1827) were deeply
anaesthetized using pentobarbital (50 mg/kg body weight) and
transcardially perfused with PBS, followed by ice-cold 4% PFA/PBS
for 3540 min. Brains were removed, postxed for 4 h in 4%
PFA/PBS, and cryoprotected with 25% sucrose. Coronal sections
(40 m) were cut at the level of the NRT, washed several times
with TBS, and blocked with 10% normal horse serum (NHS), 2%
BSA, 0.3% Triton X-100 in TBS for 2 h to minimize nonspecic
binding before incubation of slices with primary antibodies in
2% NHS, 2% BSA, 0.3% Triton X-100 in TBS at 4 C for 1618 h.
The following antibodies were used: mouse anti-RyR2 (1:500,
Dianova, Hamburg, Germany, MA3-925), rabbit anti-RyR3 (1:1000,
Chemicon/Millipore, Schwalbach/Ts., Germany), rabbit anti-CaV 3.3
(1:1000, Alomone Labs), and rabbit anti-CaV 3.2 (1:1000, Alomone
Labs). Antibodies against SK2 (guinea pig anti-SK2) were a kind
gift from Dr. Rafael Lujan (Universidad de Castilla-La Mancha,
Fig. 2. Ryanodine receptors, Ca2+ activated K+ channels, and LVA Ca2+ channels are co-expressed in NRT neurons. Fluorescence images of NRT slice preparations with
immunohistochemical labelling of proteins involved in NRT Ca2+ signalling. Photographs in (A) each show a part of the NRT anked medially (left) by the ventrobasal
thalamic complex (VB) and laterally (right) by the internal capsule (ic, see rst photograph for illustration). Photographs in (B) each show a set of superimposed confocal
images, showing individual neurons at a larger magnication (compare scale bars). RyR2 and 3 were co-expressed in the same neurons (top left) with RyR3 delivering a
much stronger signal and showing a somatic expression with particularly strong signals from outside the area where the nucleus is expected (top right). CaV 3.3 and RyR2
were also co-expressed (bottom left), as were RyR3 and SK2 (top right) and CaV 3.2 and SK2 (bottom right).
337
3. Results
3.1. Basic properties of NRT neurons
Neurons of the NRT showed one to several LTS after a hyperpolarizing current injection under current-clamp conditions (Fig. 1C,
right trace, see also Fig. 6A, upper traces, Fig. 7A, 8C, and 9A) or
a comparably long-lasting LVA Ca2+ current under voltage-clamp
conditions (Fig. 5A), as described previously [22,23]. The following
resting properties were found: membrane capacitance: 62 1.6 pF
(n = 331), input resistance: 291 7 M (n = 234), resting membrane potential: 67 0.6 mV (n = 222), width of LTS: 286 11 ms
(n = 180), number of APs crowning a rebound LTS (after recovery
from 85.8 0.4 mV to the resting membrane potential): 4.9 0.2
(n = 180), frequency of AP ring during LTS (measured from the
rst two APs): 151 3.2 Hz (n = 172). Three of these parameters
(width of LTS, number of APs, and AP frequency) are plotted in
Fig. 1D. All parameters were comparable to previously published
values [3,2325]. Some cells (38 out of 204 tested under currentclamp conditions) showed several LTS upon returning to 60 mV or
bursted spontaneously (not shown). Another subset of cells (24 out
of 204) did not show any rebound LTS under these conditions, with
6 cells not showing an LTS at all during current-clamp recordings.
These cells were excluded from further analysis.
Although there was some variety among neurons, we found
the electrophysiological data of the 180 cells investigated under
current-clamp conditions and plotted in Fig. 1D to be insufcient
to subdivide the data set into distinctive cell types, as has been done
previously [25,26].
3.2. Ca2+ signalling proteins in NRT neurons
Fig. 3. Caffeine-induced Ca2+ transients in NRT neurons. (A) Somatic Ca2+ signals
after local application of caffeine (arrows). After the addition of the SERCA blocker
CPA (30 M) basal Ca2+ levels increased and, after 1015 min, caffeine applications
ceased to have an effect. (B) Dendritic (50 m from soma) Ca2+ signals after application of caffeine. Note the reduced scale. (C) Multiple applications of 20 mM caffeine
caused comparable Ca2+ transients.
In order to verify that RyR, LVA Ca2+ channels, and SK2 channels are all co-expressed in neurons of the NRT, we performed
immunohistochemical stainings. We found that RyR2 and 3 are coexpressed alongside with CaV 3.2 and 3.3 LVA Ca2+ channels and
SK2 (see Fig. 2). Type 3 RyR gave a stronger signal than type 2
(Fig. 2A) and were typically located in or near the soma, especially
around the area where the nucleus can be expected (Fig. 2B, top
right photograph). Fast LVA Ca2+ channels (CaV 3.2, Fig. 2B, bottom
right photograph) were also found to be expressed near the soma.
SK2 channels seemed to be expressed strongly in the proximal dendrites (Fig. 2B, bottom right, red), conrming previous ndings [3].
3.4. Ca2+ entry through HVA Ca2+ channels fails to trigger CICR
In the NRT, fast LVA Ca2+ channels of the CaV 3.1 and 3.2 type
appear to be expressed on somatic regions of the plasma membrane while slowly inactivating LVA Ca2+ channels (CaV 3.3) are
preferentially expressed on proximal dendrites [27]. In a previous study, Ca2+ signals of the NRT were investigated in dendritic
regions [3] and it was found that intracellular Ca2+ stores play a
major role in a signalling triad consisting of Ca2+ activated K+ channels of the SK2 type, T-type Ca2+ channels, and SERCAs. In this work
we focused on Ca2+ signals from somatic and proximal dendritic
regions of interest and CICR through RyR. Unless stated otherwise,
we included large parts of the soma and a part of one or several
proximal dendrites (extending no more than 15 m from the soma)
in a region of interest, averaging the cellular Ca2+ signals. Bath application of 10 or 20 mM caffeine resulted in a transient rise in [Ca2+ ]i
(not shown). Local application of 20 mM caffeine in HEPES-buffered
ACSF resulted in Ca2+ transients that were reected in uorescence
changes of 20.5 1.8% FG/R (soma, n = 6, Fig. 3A) and 34.5 5.7% FG/R
(dendrite, 1050 m from soma, n = 4, Fig. 3B). When repeatedly
elicited, these transients reduced their amplitude only slightly (two
out of six cells) or not at all (Fig. 3C; on average, subsequent tran-
338
Fig. 4. Ca2+ transients evoked by HVA Ca2+ channels. (A) Voltage steps from 45 to 5 mV caused HVA Ca2+ currents. (B) Increasing step duration (pulse width) caused
increasing changes in [Ca2+ ]i . (C) Plotting the unit calcium transient (rel. (FG/R )/Q) against the pulse width rendered more or less constant values, suggesting no contribution
of CICR (black boxes, n = 9). When 50100 M ryanodine was present in the pipette solution, a separate set of neurons behaved similarly (dashed line, open boxes, n = 15).
Values were normalized to the longest pulse width. The value at 1000 ms was signicantly reduced in ryanodine (p = 0.03). (D) To verify that CICR did not contribute to the
changes in [Ca2+ ]i under these conditions, the unit calcium transient was recorded both under control conditions and in the presence of 30 M CPA in the same set of neurons
(n = 4). Again, no indication of CICR was observed. Note that the slightly increased values at the rst pulse were caused by noise in the very small uorescence signals at a
pulse width of 50 ms.
contribution of CICR to the changes in [Ca2+ ]i [29,13]. In our experiments, the normalized unit calcium transient was independent
of the (increasing) pulse width of the HVA Ca2+ current activation (Fig. 4C), indicating that all Ca2+ detected by uorescence had
entered via the plasma membrane and was thus also detected as a
charge movement. It could be argued, however, that increasing activation of the Na+ /Ca2+ -exchanger, or other Ca2+ extrusion systems,
act to mask or even reverse an effect resulting from CICR. Since CICR
was reported to occur via RyR elsewhere in the thalamus [13], we
blocked this release in a separate set of experiments by using high
concentrations of ryanodine (50100 M) in the pipette solution.
The normalized unit calcium transient was still independent of the
pulse width under these conditions (Fig. 4C, dashed line). In order
to rule out a masked contribution of IP3 receptors and to eliminate
Ca2+ sequestration by SERCAs, we measured the unit calcium transient before and after the application of CPA (10 min, Fig. 4D). Again,
the normalized unit calcium transient remained independent of the
pulse width. We conclude that intracellular Ca2+ sources do not
contribute to an increase in [Ca2+ ]i induced by HVA Ca2+ channels.
339
Fig. 5. Ca2+ transients evoked by LVA Ca2+ channels (A) Voltage steps from 100 to 45 mV caused LVA Ca2+ currents that fully inactivated within 200 ms. When activated
repeatedly at 2 Hz (with intermediate de-inactivation), LVA currents did not show any signs of run-down, as can be seen in the overlay of the rst and the last in a set of
20 traces (left and middle panel, middle panel recorded after 10 min in 30 M CPA). The overlay of currents under control conditions and in CPA shows that no direct effect
on LVA Ca2+ currents occurred (right panel). (B) Five, ten, and twenty consecutive LVA Ca2+ channel activations at 2 Hz caused Ca2+ transients of increasing amplitude. (C)
In CPA, the transients were reduced in the same neuron (left panel, dashed line/open boxes, n = 6). When ryanodine was added to the bath (50 M), the transients were
reduced in a similar way (right panel, n = 7). In another set of cells, when ryanodine (100 M) was present in the pipette solution, CPA did not have an effect (not shown,
n = 6). Fluorescence values were normalized to the response at 20 LVA activations under control conditions.
nally rendering negative slopes for a plot of the unit calcium transient against the number of activations. However, we found that
the simultaneously measured Ca2+ currents were constant over
time: The rst and last pulse in a series of 20 consecutive LVA
activations were very similar (Fig. 5A, left traces) and this was
also true after 10 min in CPA (middle traces). Thus, an approach
using the unit calcium transient is not mandatory. Instead, we
chose to directly plot the amplitude of the uorescence Ca2+ signal
(see Fig. 5B) against the number of consecutive LVA Ca2+ channel
activations (Fig. 5C; 5, 10, and 20 consecutive LVA Ca2+ current activations). Fluorescence amplitudes were normalized for each cell to
the value obtained with 20 activations under control conditions.
In order to detect a possible inuence of CICR on this relation,
we once again blocked SERCAs with 30 M CPA (10 min) or 2 M
thapsigargin (not shown). In the presence of either blocker, the
resulting Ca2+ uorescence transients were smaller in all cases,
340
indicative of CICR [14]. This build-up was reduced in the presence of CPA (Fig. 6A, lower traces). Even with just a single LTS,
a signicant effect of CPA was discernible and the effect continuously increased with an increasing number of LTS (Fig. 6B).
The application of TTX (0.51 M) caused the abolition of APs
(Fig. 6D) but left the LTS-evoked increase in [Ca2+ ]i almost unaffected. This revealed that APs contributed little to the build-up
of [Ca2+ ]i (7.8 3.8% at 0.3 Hz, n = 9, p = 0.07; 18.0 7.3% at 3 Hz,
n = 8, p = 0.04; Fig. 6C and E). These results conrm previous ndings [3] that an increase of [Ca2+ ]i by a rhythmic generation of
LTS mainly depends on LVA Ca2+ channels and not on APs. Additionally, we show that CICR can be elicited by this rhythmic burst
activity.
3.7. CICR controls repetitive low threshold activity
For the experiments shown in Fig. 6B we elicited up to 32
LTS in succession. As shown in Fig. 7A (left superimposed traces)
the delay between return from step hyperpolarization and the
rst AP of the subsequent LTS considerably decreased between
the rst and the 32nd LTS in a series. This was similar in the
presence of CPA (right traces). However, the number of APs crown-
Fig. 6. Ca2+ transients evoked by LTS and the contribution of action potentials to LTS-evoked increases in [Ca2+ ]i . (A) Repeated LTS (30 at 1 Hz, upper traces, APs truncated)
caused a build-up of [Ca2+ ]i (lower traces). In the presence of 30 M CPA this build-up was signicantly reduced (lower right trace, 73 3%, p = 5 105 , n = 8). Note the
increased number of APs evoked by a rebound burst in the presence of the blocker (see Figs. 79). (B) One or several LTS (2, 4, 8, 16, and 32) at a frequency of 1 Hz caused Ca2+
transients that were signicantly reduced in the presence of CPA (n = 5). Values were normalized to [Ca2+ ]i changes resulting from 32 LTS under control conditions. (C) In the
presence of TTX (0.51 M) Ca2+ transients evoked by 40 LTS at 0.3 Hz or 100 LTS at 3 Hz were largely unchanged when compared to control conditions. (D) LTS under control
conditions and in the presence of TTX. (E) At 0.3 Hz Ca2+ transients were not signicantly changed in the presence of TTX (n = 9), while at 3 Hz the change was signicant,
albeit small (n = 8).
341
Fig. 7. Ca2+ released from internal stores shapes the form of the LTS. (A) By release from hyperpolarizing current injections, 32 LTS were successively evoked. The delay to
the rst AP shortened between the rst and the 32nd LTS. In parallel, the number of crowning APs was reduced (left superimposed traces). The differences between the rst
and the 32nd LTS were very similar in CPA (right superimposed traces), but the number of APs was increased in both cases when compared to controls. APs are truncated.
(B) Statistical analysis revealed a signicantly shorter delay (p = 0.03) in ve out of six cells under control conditions (left panel, black bars) which was probably due to an
accompanying depolarization by 5.5 1.0 mV (n = 5). Although similar, the difference was not signicant in CPA (grey bars). The depolarization between the rst and last LTS
was only slightly reduced here (5.1 2.2 mV). The number of APs crowning the 32nd LTS was signicantly increased in CPA (right panel, p = 0.025).
ing each LTS was increased. The rst LTS in such a series of
32 showed an average delay between the onset of the LTS and
the rst AP of 170 38 ms (n = 6; 1 Hz; Fig. 7B, left panel). In
ve out of six cells, the last LTS showed a signicantly shorter
delay (78 15 ms, n = 5, p = 0.03). The delays between release from
hyperpolarization and the rst AP were similar in the presence
of CPA (1st LTS: 164 62 ms, last LTS: 82 25 ms, n = 5, p = 0.10,
Fig. 7B, left panel). The number of APs elicited during the rst
LTS was only slightly increased in CPA, whereas the 32nd LTS
generated signicantly more APs (3.3 0.8 under control conditions, 4.0 0.9 in the presence of CPA; n = 6, p = 0.025; Fig. 7B, right
panel).
In order to simulate oscillatory activity, and to evoke a usedependent cumulative activation/inactivation of the LVA Ca2+
channels, we repeatedly hyperpolarized NRT neurons at varying frequencies (1, 2, and 3 Hz) after an initial depolarizing step
(Fig. 8A). We chose parameters that would just sufce to evoke a
rebound burst at 1 Hz (i.e. 100 or 150 pA, see lower trace) in
order to assess the frequency-following capability and low threshold activity. We simultaneously recorded the [Ca2+ ]i , showing a
build-up as described in Fig. 6. After each set of hyperpolarizations,
Ca2+ was allowed to return to resting values before continuing the
experiments. Some hyperpolarizations failed to evoke a rebound
burst even at 1 Hz (see Fig. 8B, left trace), and the number of LTS per
hyperpolarization decreased as the frequency increased. This was
true both for the rst ve LTS in a series, that were usually most
reliably evoked, as well as for the last ve LTS, that were evoked
342
Fig. 8. Released Ca2+ modulates burst discharges. (A) Current-clamp recording of an NRT neuron (Em : upper trace, injected current: lower trace). Neurons were set to an Em of
60 mV by DC current injection. The dashed line marks 90 mV. To evoke rebound LTS, currents of 100 or 150 pA were injected after positive current injection, mimicking
a shift from tonic to burst activity. The highlighted areas mark sections shown in (B) and (C) at an expanded timescale. (B) Release from the rst ve hyperpolarizations most
reliably evoked a rebound LTS. In this case, three out of ve releases from hyperpolarization caused an LTS under control conditions (left trace), whereas all ve releases from
hyperpolarization were successful in the presence of ryanodine (right trace). Voltage scale as in A. (C) The form of the LTS in the presence of ryanodine differed from that
under control conditions, most notably the number of APs. (D) Statistical analysis of the rst and last ve events. Under control conditions (black bars) at 3 Hz, the number of
LTS per hyperpolarization was signicantly reduced when compared to 1 Hz for the rst ve events (p = 0.026, unpaired t-test). In the presence of ryanodine this was lessened
and no longer signicant (n = 7 and 10). (E) The number of APs per trace was compared between control conditions (black bars) and in the presence of ryanodine (grey bars).
It was increased in the presence of ryanodine, which was most obvious at 1 Hz and at 3 Hz (1 Hz: p = 0.025, n = 10, 3 Hz: p = 0.046, n = 7). APs are truncated.
343
Fig. 9. The ryanodine-evoked modulation of burst discharges does not rely on SK2 channel function. (A) The application of apamin (100 nM) abolished the afterhyperpolarization following an LTS and suppressed spontaneous repetition of LTS generation as was reported previously [3]. (B) Representative trace taken under similar conditions
as shown in Fig. 8B with apamin present in the bath solution. In the presence of ryanodine a similar modulation of burst discharges occurred. The example shows the last
ve traces of a neuron that was repeatedly hyperpolarized at 3 Hz with a current of 150 pA. (C) Both the rst and the last ve events showed signicantly fewer LTS per
hyperpolarization at 3 Hz when compared to 1 Hz (in the presence of 100 nM apamin; rst ve events: p = 0.003, n = 7; last ve events: p = 0.043, n = 7). After ryanodine was
added to the bath solution this effect was reduced in much the same way as shown in Fig. 8D. Here too, the remaining decrease was not signicant (n.s.; p = 0.12). (D) The
number of action potentials elicited in a sweep (see Fig. 8A) were similarly increased in the presence of apamin. APs are truncated.
hyperpolarizations was 90: 29.7 9.6 failures under control conditions with apamin, 11.6 7.1 failures in ryanodine with apamin,
p = 0.009). In ryanodine and apamin, the number of LTS per hyperpolarization for the rst and last 5 events was increased at all
frequencies (with the exception of the rst 5 traces at 1 Hz, Fig. 9B;
Fig. 9C, grey bars; 2 Hz not shown). Again, ryanodine rendered the
remaining frequency-dependent decrease of the number of LTS per
hyperpolarization not signicant. Although ryanodine did not seem
to alter the shape of the rebound LTS in the presence of apamin by
as much as in its absence, here too, more APs were observed per
LTS (Fig. 9D). Combined with the increased low threshold activity
(LTS per hyperpolarization), more APs per trace were generated
at all frequencies, with 1 and 2 Hz showing a signicant effect
(Fig. 9D).
4. Discussion
While the inputoutput connections as well as cellular electrogenic mechanisms of the NRT have been studied extensively
[3,6,25], the dynamics of intracellular Ca2+ signalling are far from
being completely understood. To our knowledge, the present study
is the rst to provide evidence for Ca2+ release mechanisms and
their functional signicance in NRT neurons. The results can be
summarized as follows: (1) We demonstrated Ca2+ release in NRT
neurons. (2) CICR via RyR was coupled to LVA Ca2+ channels, could
be evoked by repetitive LTS generation, and contributed 30% to
stimulus evoked transients and Ca2+ build-up during repetitive
stimulation. It was abolished in the presence of CPA or ryanodine.
(3) HVA Ca2+ currents failed to evoke CICR and APs contributed little
344
Ca2+ channels failed to elicit CICR, although the overall HVA Ca2+
channel-induced Ca2+ increase was generally larger. The spatial
coexpression of LVA Ca2+ channels and SERCAs was shown previously [3], suggesting a close proximity of these channels to the
respective Ca2+ release sites.
4.3. CICR and LVA Ca2+ channels in NRT neurons
It could be argued that an amplication mechanism such as CICR
would result in a supra-linear dependence of [Ca2+ ]i on the number of LTS- or LVA Ca2+ channel activations, comparable to what
has been reported for HVA Ca2+ channel-induced Ca2+ transients
[29,13]. However, HVA Ca2+ channel stimulation is continuous,
since these channels do not completely inactivate, while LVA Ca2+
channel activation has to occur repetitively to de-inactivate the
channels in between stimuli [14,23]. This mimics the putative physiological scenario of oscillatory bursting but limits the amount
of Ca2+ that can be accumulated within a given amount of time,
because of the counteracting effects of Ca2+ sequestration and
extrusion and the time consuming de-inactivation process. As a
result, to induce Ca2+ accumulations of comparable amplitude, LVA
Ca2+ currents must be activated over a period of time that is 10 to
20 times longer than for HVA Ca2+ currents (HVA Ca2+ channel activation of 500 ms: 0.31 0.04 FG/R , n = 9; 20 LVA Ca2+ channel
activation @ 2 Hz: 0.32 0.06 FG/R , n = 6). During this increased
time, larger amounts of Ca2+ can be expected to be passively or
actively extruded from the neuron or transported back into the
stores. Thus, the effect of CICR will not lead to a supra-linear Ca2+
accumulation but may instead be a prerequisite for an increase of
[Ca2+ ]i to occur, as has been proposed previously [14].
4.4. Physiological and pathophysiological relevance of CICR in the
NRT
The interplay between NRT neurons and TC relay neurons during the early phases of sleep has been studied extensively. Here, the
physiological function of the NRT is believed to lie in the generation of sleep spindles and the structuring of slow oscillatory activity
[6]. During attentive wakefulness the GABAergic NRT neurons are
tonically active at low frequencies [38]. By suppressing background
activity, the projections into the dorsal thalamus enhance the transfer of afferent information via relay neurons. However, since NRT
neurons reach intraburst frequencies during slow-wave sleep that
are higher than the fastest tonic discharges during steady states
of wakefulness [38], the nucleus primary function seems to be
reected in its ability to produce burst discharges. This idea is further strengthened by the ndings of this study, since CICR seems
to play a role only during burst activity and modulates it in return.
Burst activity in the NRT has been implicated in the generation and/or maintenance of generalized nonconvulsive seizures in
rodent models [39,40]. Moreover, an increase of LVA Ca2+ current in
the NRT, relating to an increase of the expression of CaV 3.2, has been
observed in a rat model of absence epilepsy [41,42]. Under both
physiological and pathophysiological conditions, burst ring in the
NRT and associated thalamic synaptic rebounds are characterized
by a typical temporal pattern, indicated by the waxing and waning of the LTS burst oscillation and the acceleration/deceleration
sequence of APs within each burst [2]. Important differences of
pathophysiological activity, such as absence seizures, are the high
degree of synchronization, more stereotyped oscillations at low
frequencies, and the more sustained nature of the synchronized
oscillatory activity [43]. While the exact contribution of CICR to
these phenomena is not clear, the present results demonstrate that
CICR signicantly contributes to the frequency-dependent characteristics of the LTS activity pattern and the APs generated by
345
346
[35] T. Murayama, Y. Ogawa, RyR1 exhibits lower gain of CICR activity than RyR3 in
the SR: evidence for selective stabilization of RyR1 channel, Am. J. Physiol. Cell
Physiol. 287 (2004) C3645.
[36] C. Franzini-Armstrong, F. Protasi, Ryanodine receptors of striated muscles:
a complex channel capable of multiple interactions, Physiol. Rev. 77 (1997)
699729.
[37] M. Stocker, P. Pedarzani, Differential distribution of three Ca2+ -activated K+
channel subunits, SK1, SK2, and SK3, in the adult rat central nervous system,
Mol. Cell. Neurosci. 15 (2000) 476493.
[38] M. Steriade, L. Domich, G. Oakson, Reticularis thalami neurons revisited: activity changes during shifts in states of vigilance, J. Neurosci. 6 (1986) 6881.
[39] G. Avanzini, M. Vergnes, R. Spreaco, C Marescaux, Calcium-dependent regulation of genetically determined spike and waves by the reticular thalamic
nucleus of rats, Epilepsia 34 (1993) 17.
[40] V. Crunelli, N. Leresche, Childhood absence epilepsy: genes, channels, neurons
and networks, Nat. Rev. Neurosci. 3 (2002) 371382.
doi:10.1111/j.1460-9568.2010.07081.x
SYNAPTIC MECHANISMS
Keywords: Ca2+-induced Ca2+ release, Ca2+ signalling, high-voltage-activated Ca2+ channels, rat, ryanodine receptors,
thalamic function
Abstract
Neuronal Ca2+ channels are rapidly inactivated by a mechanism that is termed Ca2+-dependent inactivation (CDI). In this study we
investigated the influence of intracellular Ca2+ release on CDI of high-voltage-activated Ca2+ channels in rat thalamocortical relay
neurons by combining voltage-clamp, Ca2+ imaging and immunological techniques. Double-pulse protocols revealed CDI, which
depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was
accompanied by an increase in the duration of Ca2+ transients. Inhibition of ryanodine receptors and endoplasmic Ca2+ pumps (by
thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by
intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential
channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The
central role of L-type Ca2+ channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when
Ca2+ was replaced by Ba2+ and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N,N,-tetraacetic acid (BAPTA). Trains of action
potential-like stimuli induced a strong reduction in high-voltage-activated Ca2+ current amplitude, which was significantly reduced
when intracellular Ca2+ stores were made inoperative by thapsigargin or Ba2+ BAPTA. Western blotting revealed expression of Ltype Ca2+ channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling
between L-type Ca2+ channels and ryanodine receptors that controls the amount of Ca2+ influx during neuronal activity.
Introduction
Voltage-gated Ca2+ channels of the plasma membrane have three
subfamilies (CaV1, CaV2 and CaV3) (Lacinova, 2005). They are
composed of 10 pore-forming a1 channel subunits and are important
components of a universal cellular Ca2+ signalling tool kit (Berridge
et al., 2000). Depending on the specic voltage-gated Ca2+ channel
subtype, Ca2+-induced Ca2+ release (CICR) from intracellular stores is
a mechanism that amplies the Ca2+ entry through plasma membrane
Ca2+ channels, whereas Ca2+-dependent inactivation (CDI) of these
plasma membrane channels represents an important negative feedback
mechanism (Budde et al., 2002; Bardo et al., 2006). With respect to
the thalamus, only recently has a role for Ca2+ ions beyond being
charge carriers started to evolve, and contributions of low-voltageactivated (CaV3) as well as high-voltage-activated (HVA) (CaV1 and
CaV2) Ca2+ channels to intracellular Ca2+ signals have been found
Tissue preparation
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
Ca 2+ imaging
Patch-clamp recordings
Whole-cell recordings were performed on visually identied TC
neurons of the dLGN at room temperature (2123C), using glass
microelectrodes pulled from borosilicate glass capillaries (GC150TF10; Clark Electromedical Instruments, Pangbourne, UK) that were
connected to an EPC-9 2 amplier (double patch clamp; HEKA,
Lambrecht, Germany). The typical electrode resistance was 24 MX,
whereas access resistance was 515 MX. Series resistance compensation was routinely used (> 30%). Voltage-clamp experiments were
governed by Pulse software (HEKA Electronics). For standard recordings, the following solutions were used: (i) extracellular solution
(in mm): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2226 NaHCO3, 2
MgSO4, 2 CaCl2, 10 glucose, 0.001 tetrodotoxin and 4 4-aminopyridine, pH 7.35 with NaOH; and (ii) intracellular solution (in mm): 85
Cs-gluconate, 10 Cs3-citrate, 10 NaCl, 1 KCl, 1.1 EGTA, 0.1 CaCl2,
0.25 MgCl2, 10 HEPES, 15 tetraethylammonium (TEA)-Cl, 3 Mg-ATP
and 0.5 Na-GTP, pH 7.25 with CsOH. For measurements with trains of
action potential-like stimuli, the following intracellular solution was
used (in mm): 95 CsMeSO4, 10 NaCl, 1 KCl, 1.1 EGTA, 0.1 CaCl2, 0.25
MgCl, 10 HEPES, 15 TEA-Cl, 3 Mg-ATP, 0.5 Na-GTP, 5
4-aminopyridine, 3.35 QX-314-Cl and 15 phosphocreatine, pH 7.25
with CsOH.
The HVA Ca2+ currents were evoked from a holding potential of
)40 mV. For conditioning pulses, the voltage was stepped to varying
potentials ()40 to +60 mV, 200 ms duration; in some experiments the
conditioning pulse length was varied between 50 and 1000 ms),
followed by a brief gap ()40 mV, 50 ms) and a subsequent analysing
test pulse to a xed potential of +10 mV (200 ms). Double pulses were
applied every 6.4 s. In one set of experiments, double pulses were
repeated every 60 s. For standard recordings, Ca2+ was used as the charge
carrier and 1.1 mm EGTA was included in the intracellular solution. In
one set of experiments Ba2+ was used as the charge carrier and 11 mm
BAPTA was included in the intracellular solution. The degree of
inactivation (Dinact) (see bracket in Fig. 1B) was determined as follows
Dinact 1I min =I max 100%
with Imin representing the minimal test pulse amplitude after a preceding conditioning pulse, as measured at the beginning of the voltage step
to +10 mV (upper arrowhead in Fig. 1B), and Imax representing the
maximal test pulse amplitude (lower arrowhead), as measured without a
preceding conditioning pulse. Time-dependent inactivation of the
conditioning pulse to +10 mV was assessed by calculating the
inactivation ratio (Rinact), which was determined as follows
Rinact I 200 =I peak
with I200 and Ipeak representing the current amplitude at the end of
the 200 ms pulse and the peak amplitude, respectively. Rinact values
can be expected to decrease as time-dependent inactivation becomes
faster.
Results
Ca 2+-dependent inactivation is active in thalamocortical relay
neurons of acute brain slice preparations
To conrm the presence of CDI in dLGN TC neurons of thalamic
slices, HVA Ca2+ currents were recorded from more than 150 cells.
A double-pulse voltage protocol (Fig. 1A; see Materials and methods)
was used that effectively discloses CDI (Budde et al., 2002). If CDI is
operative, the current evoked by the test pulse should exhibit an
inverted U-shaped dependence (due to the normalization procedure
used in the present study) on the conditioning pulse potential, with
maximal inactivation occurring at the peak of the conditioning pulse
currentvoltage (IV ) relationship. Under standard conditions, the IV
relationship of the conditioning pulse (Fig. 1C, open squares)
demonstrated HVA Ca2+ currents (Fig. 1B) with an activation
threshold negative to )30 mV, a maximal inward current at around
+10 mV and an apparent reversal potential at around +40 mV. The test
pulse IV (peak amplitude of the test pulse current plotted vs.
conditioning pulse voltage) showed an inverted U-shape with the
minimal current occurring at +10 mV (Fig. 1C, closed squares), as
expected for a CDI mechanism. In the following the Dinact was used as
a simple measure for the strength of CDI. This parameter was
determined by dividing the minimal test pulse amplitude (following
the conditioning pulse to +10 mV; upper arrowhead in Fig. 1B) by the
maximal test pulse amplitude (occurring when no preceding conditioning pulse was applied; lower arrowhead in Fig. 1B) and calculating the percent change in current amplitude. With respect to the
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
conditioning pulse
HVA Ca 2+ currents
+60 mV
1 nA
200 ms
test pulse
+10 mV
Dinact
-40 mV
200ms
200ms
D
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
***
60
***
50
*** (120)
40
30
20
-40 -20
0
20
40
60
Conditioning pulse potential (mV)
(5)
(5)
Dinact (%)
I/Imax
(5)
(5)
100
1000
Conditioning pulse length (ms)
Fig. 1. Identication of CDI in TC neurons in the slice preparation. (A) Scheme of the double-pulse protocol used to elicit HVA Ca2+ currents. TC neurons were
held at -40 mV and stepped to varying potentials (conditioning pulse, )40 to +60 mV, 200 ms duration). This was followed by a brief pause ()40 mV, 50 ms) and a
subsequent analysing test pulse to a xed potential of +10 mV (200 ms). (B) Representative current traces elicited by the pulse protocol shown in A. The Dinact,
which was taken as a measure of the strength of CDI, is indicated by the two arrowheads and bracket. (C) Mean IV relationship evoked by the conditioning pulse
(open squares; data from 10 randomly chosen cells were averaged) and the test pulse depending on conditioning pulses of increasing depolarization (closed squares;
data from 120 cells were averaged) are shown. Normalized current amplitudes are plotted vs. the conditioning pulse potential. (D) Dependency of Dinact on the length
of the conditioning pulse. Signicance was tested against values obtained by the shortest conditioning pulse duration of 50 ms (the number of observations is
indicated in brackets) ***P < 0.001.
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
control
1
-0.4
10 mM
caffeine
-0.6
2
+10 mV
I/Imax
caffeine
1
2
-40 mV
2
1
-0.8
control
-1.0
1nA
-30
0
30
60
Conditioning pulse potential (mV)
200ms
***
*
**
*
(5)
(6)
40
(16)
(5)
**
40
30
(10)
10
15
20
(14)
***
(14)
(10)
10 (14)
(120)
***
(15)
50
20
***
(14)
***
(14)
10 mM caffeine
60
(5)
Dinact (%)
50
35
70
***
55
45
control
80
60
Dinact (%)
***
***
******
**
******
***
(22)
(14)
**
100
10 M ryanodine
1000
Fig. 2. Effect of caffeine on CDI. (A) Scheme of the double-pulse protocol (left) and representative current traces recorded under control conditions (upper right
panel) and during extracellular application of caffeine (10 mm; lower right panel) with no conditioning pulse (1) and a conditioning pulse to +10 mV (2) are shown
(traces were taken from the same cell). (B) Mean IV relationship of normalized currents evoked by the test pulse under control conditions (closed squares) and in the
presence of 10 mm caffeine (closed circles). (C) Concentration-dependent effect of caffeine on Dinact. (D) Dependency of Dinact on the length of the conditioning pulse
in the presence of caffeine (10 mm; closed squares) and ryanodine (10 lm; closed triangles). Signicance was tested against values obtained with the shortest prepulse
duration (50 ms) for each substance. The dashed line indicates the t through the control data points shown in Fig. 1D. *P < 0.05, **P < 0.01, ***P < 0.001.
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
0.75
+60 mV
2, 2
Ratio 350/380
3, 3
2
1
3
20 m
0.60
control
0.50
20
100
0.05
60
80
100
0.10
1, 1
40
Time (s)
0.15
0.00
-40 mV
caffeine
Ratio 350/380
0.65
control
caffeine
0.20
+10 mV
1, 1
0.55
(1) soma
(2) somato-dendritic junction
(3) proximal dendrite
(4) background
0.70
2, 2
3, 3
control
caffeine
**
80
**
*
60
40
20
0
1, 1
2, 2
3, 3
Fig. 3. Ca2+ imaging in TC neurons. (A) Typical TC neuron loaded with 100 lm bis-Fura-2. Regions of interest in different cellular compartments are indicated.
(B) Recording of somatic uorescence ratio changes in TC neurons under control conditions (black trace) and during application of 10 mm caffeine (grey trace).
Ca2+ transients were elicited by three double pulses (see scheme) causing three Ca2+ peaks. In this set of experiments, each double-pulse application consisted of a
variable conditioning pulse ()40, +10 and +60 mV, 500 ms; 1, 2 and 3), a pause of 50 ms and a test pulse xed to +10 mV (200 ms; , and ). The resulting
three Ca2+ transients are labelled accordingly (rst double pulse: 1, ; second double pulse: 2, ; third double pulse: 3, ). For clarity, ratio traces were shifted by
2.5 s in time. The beginning of electrical stimulation is indicated by the arrowheads. Note that the recovery of intracellular Ca2+ concentration back to baseline levels
is delayed in caffeine after termination of electrical stimulation. (C) Bar graph representation of Ca2+ transient peak amplitudes (measured from the basal Ca2+ level
for each recording condition) under control conditions (black bars) and in the presence of caffeine (10 mm; white bars). (D) Relative recovery of uorescence ratio
back to baseline levels with respect to the transient peak amplitude at 10, 20 and 30 s after starting stimulation of the cell (control, black bars; 10 mm caffeine, open
bars). Signicance was calculated for the comparison of the two recording conditions. *P < 0.05, **P < 0.01.
Discussion
2+
of CDI. (vi) Trains of action potential-like stimuli induced an ERdependent reduction in HVA Ca2+ current amplitude. (vii) The degree
of CDI in the presence of the specic L-type Ca2+ channel blocker
nifedipine was strongly reduced and close to the value that was
reached when Ca2+-dependent signalling was completely disabled by
using Ba2+ BAPTA. (viii) CaV1.2 channels were expressed in dLGN
tissue.
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
B
control
thapsigargin
CPA
ryanodine
heparin
2APB
1 nA
+10 mV
200 ms
60
-40 mV
Dinact (%)
50
40
(120)
30
***
***
(4)
(4)
(6)
***
(4)
(22)
20
10
He p a ir n
ar
in
in
e
ry
an
od
CP
A
n
th
ap
si
ga
rg
i
l
nt
ro
co
APB
Ry
CPA
he
p
haps i
T
c o n VC
2A
PB
Fig. 4. Inuence of the ER on CDI. Representative current traces (upper panels; see pulse protocol in the inset) and bar graph representation of Dinact (lower panels)
obtained under control conditions (A) and in the presence of thapsigargin (10 lm; extracellular application) (B), CPA (30 lm; extracellular application) (C),
ryanodine (10 lm; extracellular application) (D), 2APB (100 lm, intracellular application) (E) and heparin (2 mg mL; intracellular application) (F). The scale bars
in F apply to all current traces. ***P < 0.001.
-0.4
60
-0.6
I/Imax
control
* * * *
-0.8
2APB
* *
2APB
Dinact (%)
50
control
40
(120)
(6)
30
20
c o n VC
AP Be xt
2A
PB
-40 -20
0
20
40
60
Conditioning pulse potential (mV)
nt
ro
200 ms
-1.0
co
1 nA
10
Fig. 5. Inuence of store-operated Ca2+ inux on CDI. (A) Representative current traces recorded under control conditions (middle panel) and during extracellular
application of 2APB (100 lm; lower panel) elicited by the indicated pulse protocol (upper panel; all traces were taken from the same cell). (B) Mean IV relationship
of normalized currents evoked by the test pulse under control conditions (closed squares) and in the presence of 2APB (closed circles). (C) Bar graph representation
of Dinact under different recording conditions (as indicated). *P < <0.05.
cytoskeleton (Meuth et al., 2001, 2002, 2005). The present study adds
to these ndings by demonstrating an inuence of intracellular Ca2+
release on the degree of CDI. Although acutely isolated cell
preparations have the advantage of a low run-down of HVA Ca2+
currents (Budde et al., 1998), the cells are devoid of most of their
dendritic tree and synaptic connections. Here, we conrmed the
occurrence of CDI in TC neurons in the slice preparation by using the
well-established double-pulse protocol (Eckert & Tillotson, 1981;
Armstrong, 1989) and found a degree of CDI of 39% under
comparable control conditions. Although this is very similar to our
previous ndings, the data interpretation may be complicated by the
occurrence of HVA Ca2+ current run-down and a partial U-shape of
the test pulse IV. As a reduction of the driving force of Ca2+ currents
has only very little effect on the degree of CDI in TC neurons in slices
(this study) and after acute isolation (Meuth et al., 2001), an inuence
of run-down on the inactivation process seems unlikely. The partial
U-shape of the inactivation curve may be explained as follows. (i) The
Ca2+ imaging data presented here indicate that removal of Ca2+ from
the cytoplasm between voltage steps is incomplete with stimulation
frequencies around 0.1 Hz. Indeed, when the stimulation frequency
was reduced to 0.0167 Hz, the inactivation curve returned to baseline
with the most depolarized conditioning pulses. This view is in
agreement with the slow Ca2+ transients found under low-afnity but
high-capacity Ca2+-binding conditions (EGTA, citrate and gluconate)
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
B
nifedipine
nifedipine
+ CPA
E
nifedipine
+ ryanodine
Ba2+/BAPTA
1 nA
control
200 ms
60
+10 mV
Dinact (%)
50
40
(120)
-40 mV
30
20
***
***
***
(8)
(5)
(6)
***
(7)
10
Ba 2 + BAPTA
2+
/B
AP
TA
Ni fRy
Ba
ne
ni
fe
di
pi
l
nt
ro
co
Ni fCP A
n
+ ifed
ry ip
an i n
od e
in
e
Nfi
c o n VC
n
+ ifed
CP ip
A ine
Fig. 6. Inuence of L-type Ca2+ channels on CDI. Representative current traces (upper panel; see inset for the pulse protocol) and bar graph representation of Dinact
(lower panel) obtained under control conditions (A), in the presence of nifedipine (1 lm; extracellular application) (B) and in the presence of nifedipine and CPA (C),
nifedipine and ryanodine (D), and Ba2+ (3 mm, as charge carrier) with BAPTA (11 mm, as Ca2+ chelator) (E). Scale bars in E apply to all current traces.
***P < 0.001.
(Meuth et al., 2002), which were also used in the present study.
(ii) The partially U-shaped curve is consistent with the dominance of
VDI over CDI at strongly depolarized potentials (Hadley & Hume,
1987; Hadley & Lederer, 1991). However, the shift in hysteresis of the
inactivation curve when the order of the conditioning pulses was
reversed (from the most depolarized potentials to the most hyperpolarized potentials) indicates only a small contribution of VDI with
200 ms pulses. The rather strong Dinact with long conditioning pulses
(5001000 ms) indicates that a signicant part of the inactivation can
be attributed to the slow VDI process during prolonged depolarization
(Lacinova, 2005; Lacinova & Hofmann, 2005).
Interaction between Ca 2+-induced Ca 2+ release and
Ca 2+-dependent inactivation
A prototypical feature of Ca2+ signalling in cardiac cells and neurons
is the release of Ca2+ from intracellular stores via the activation of
RyR by Ca2+ inux through closely connected L-type Ca2+ channels
(Berridge et al., 2000). In ventricular myocytes, a signicant fraction
of CDI depends on CICR and is therefore termed CICR-dependent
CDI (Grantham & Cannell, 1996; Takamatsu et al., 2003). Thus, the
negative feedback of intracellular Ca2+ release on CDI in TC neurons
is in good agreement with the detection of RyR2, RyR3 and CaV1.2 in
TC neurons and dLGN tissue by specic uorescent blockers (Budde
et al., 1998) and antibodies (Budde et al., 2000; Meuth et al., 2001).
In a previous study on isolated TC neurons it was assumed that 10 mm
of the low-afnity Ca2+ buffer citrate interfered with intracellular
signalling such that Ca2+ entry via the plasma membrane and the
subsequent release via the ER were separated in time (Meuth et al.,
2002). Although this may indeed be true for uorescent measurements
of bulk intracellular Ca2+ levels (Budde et al., 2000), the following
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
test pulse
control pulse
0 mV
99 - 101
1-3
-40 mV
198 - 200
B
control
C
thapsigargin
Ba2+/
BAPTA
40
(5)
30
(8)
-45 to +50 mV
3ms
***
20
(6)
***
-50 to -45 mV
1 ms
10
0
+50 mV, 1 ms
c o n rt o
l
control
Ba B
/ AP TA
t a
hps i
thapsi
+50 to -50 mV
3 ms
2+
Ba /BAPTA
Fig. 7. Interaction between CDI and intracellular Ca2+ stores during trains of action potential-like stimuli. (A) Scheme of the stimulation protocol. A voltage
step (200 ms, from )40 to 0 mV) was applied 1 min before (control pulse) and immediately after (test pulse) a train (200 pulses of 30 Hz) of short
depolarizations. Current traces evoked by pulses before (left column), during (three middle columns; responses to the rst, second, third, 99th, 100th, 101st,
198th, 199th and 200th pulses in a train are shown) and following (right column) train stimulation under control conditions (B), during extracellular
application of thapsigargin (10 lm) (C) and using Ba2+ (3 mm, as charge carrier) with BAPTA (11 mm, as Ca2+ chelator) (D). (E) Mean bar graph
representation of the reduction of the test current peak amplitude in relation to the control current. (F) Scheme of the action potential-like voltage protocol.
***P < 0.001.
thereby supporting the recruitment of CICR with longer conditioning pulse durations. It is interesting to note that the block of
transient receptor potential channels indicates that relling of
intracellular Ca2+ stores depends on Ca2+ inow from the
extracellular space.
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
Supporting Information
Additional supporting information may be found in the online version
of this article:
Fig. S1. Western blot analysis of the membrane fraction of rat
hippocampus, thalamus, and liver.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset by Wiley-Blackwell.
Technical support issues arising from supporting information (other
than missing les) should be addressed to the authors.
Acknowledgements
This work was supported by DFG (BU 1019 8-1 and GRK 1167-P1). P.E. was
a fellow of the Otto Creutzfeldt Center for Cognitive and Behavioral
Neuroscience Munster. Thanks are due to A. Jahn, M. Marunde, E. Na and
R. Ziegler for excellent technical assistance. This work was performed in partial
fulllment of the PhD theses of P.E. and V.R.
Abbreviations
2APB, 2-aminoethoxy diphenyl borate; CDI, Ca2+-dependent inactivation;
CICR, Ca2+-induced Ca2+ release; CPA, cyclopiazonic acid; Dinact, degree of
inactivation; dLGN, dorsal part of the lateral geniculate nucleus; ER,
endoplasmic reticulum; HVA, high-voltage-activated; IV, current-voltage;
Rinact, inactivation ratio; RyR, ryanodine receptors; SERCA, sarcoplasmic endoplasmic Ca2+ ATPase; TC, thalamocortical relay; VDI, voltagedependent inactivation.
References
2+
It has been suggested previously that HVA Ca2+ channels and their
inherent CDI process are most relevant for neuronal activity of dLGN
TC neurons seen during depolarized membrane states, including tonic
ring of action potentials, plateau-like depolarizations evoked by optic
tract stimulation during early development and fast high-threshold
Armstrong, D.L. (1989) Calcium channel regulation by calcineurin, a Ca2+activated phosphatase in mammalian brain. Trends Neurosci., 12, 117122.
Bardo, S., Cavazzini, M.G. & Emptage, N. (2006) The role of the endoplasmic
reticulum Ca2+ store in the plasticity of central neurons. Trends Pharmacol.
Sci., 27, 7884.
Berridge, M.J., Lipp, P. & Bootman, M.D. (2000) The versatility and
universality of calcium signalling. Nat. Rev. Mol. Cell Biol., 1, 1121.
Bertram, R., Smith, G.D. & Sherman, A. (1999) Modeling study of the effects
of overlapping Ca2+ microdomains on neurotransmitter release. Biophys. J.,
76, 735750.
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
The Authors (2010). Journal Compilation Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 31, 439449
a
Westflische Wilhelms-Universitt Mnster, Institut fr Physiologie I,
Robert-Koch-Strasse 27a, D-48149 Mnster, Germany
b
C. u. O. Vogt Institut fr Hirnforschung, Universittsstr. 1, D-40225
Dsseldorf, Germany
The initial step in cortical processing of sensory information is the arrival of afferent information via the thalamus.
Except for olfactory signals, all sensory information is relayed to primary sensory cortical areas through specific
thalamic nuclei. From the primary sensory areas the information is thought to be projected to second and higher
order cortical areas, initializing a complex activity pattern,
including parallel processing and feedback projections.
Thalamocortical brain slice preparations have proven
to be valuable tools in the study of thalamocortical interactions. Previous in vitro studies relied on stimulation on
the border between layer 6 and the white matter, or of layer
4, to mimic thalamic input to the cortex. To date, three
different thalamocortical brain slice preparations have
been described: one somatosensory (Agmon and Connors, 1991), one auditory (Cruikshank et al., 2002), and
one visual (MacLean et al., 2006). The most thoroughly
studied is the somatosensory thalamocortical slice, in
which optical imaging has been successfully used to visualize cortical responses to thalamic input. Using these
methods, it has been shown that thalamic input to the
barrel cortex evokes cortical excitation initially in layers 4
and 5, from where the activity spreads in vertical and
horizontal directions (Laaris et al., 2000; Llinas et al.,
2002).
The primary auditory cortex (A1) is believed to differ
from other well studied primary sensory areas (visual, V1,
and somatosensory, S1) in terms of localization of
thalamocortical input and intracortical circuitry (Atzori et
al., 2001; Linden and Schreiner, 2003; Winer and Lee,
2007; Barbour and Callaway, 2008). In contrast to the
concentrated thalamocortical innervation of spiny stellate
neurons of layer 4 in S1 and V1, the auditory cortex seems
to receive afferents in a more graded fashion, with the
majority of terminations being located in layers 3 and 4.
Furthermore, the thalamorecipient cell types differ between the cortices, due to the absence of spiny stellate
cells in A1 (Smith and Populin, 2001; Linden and Schreiner, 2003; Winer et al., 2005; Barbour and Callaway,
2008).
0306-4522/10 $ - see front matter 2010 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2009.10.025
371
372
EXPERIMENTAL PROCEDURES
Unless stated otherwise, the experimental procedures were performed on adult mice (C57BL/6, 9 14 weeks) of both genders and
all chemicals were obtained from Sigma (Sigma-Aldrich, Munich,
Germany). All animal procedures were approved by the local
authorities.
Fig. 1. Agar ramp used for preparation of angled horizontal slices. (A)
Teflon form used to produce agar ramps. The depicted form yields 20
ramps. (B) Posterior view of mouse brain glued on a 25 agar ramp.
The ramp is glued onto the vibratome mounting platform. (C) Birds eye
view of a mouse brain glued on a 25 agar ramp in the vibratome.
Blade used to cut slices is visible in the upper right corner. The
cerebellum and the bulbus olfactorius have been removed. Scale Bars
indicate 1 cm.
Patch-Clamp
Patch-Clamp recordings were performed as described previously
(Broicher et al., 2008). Briefly, whole cell recordings in brain slice
preparations were done using glass microelectrodes pulled from
373
Histology
For biocytin staining, slices were cut as described above and
placed into an interface chamber at 30 C in standard ACSF.
Biocytin crystals were applied to the primary auditory cortex using
the tip of a needle. After biocytin application, slices were incubated
for 6 h in the interface chamber. Then, slices were fixed in 4%
paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight. For immunohistochemistry of slices used in physiological
experiments, a 4% formamide (FA) fixation was used. After fixation, the brain slices (500 m) were soaked in PBS (0.1 M, pH 7.4)
containing 25% sucrose (w/v) and 10% glycerol (v/v) for 24 h at
4 C. The slices were oriented flat onto the tissue holder of the
Frigomobil (Leica, Bensheim, Germany) combined with a microtome (Model SM 2000 R; Leica) and frozen. The slices were
resectioned to a thickness of 50 m, rinsed in PBS three times for
10 min and endogenous peroxidase activity was blocked by incubating all slices in PBS containing 1% H2O2 (Sigma) for 20 min.
Slices were rinsed three times in PBS and alternate slices were
either used for immunohistochemistry or for biocytin tracing.
Biocytin tracing
For biocytin labeling we followed a previously described protocol
(Staiger et al., 1999) with minor modifications. The 50 m thick
slices were incubated in PBS containing 10% normal goat serum
(NGS, Vector Laboratories, Burlingame) and 0.3% saponin
(Sigma) for 30 min followed by an incubation in AB-Complex (ABC
elite kit, Vector Laboratories, Peterborough, UK) at a final dilution
of 1:300 with PBS for 90 min, according to manufacturers instructions. Slices were consequently rinsed in PBS for 5 min in PBS
1, TrisHCl (0.05 M; pH 7.6) 2, and TrisHCl (pH 8.1) 2
followed by a pre-incubation in Ni-DAB solution (50 ml TrisHCl,
pH 8.1, containing 7.5 mg 3=-3-diaminobenzidine tetrahydrochloride (DAB, Sigma) and 0.2 g ammonium nickel sulfate (Ni, Fluka,
Buchs, Switzerland)) for 10 min. Afterward the Ni-DAB solution
was replaced by a freshly prepared solution containing in addition
0.01% H2O2 to induce the peroxidase reaction. The reaction was
terminated according to visual inspection after approximately 10
min. Sections were rinsed in TrisHCl (0.05 M, pH 7.6) mounted
on glass slides, air-dried, cleared in xylene, and coverslipped with
Entellan (Merck, Darmstadt, Germany).
Cortical parcellation
Sections collected for the identification of functional cortical
subdivisions were immunohistochemically and histochemically
stained according to previous protocols (Bidmon et al., 1997)
using antibodies SMI 31 (Sternberger monoclonal Inc., Baltimore,
MD, USA) for the labeling of axons, SMI 311 for the labeling of
non-phosphorylated neurofilaments, or Wisteria floribunda agglutinin (WFA, Sigma) histochemistry for the region-specific presence
of the so-called perineuronal nets (Celio and Blumcke, 1994). In
brief, for immunohistochemistry, sections were incubated in 2%
NGS (Vector Laboratories) containing 10% fetal calf serum, dissolved in PBS containing 0.3% saponin for 1 h, followed by an
incubation either in antibody SMI 31 (final dilution 1:500), or in
374
RESULTS
We recorded stimulus evoked cortical activity in auditory
thalamocortical preparations (n24 slices with intact connections, slices derived from 23 animals aged p 6597). To
verify connectivity in our preparations, we injected biocytin
into the primary auditory cortex (A1) of thalamocortical
slices (n11 animals), which led to a retrograde staining of
thalamocortical fibers and cell bodies within the MGN (Fig.
2AC). To further identify cortical areas excited by stimulation of the MGN, we performed immunohistochemical
stainings of the pan-neuronal neurofilament (SMI-311; Fig.
2D). In the plane of the slices, the primary auditory cortex
was flanked by the ventral secondary auditory cortex (AuV)
and the association area of the temporal lobe (TeA, Fig.
2D). To control for possible unspecific effects of the voltage
sensitive dye, we performed whole cell patch-clamp recordings in thalamocortical projection neurons of the MGN and
neurons in A1. The recorded neurons (MGN, n5; A1, n7)
displayed resting parameters (resting membrane potential:
Fig. 2. Biocytin and immunohistochemical staining of angled horizontal brain slices. (A) Overview of an angled horizontal brain slice after
peroxidase staining of biocytin. The inset on the upper left shows the
entire slice, which was trimmed frontally and along the midline. The
medial geniculate nucleus (MGN), the lateral geniculate nucleus
(LGN), the nucleus reticularis thalami (NRT), the ventrobasal nuclear
complex (VB), and the primary auditory cortex (A1) are indicated.
Application of biocytin into the primary auditory cortex led to a retrograde staining of projection neurons in the MGN. Labeled neurons are
375
376
Fig. 3. Effects of high frequency stimulation, removal of extracellular Ca2, CNQX, and TTX on evoked activity. (A) Voltage sensitive dye signals
(VSD, upper traces) and field potential recordings (Field, lower traces) under control conditions (single pulse stimulation, Ctrl), upon stimulation with
three pulses at 100 Hz (Ctrl HF), in nominal Ca2 free ACSF (0 Ca2), and in 0 Ca2 with 0.5 M TTX added to the bath solution (0 Ca2 TTX). In
field potential traces, black arrows denote the presumed afferent thalamocortical fiber volley, white arrows denote the first presumed postsynaptic
population spike. Note the absence of compound action potentials only in the presence of TTX. All recordings derive from the same slice. Arrows below
VSD signals indicate time of stimulus in A and B. (B) Same conventions as in A. Next to control responses to single and high frequency stimulation, the
responses in the presence of 10 M CNQX are shown for low and high frequency stimulation (HF). All recordings derive from the same slice. All VSD signals
shown were recorded in the supragranular layers of A1. Changes in the size of the excited area due to high frequency stimulation (HF) are shown in (C),
changes due to the removal of extracellular Ca2 are shown in (D), and the effects of 10 M CNQX on the size of the excited area are shown in (E). The
depicted points in C, D, and E correspond to time bins: left points 0 26 ms after stimulus, middle points 2751 ms after stimulus, right points 52102 ms
after stimulus. Filled symbols represent control conditions while open symbols represent alterations in stimulus or pharmacological manipulation. * P0.05.
Fig. 4. (A) Maximal amplitudes per cortical layer and slice under control
conditions (Ctrl) and with high frequency stimulation (Ctrl HF). No responses in a given layer and slice were included with an amplitude of zero
(see text). (B) Age dependence of normalized averaged optical signal
amplitudes in the infragranular layers. Infragranular activity was measured as the averaged amplitude of the optical signal in the infragranular
layers normalized to the averaged amplitudes in granular and supragranular layers and compared between four age groups (see methods).
Differences between p 65 82 and p 83100 compared to the youngest
group were significant (one-way ANOVA, * P0.05).
377
im
St
Stim
Scaling
Ctrl
Ctrl HF
AP5
5 10-4
I/I
378
64 ms
AP5 HF
3.8 ms
3.8 ms
3.8 ms
3.8 ms
7.6
7.6
7.6
7.6
10.2
10.2
10.2
10.2
12.7
12.7
12.7
12.7
15.3
15.3
15.3
15.3
17.8
17.8
17.8
17.8
20.4
20.4
20.4
20.4
31.9
31.9
31.9
31.9
42
42
42
42
52.2
52.2
52.2
52.2
62.4
62.4
62.4
62.4
72.6
72.6
72.6
72.6
Fig. 5. Pseudocolor maps of evoked activity. (A) Overview of the slice from the top. Slices were trimmed frontally and along the midline. The
stimulation electrode is indicated (Stim) and was placed in the medial geniculate nucleus. The electrode to the left was used for fixation. The white
hexagons indicate the positions of the photodiode array (10 objective). (B) Composite overview picture of the slice from below. The position of the
stimulation electrode is visible as a black dot and is indicated (Stim). (C) Voltage sensitive dye trace used for scaling. Period used for scaling of
the pseudocolor images is indicated by the horizontal line below the VSD trace. Trace used for scaling was recorded in the supragranular layers of
the primary auditory cortex under high frequency stimulation. (D) Enlarged sections containing the three photodiode array positions, displaying the
evoked activity under control conditions using single pulse stimulation. Time after stimulus is indicated on the lower right of each frame. (E) Evoked
activity using high frequency stimulation (three pulses at 100 Hz). (F) Evoked activity using single pulse stimulation in the presence of 50 M AP5.
(G) Evoked activity using high frequency stimulation under the influence of 50 M AP5. Scale bars in A and B, as well as in D through G represent
1 mm.
379
Latency
A
AuV
A1
TeA
I
G
S
6-7 ms
7-8 ms
8-9 ms
AuV
A1
TeA
I
G
S
9-10 ms
>10 ms
No signal
Amplitude
C
AuV
A1
TeA
I
G
S
AuV
A1
I
G
S
TeA
Fig. 6. Summary of minimal onset latencies (A and B) and maximal amplitudes (C and D) per slice using single pulse stimulation. Slices were grouped
according to lateral spread of activity into extended response slices (A and C) and local response (B and D) slices. The cortex was subdivided into areas
(ventral secondary auditory cortex, AuV; primary auditory cortex, A1; association area of the temporal lobe, TeA) and layers (infragranular, I; granular, G,
shaded line; supragranular, S). Onset latencies and amplitudes were binned, color coded (scale on the right), and plotted on the corresponding regions of
the slice.
380
mediated inhibition limits the spatiotemporal spread of activity in auditory thalamocortical slices.
DISCUSSION
The present study employed the auditory thalamocortical
brain slice preparation in adult mice (Rose and Metherate,
2001, 2005; Cruikshank et al., 2002; Kaur et al., 2005;
Kotak et al., 2008; Lee and Sherman, 2008). We used this
preparation to study cortical activity evoked by activation of
thalamic afferents via stimulation of the MGN. Optical imaging using voltage sensitive dyes in combination with
conventional field potential recordings allowed us to study
the spatiotemporal pattern of evoked activity at high temporal and spatial resolution. To the best of our knowledge,
this is the first study to report the spatiotemporal characteristics of thalamically evoked activity in the auditory cortex of adult mice.
The major findings of the present study are (1) a prominent supragranular and granular excitation with pronounced horizontal spread, (2) a dramatic developmental
decrease of activity in the infragranular layers, (3) a predominance of non-NMDA receptor mediated activity, (4)
comparatively small effects of high frequency stimulation
and NMDA receptor blockade, (5) a pronounced influence
of GABAA receptor-mediated inhibition on the spatiotemporal spread of activity.
The basic spatiotemporal profile of evoked activity in
slices displaying activity only in A1 (local response slices)
matches previous descriptions (Kubota et al., 1997, 1999;
Cruikshank et al., 2002; Kaur et al., 2005), while the activity in slices with activity spreading beyond the boundaries of A1 into AuV and TeA (extended response slices)
differed from previous reports in several aspects. In this
subset of samples, minimal onset latencies were located in
the supragranular layers of A1. The activity in areas lateral
to A1 (AuV and TeA) displayed shortest onset latencies in
the granular layer, while the largest signal amplitudes were
found in the supragranular layers. Notably, the evoked
activity in the supragranular layers of the TeA was of
comparable amplitude to the activity in the supragranular
layers of A1, while displaying significantly longer onset
latencies. The layer distribution of evoked activity, as well
as the influence of NMDA receptors and high frequency
stimulation, differed from observations made in somatosensory thalamocortical slices after stimulation of the ventrobasal nuclear complex (Laaris et al., 2000; Beierlein et
al., 2002; Llinas et al., 2002), indicating differences in
cortical processing of thalamic inputs in rodent A1 as compared to S1.
Technical considerations
When interpreting the results of the present study, some
technical issues need to be addressed. The optical signals
described here reflect average membrane potential
changes in columns of tissue extending 100 m into the
slice (Salzberg et al., 1977; Cohen and Salzberg, 1978;
Cohen et al., 1978; Wu and Cohen, 1993; Grinvald and
Hildesheim, 2004; Baker et al., 2005). Furthermore, optical
381
Fig. 7. Effects of AP5, CNQX, and bicuculline on evoked activity. (A) Voltage sensitive dye signals (VSD, upper traces) and field potential recordings
(Field, lower traces) under control conditions (Ctrl), in the presence of 50 M AP5 (AP5), and in the presence of 50 M AP5 in combination with 10
M CNQX (AP5 CNQX). All recordings derive from the same slice. (B) VSD and field potential recording from the same slice shown in A using high
frequency stimulation (three pulses at 100 Hz, HF), under control conditions (Ctrl HF), in the presence of 50 M AP5 (AP5 HF), as well as of 50 M
AP5 in combination with 10 M CNQX (AP5 CNQX HF). Scale bars apply to A and B. Arrows below VSD signal denote the time of stimulation. (C)
VSD and field potential recordings under control conditions (Ctrl, upper panels) and in the presence of 10 M bicuculline (Bicu, lower panels). VSD
signals in A-C derived from the supragranular layers of A1. (D) Maximal amplitudes under control conditions (Ctrl) and after application of 50 M AP5
(AP5) under single pulse and high frequency stimulation (HF) in the infragranular layers (I), the granular layer (G), and the supragranular layers (S).
(E, F, and G) show changes in the excited area due to application of 50 M AP5 (E), 50 M AP5 in combination with 10 M CNQX (F), and 50 M
AP5 under high frequency stimulation (G). (H) Maximal amplitudes before (Ctrl) and after application of 10 M bicuculline (Bicu) for the infragranular
layers (I), the granular layer (G), and the supragranular layers (S). (I) Changes in excited area induced by 10 M bicuculline (Bicu). The depicted points
in E, F, G, and I correspond to time bins: left point 0 26 ms after stimulus, middle point 2751 ms after stimulus, right point 52102 ms after stimulus.
Closed symbols represent control conditions, while open symbols represent alterations in stimulation or pharmacological manipulation.
im
t
S
50 ms
scaling interval
7.6 ms
17.8 ms
10.2 ms
22.9 ms
12.7 ms
26.8 ms
5 10-4 I/I
382
Fig. 8. Pseudocolor maps of evoked activity in the presence of 10 M bicuculline. (A) Top view of the slice. The stimulation electrode located in the
medial geniculate nucleus is indicated (Stim) and the positions of the photodiode array are displayed as shaded hexagons (20 objective). (B) Voltage
sensitive dye trace used to scale pseudocolor images. Period used for scaling is indicated by the horizontal line below the VSD trace. (C) Enlarged
sections of the slice containing all photodiode array positions, showing the evoked activity at the indicated time points after stimulation. Scale bar in
A and C indicates 1 mm.
signal amplitudes are directly proportional to the magnitude of voltage deflections as well as the amount of membrane experiencing a potential change, resulting in a
strong influence of dendritic potential changes (Cinelli and
Salzberg, 1990, 1992). This implies that the strongest
voltage sensitive dye signals may be located in a different
layer than the somata of the excited neurons. Furthermore,
the density of neuronal membrane (somata and dendrites)
will differ between different layers of cortex, which will
affect the amplitudes of voltage sensitive dye signals. In
particular, neuronal density is likely to be higher in the
supragranular layers in comparison to the infragranular
layers (Weedman and Ryugo, 1996), which could lead to
an overestimation of signal amplitudes in the supragranular layers compared to the infragranular layers. In addition,
dye concentrations may vary between different layers of
cortex, and non-neuronal signals may add to neuronal
signals, distorting the results. Differences in dye concentrations are unlikely to be of influence to our data, as all
signals are given as fluorescence changes relative to the
resting light intensity. Non-neuronal voltage changes have
marily by orthodromic activation of thalamocortical fibers, as was previously demonstrated by Rose and
Metherate (2001).
The pseudocolor maps constructed to study the spatiotemporal distribution of evoked cortical activity were assembled from successive recordings, in order to cover
larger areas of the slice by repositioning the photodiode
array. In most cases evoked activity recorded at different
points in time could be combined into a single map without
abrupt changes at the photodiode array borders. Additionally, all optical signals analyzed in this study are averages
from three successive recordings separated by 30 s. This
indicates that the activity described here represents stereotypical behavior of our preparations, which can be
evoked repetitively.
Spatiotemporal distribution of evoked activity in the
auditory cortex
The most robust finding of the present study was a graded
distribution of optical signal amplitudes. The largest amplitudes were located in the supragranular layers, from where
amplitudes progressively decreased with increasing distance from the pial surface. Furthermore, we detected a
developmental decrease of activity in the infragranular
layers. At age p 65 and above, very little or no activity in the
infragranular layers was found in most of our preparations.
Taking the results of previous studies into account (Rose
and Metherate, 2001; Cruikshank et al., 2002; Kaur et al.,
2005), infragranular activity due to thalamic stimulation
seems to decrease during development. While the previous studies used younger animals (p 1319) and found
moderate levels of infragranular activity, our study shows a
very low level of activity from p 65 onwards, and a graded
decrease between p 31 and p 64 (Fig. 4B).
The earliest signal onsets were located in the supragranular layers, in the granular layer, or both, while infragranular activity had longer onset latencies. The amplitude
and latency results match the anatomical descriptions of
the terminations of thalamocortical synapses coming from
the ventral division of the medial geniculate body (Linden
and Schreiner, 2003; Winer and Lee, 2007).
While the low amplitude activity in the infragranular
layers was a novel and very robust finding in our experiments it should be interpreted with caution, as a lower
neuronal density in the infragranular layers might confound
this result. In any case, signal onsets are unlikely to be
influenced by this. We consistently observed the longest
onset latencies in the infragranular layers. This hints at an
intracortical as opposed to a thalamocortical activation
mechanism and points to a more efficient interconnection
between the granular and supragranular layers in comparison to the interconnection between the infragranular and
the upper layers in A1, AuV, and TeA. Furthermore, the
spread of activity was independent of the presence or
absence of infragranular excitation, which indicates that, in
the auditory cortex, lateral spread of activity can be
achieved by the supragranular and granular layers alone.
An isolated spread of activity in the infragranular layers
was not observed in our experiments. In slices in which the
383
evoked activity extended beyond the borders of A1, average minimal onset latencies of the entire slice were located
in the supragranular layers of A1. In the AuV and the TeA,
average minimal onset latencies were located in the granular layer, while largest signal amplitudes were found in the
supragranular layers. To the best of our knowledge, this
peculiar spatiotemporal activity pattern has not been observed before. It is consistent with a highly focal localization of thalamocortical projections in the granular and/or
supragranular layers of A1 as well as with a more distributed localization of thalamic input in the supragranular
layers of A1 and the granular layers of A1, AuV, and
possibly the TeA. The former scenario implies a stronger
horizontal spread of activity, while the latter implies a
larger area of thalamic input with a predominant upward spread of activity to the supragranular layers.
Interestingly, the average overall shortest onset latency
was located in different layers depending on the extent
of the lateral spread of activity (extended response
slices vs. local response slices). Possibly, the differences in lateral spread of activity were caused by differences in the localization of thalamic input and the integrity of intracortical circuitry, which are likely to vary with
small differences in the individual slicing procedures and
between different animals.
Effects of glutamate and GABAA receptor blockade
and high frequency stimulation
Our pharmacological experiments indicated a strong influence of non-NMDA glutamate receptors along with a modest contribution of NMDA receptors. These findings are in
agreement with a previous study in rats (Kubota et al.,
1997). Interestingly, an increase of stimulation frequency
neither markedly changed the spatiotemporal activity pattern, nor did it increase the relative effects of AP5, indicating that under our recording conditions there was no
marked increase in NMDA receptor recruitment by high
frequency input. Notably, this result differs from NMDA
receptor dependent high frequency induced long-lasting
activity observed in A1 of slices of juvenile mice (Rose and
Metherate, 2005). This discrepancy might be explained by
a difference in the number of pulses and the stimulation
frequency (mostly 10 pulses at 40 Hz by Rose and Metherate (2005), vs. three pulses at 100 Hz in the present
study) and/or the age of the animals used in the experiments. Blockade of GABAA mediated inhibition substantially restructured evoked activity. In the presence of bicuculline, stimulation of the thalamus generated high amplitude activity, which spread horizontally in both directions.
Although signals in the infragranular layers still displayed
the lowest amplitude, the inter-laminar differences were
smaller in the absence of GABAA receptor-mediated inhibition. This might be explained by an unopposed vertical
spread of activity. Furthermore, supragranular activity led
the spreading excitation, pointing at a more efficient horizontal interconnection within the supragranular layers
compared to the deeper layers.
384
impact of conductance velocity changes induced by demyelination on the thalamocortical system. The goal of the
present study was to provide solid ground for future investigations by describing the basic patterns of evoked cortical
activity in adult mice. A combination of voltage sensitive
dye imaging and field potential recordings, as employed
here, will allow investigating changes in evoked activity in
both temporal and spatial dimensions. We believe that the
auditory thalamocortical slice is well suited for such investigations, as the fiber tract from MGN to A1 is sufficiently
long to detect changes in onset latency and it allows the
effects of intracortical and subcortical demyelination to be
addressed separately.
AcknowledgmentsThe Authors would like to thank Birgit Herrenpoth for excellent technical assistance. This work was supported by Interdisziplinres Zentrum fr Klinische Forschung (Bud
3/005/07), Deutsche Forschungsgesellschaft (BU1014/8-1/9-1;
PA336/17-1), and the Max-Planck-Gesellschaft (Max-PlanckResearch-Award to HCP).
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385
CELLULAR NEUROPHYSIOLOGY
Received: 7 November 2007 / Revised: 12 February 2008 / Accepted: 21 February 2008 / Published online: 14 May 2008
# Springer-Verlag 2008
Abstract By combining electrophysiological, immunohistochemical, and computer modeling techniques, we examined the effects of halothane on the standing outward current
(ISO) and the hyperpolarization-activated current (Ih) in rat
thalamocortical relay (TC) neurons of the dorsal lateral
geniculate nucleus (dLGN). Hyperpolarizing voltage steps
elicited an instantaneous current component (Ii) followed
by a slower time-dependent current that represented Ih.
Halothane reduced Ih by shifting the voltage dependency of
activation toward more negative potentials and by reducing
the maximal conductance. Moreover, halothane augmented
Ii and ISO. During the blockade of Ih through Cs+, the
currentvoltage relationship of the halothane-sensitive
current closely resembled the properties of a current
through members of the TWIK-related acid-sensitive K+
(TASK) channel family (ITASK). Computer simulations in a
Introduction
Thomas Budde and Philippe Coulon are equally contributing first
authors.
T. Budde (*) : P. Coulon : M. Pawlowski : P. Meuth :
T. Kanyshkova : H.-C. Pape
Institut fr Physiologie I,
Westflische Wilhelms-Universitt Mnster,
Robert-Koch-Str. 27a,
48149 Mnster, Germany
e-mail: tbudde@uni-muenster.de
A. Japes
Institut fr Anorganische und Analytische Chemie,
Westflische Wilhelms-Universitt Mnster,
Corrensstr. 30/36,
48149 Mnster, Germany
S. G. Meuth
Klinik fr Neurologie, Julius-Maximilians-Universitt Wrzburg,
Josef-Schneider-Strae 11,
97080 Wrzburg, Germany
1062
1063
1064
intracellular application
extracellular application
Ih
halo 0.1 %
control
-43 mV
1.0
1.0
0.8
0.8
control
control
0.6
p(V)
p(V)
0.6
0.4
halo 0.1 %
control
halo 0.1 %
0.2
-133mV
0.0
0.4
halo 0.1 %
0.2
0.0
-80
-60
-40
V (mV)
4
2
Ihc on
Ihhal o
Ih
halo
I Lc on
con
ILhalo
Ii
halo
con
-40
**
12
10
-60
V (mV)
12
-80
10
8
**
6
4
2
0
con
Ih
halo
con
Ii
halo
Fig. 1 Halothane effect on hyperpolarization-activated inward currents in TC neurons. Representative current traces and analyses
obtained from experiments with intracellular (a) and extracellular (b)
application of halothane are shown. Upper panels current traces
obtained by stepping from a holding potential of 43 to 53, 73,
93, 113, and 133 mV (inset, middle panel) are shown (left
superimposed traces control conditions, right superimposed traces
presence of 0.1% halothane). The tail current voltage was 103 mV.
The duration of each hyperpolarizing step was shortened as command
potentials became more negative. This accounts for increasingly fast
activation kinetics of Ih and improves cell viability. The total trace
1065
Immunohistochemistry
LongEvans rats (postnatal days 2527) were deeply
anesthetized using pentobarbital (50 mg/kg body weight)
and transcardially perfused with PBS, followed by an icecold 4% PFA/PBS for 3540 min. Brains were removed,
postfixed for 4 h in 4% PFA/PBS, and cryoprotected with
30% sucrose. Coronal sections (20 m) were cut at the
level of the dLGN, mounted onto Polysine slide glass
(Menzel, Germany), and air dried. For detection of HCN2,
fresh-frozen sections were used. In this case, brains from
isoflurane-anesthetized rats were removed and frozen in
50C isopentane. Cryostat coronal sections of 20 m
thickness were cut at the level of the dLGN, thaw-mounted
onto Polysine slide glass, air dried, and fixed in 4% PFA/
PBS for 10 min.
After permeabilization with 0.1% Triton X-100 in PBS
for 10 min and several washings with PBS, sections were
blocked with 10% normal horse serum (NHS), 2% BSA in
PBS for 3 h to minimize nonspecific binding before
incubation of slices with primary antibodies: rabbit antiHCN1 (1:500, Alomone Labs, Israel), goat anti-HCN2
(1:250, Santa Cruz Biotechnology, USA) and rabbit antiHCN4 (1:100, Alomone Labs, Israel) in 2% NHS, 2% BSA
in PBS at 4C for 1618 h. After washing (310 min with
PBS), sections were exposed to Cy3-conjugated donkey
antirabbit IgG or Cy2 donkey antigoat IgG (1:400 in 2%
NHS, 2% BSA in PBS, Dianova, Germany) for 1.5 h,
washed again, and coverslipped with Immumount. For the
negative controls, occlusion of the primary antibody from
the staining procedure was routinely performed with no
positive immunological signal detected (see Fig. 6, insets).
Results
Intracellular and extracellular modulation of Ih
We examined effects of halothane on the hyperpolarizationactivated current (Ih) in rat TC neurons in dLGN. In Fig. 1,
we compare the controls (upper left panels) to the results
obtained in the presence of halothane (upper right panels).
First, halothane was added to the pipette solution, thus
allowing diffusion into the cytosol (Fig. 1a). Ih was
activated from a holding potential of 43 mV by using
hyperpolarizing voltage steps of increasing (V=10 mV)
amplitude and decreasing (t=1500 ms) duration (3.5 s at
133 mV, see the Materials and methods section)
followed by a constant step to 93 mV (Fig. 1a, upper
panel; the inset shows a scheme of the voltage protocol).
An analysis of the deactivating currents revealed a halfmaximal value of Ih activation (Vh) at a membrane potential
of 861 mV (n=16) under control conditions (Fig. 1a
1066
-93 mV
halo 0.1 %
control
-113 mV
1.0
1.0
0.8
0.8
control
0.4
0.6
p(V)
p(V)
0.6
halo 0.1 %
0.4
0.2
0.2
0.0
0.0
-80
-60
-40
control
halo 0.1 %
V (mV)
1.2
**
con
C onR e v
normalized Gh
1.0
I hco n
Ih hal o
Ih
halo
-40
I Lc on
con
I Lha lo
Ii
halo
0.6
H a lo R ev
-20
0.4
0.0
halo
-10
0.8
-30
0.2
con
-60
Erev
-80
V (mV)
C on S l
con
H a lo S l
-40
halo
Fig. 2 Halothane effect on isolated Ih in TC neurons. a Representative current traces and analyses obtained from experiments with
intracellular application of halothane in the presence of Ba2+ (in the
bathing solution) are shown. Current traces (upper panel; long
protocol; scale bar represent 3 s and 100 pA), mean steady-state
activation curves (middle panel), and mean bar graph representations
of current density (lower panel) were obtained as described in Fig. 1.
b Superimposed current traces (upper panel; short protocol) recorded
in Ba2+-containing extracellular solution in the presence (gray traces)
1067
halothane
1%
1.6
0.2%
1.4
Inor
0.1%
1.2
1.0
0.8
0.01%
1000
60
40
20
0
0.01
2000
0.1
time (s)
d
-28 mV
800 ms
300
-138 mV
0.2% in Cs+
250
halo 0.2% in
200
Cs+
I (pA)
control
150
100
0.02%
50
0
1068
-55
halo 0.1%
-60
V (mV)
-65
-70
1000
2000
time (s)
control
-60 mV
halo 0.1%
-66 mV
control
-10 mV shift in Ih
30% increase in ITASK
model cell
-60 mV
-66 mV
1069
4
-60 mV
10
mV
0.2% halothane
30 s
-60 mV
2s
Discussion
Effect of halothane on HCN channels in TC neurons
We have shown previously that the halothane-sensitive
current in TC neurons had a reversal potential positive to
the K+ equilibrium potential and wasin addition to the
TASK channelscarried by as yet uncharacterized Na+
and/or Ca2+ channels [27]. This conclusion is corroborated
by the finding that Na+-permeable HCN channels are
modulated by halothane. Inhibition of Ih by halothane in
native TC neurons is characterized by two effects: a
hyperpolarizing shift in the activation curve and a
decrease in the maximal available current. Recent studies
on cloned channels in expression systems indicate subunit-specific effects of clinically relevant concentrations of
halothane with the shift in Ih voltage dependency and the
reduction in the maximal available current being mediated
by HCN1 and HCN2, respectively [6], and both effects
can be observed concurrently in HCN1HCN2 heteromeric channels. Therefore, our results suggest that Ih in
TC neurons is critically based on functional HCN1HCN2
heterodimers, which is corroborated by the findings that
HCN1 and HCN2 mRNA expression was found in TC
neurons [4, 28] and the respective proteins are present in
the plasma membrane (this study). Although HCN2 was
found to be the dominant isoform in TC neurons [20, 28],
only a small reduction in the maximal available current
was observed (Fig. 2b, lower panel). This discrepancy
may be explained by the finding that the predominant
action of halothane on HCN1HCN2 heterodimers
depends on the relief of the C-terminal-dependent basal
inhibition by cAMP [6].
1070
upper row low resolution; lower row high resolution). The insets
(upper row) show negative controls (occlusion of the primary
antibody). Images were deconvoluted using the Zeiss AxioVision
(4.6.3) software package
1071
1072
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Adenosine and sleepwake regulation. Prog Neurobiol 73:379396
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Dendritic excitability of mouse frontal cortex pyramidal neurons
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Differential effects of isoflurane on excitatory and inhibitory
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10. Fanselow EE, Sameshima K, Baccala LA, Nicolelis MA (2001)
Thalamic bursting in rats during different awake behavioral states.
Proc Natl Acad Sci U S A 98:1533015335
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14. Hines ML, Carnevale NT (2001) NEURON: a tool for neuroscientists. Neuroscientist 7:123135
15. Honore E (2007) The neuronal background K2P channels: focus
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16. Huguenard JR, McCormick DA (1992) Simulation of the currents
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17. Jones MV, Harrison NL (1993) Effects of volatile anesthetics on
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18. Linden A-M, Aller MI, Leppa E, Vekovischeva O, Aitta-aho T,
Veale EL, Mathie A, Rosenberg P, Wisden W, Korpi ER (2006)
The in vivo contributions of TASK-1-containing channels to the
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19. Linden A-M, Sandu C, Aller MI, Vekovischeva OY, Rosenberg
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49.
50.
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52.
53.
54.
55.
56.
57.
58.
CELLULAR NEUROPHYSIOLOGY
Received: 18 October 2007 / Revised: 16 January 2008 / Accepted: 7 February 2008 / Published online: 19 March 2008
# Springer-Verlag 2008
=
(n=5),
. and 21 mV (n=9) in wild type, G11 , and
=
Gq G11 , respectively. This depolarization was associated
with a change in TC neuron activity from burst to tonic firing
=
in wild type and G11 , but not in Gq =G=
11 . The use of
specific antibodies and of pharmacological agents with
preferred affinity points to the contribution of m1AChR and
m3AChR. In conclusion, we present two novel aspects of the
physiology of the thalamocortical system by demonstrating
that the depolarization of TC neurons, which is induced by
the action of transmitters of ascending brainstem fibers, is
governed roughly equally by both m1AChR and m3AChR
and is transduced by Gq but not by G11.
Keywords Thalamic function . Gene knock out .
TASK channels . Thalamocortical relay neurons .
G-proteins . Muscarinic ACh receptors
Introduction
The thalamocortical system is characterized by highly
synchronized oscillatory burst activity (<15 Hz) during
states of slow wave sleep and tonic generation of action
potentials and high-frequency oscillations (~40 Hz) during
wakefulness and rapid eye movement (REM) sleep [36].
Neurons located in brainstem cholinergic nuclei, the locus
coeruleus, and the raphe nuclei release acetylcholine (ACh),
noradrenaline (NA), and serotonin (5-HT) into the thalamus
and mediate the switch between activity modes [24]. A
common action of these transmitters is a depolarizing shift
of the membrane potential of thalamocortical relay (TC)
neurons, leading to cessation of rhythmic bursts and
occurrence of tonic activity. One crucial step of membrane
depolarization is the decrease in a leak K+ conductance
1050
1051
Results
Cre recombinase-mediated inactivation of Gq and G11
in the thalamus
To determine the degree of Cre-mediated recombination in
the thalamus of forebrain-specific Gq/G11-double-deficient
mice, we analyzed the expression of Gq/G11 by Western
blotting (Fig. 1a). Thalami from mutant mice showed a
massive reduction of Gq/G11 immunoreactivity, comparable to that observed in the cerebral cortex of these mice.
The residual Gq/G11 immunoreactivity observed in
mutant thalami is probably due to non-recombined interneurons, glial cells, and thalamic blood vessels. In contrast
to the thalamus, no recombination was observed in the
brainstem (Fig. 1a, lower panel). In hippocampus and
cortex, Camkcre4-mediated recombination has been shown
to be restricted to principal neurons [22]. To test whether
this was also true in the thalamus, we performed -
1052
b
a
-gal
cortex
G11
Gq
40 kD
nRet
nRet
40 kD
brainstem
G11
Gq
Fb- Gq/G11-/-
G11-/-
Gq-/-
Wild-type
40 kD
nRet
dLGN
-gal-FITC /
Parv-TRITC / DAPI
G11
Gq
dLGN
GAD67 /
gal
thalamus
Muscarinic
AChR-mediated signaling
in Gq G11 = mice
The standing outward current (ISO) in TC neurons is carried
in part by TASK-1, TASK-3, and inward rectifier K+
channels which have recently been shown to be inhibited
by activation of mAChR [2628]. To assess the contribution of Gq/G11-mediated signaling, the effect of muscarine
=
was compared in wild type, G11 , and Gq/G11deficient mice. TC neurons were kept at a potential of
28 mV by using the whole-cell configuration of the patchclamp technique. Under steady-state conditions, ISO was
45479 pA (n=10) in wild-type mice and 40625 pA in
1053
b
1500
-28 mV
1500
800 ms
I (pA)
1000
-138 mV
500
0
-500
Gq/G11-/-
Inor
G11-/-
Vhold = -28 mV
1000
2000
time (s)
3000
0.5
1.0
q /G
1.5
2.0
300
200
G11-/-
-10
n = 15
-20
100
0
-30
Gq/G11-/-
-100
-40
n=8
-50
Fig. 2 Muscarine effect on ISO. a, b Current responses to hyperpolarizing ramp protocols (the holding potential was ramped from
28 mV to 138 mV in 800 ms; see inset) under control conditions
(black traces) and during application of muscarine (gray traces) in
=
=
G11 (a) and Gq =G11 (b). Note that the outward current
deflection following the hyperpolarizing ramp depends on the
+
activation of voltage-dependent K currents with the early phase
mainly carried by IA [3]. c Normalized mean ISO amplitude vs. time
=
muscarine (50 M)
0.0
11 -/-
11 -/-
1.2
0.8
muscarine
time (s)
time (s)
0.6
500
1.0
ISO
1000
muscarine
-500
Gq/G11-/-
I (pA)
2000
G11-/-
I (pA)
2000
n=5
-120
-80
-40
V (mV)
.
=
=
plot (black squares, G11 ; grey circles, Gq G11 ). Horizontal
bar indicates the period of substance application. d Mean bar graph
representation of the reduction in ISO amplitude induced by muscarine
=
application
(black bar, G11 ; white bar, wild type; grey bar,
.
=
Gq G11 ). e The mean IV relationship of the muscarine-sensitive
current was calculated by graphical subtraction of currents during drug
action from those under control conditions (i.e., control minus
muscarine)
=
1054
muscarine
control
40
control
muscarine
20
20
0
G
11-/-
V (mV)
V (mV)
40
-20
-40
G
q/G
11-/-
-20
-40
-60
-60
-80
-80
500 ms
500 ms
d
-20
muscarine (50 M)
depolarization (mV)
-30
V (mV)
-40
G
11-/-
-50
-60
-70
G
q/G
11-/-
-80
1000
2000
3000
time (s)
Fig. 3 Muscarine effect on firing properties. Functional consequences
of muscarine administration were tested under whole-cell current=
=
clamp conditions in G11 (a) and Gq =G11 (b). Cells of both
genotypes were held at a potential of approximately 72 mV by DC
current injection. This holding current was not changed during the
course of the experiment. Cells were challenged using 100200 pA
depolarizing current pulses (800 ms duration; see inset in a). Under
control conditions, depolarizing current pulses starting from hyperpolarized membrane potentials evoked typical burst firing (left traces).
Muscarinic receptor subtypes involved in Gq G11
signaling
In an effort to distinguish between receptor subtypes, we
tested the muscarinic agonist OxoM with some preference
=
to m1AChR [12, 37]. In G11 , application of OxoM
(10 M) induced a 292% (n=9; Fig. 4a,c) decrease in ISO
amplitudes under voltage-clamp conditions and a 122 mV
depolarization (from Vh) of the membrane potential under
current clamp conditions (n=7,
. Fig. 4d). Both effects were
significantly reduced in Gq G=
(ISO reduction: 13
11
3%, n=8, p<0.001; Fig. 4b,c; depolarization: 41 mV, n=
5; Fig. 4d, p<0.03). In all. control mice (Fig. 4e, upper
=
panel) but none of the Gq G11 (Fig. 4e, lower panel),
the OxoM-induced depolarization was accompanied by a
switch to tonic firing.
To further identify mAChR subtypes, we used antagonists with preferred affinity to one of the known Gqcoupled receptors. Application of the m1AChR-affinitive
antagonist pirenzepine (10 M) [14] induced a non=
significant increase in ISO amplitude in G11 (32%,
30
n=6
n=5
20
10
n=9
0
G
11-/-
WT
G
q/G
11-/-
.
=
n =7) and Gq G11 (5 3%, n =5) mice, possibly
indicating the activity of ambient ACh or basal receptor
activity (Fig. 5a,d). Subsequent application of muscarine in
=
G11 mice induced a reversible reduction in ISO amplitude which was significantly (p<0.0002) smaller (183%,
n=7) compared to the control effect (34%, see above).
When muscarine
was applied in the presence of pirenzepine
.
=
in Gq G11 mice, the reduction in ISO amplitude was
62% (n=5; Fig. 5a,b) which was significantly (p<0.02)
=
smaller compared to G11 . The IV relationship of the
muscarine-sensitive current in the presence of pirenzepine
=
in G11 revealed inwardly and outwardly rectifying
components
and reversed at 1041 mV (n=7, Fig. 5c).
.
=
In Gq G11 mice, the small rather inwardly rectifying
muscarine-sensitive current in the presence of pirenzepine
reversed at 1032 mV (n=5; Fig. 5c).
Next, we used the m3AChR-affinitive antagonist 4-DAMP
[29]. Application of 4-DAMP (1 M) induced an increase
in
.
=
=
ISO amplitude in G11 (5 4%, n = 6) and Gq G11
(71%, n=3) mice, which was significant in the latter case
=
(Fig. 5b,d). Subsequent application of muscarine in G11
-500
I (pA)
Vhold = -28 mV
500
0.2
1.0
-500
1.2
d
G
q/G
11-/-
1.0
0.8
11-/G
0.6
Vhold = -28 mV
1000
2000
time (s)
e
15
OxoM (10 M)
0.0
n=7
V (mV)
c
1.2
OxoM
OxoM
0.0
G
q/G
11-/-
1000
depolarization (mV)
I (pA)
500
G
11-/-
1000
10
0.2
co n
KO
G
q/
G
11-/-
0.6 0.8
time (s)
1.0
1.2
G
11-/-
-40
40
G
11-/-
0.4
40
-80
n=5
V (mV)
I (pA)
1055
control
OxoM
G
q/G
11-/-
-40
-80
1056
pirenzepine ( 10 M)
1.2
1.2
muscarine (50 M)
1.1
Gq/G11-/-
0.9
0.8
G11-/-
0.7
Gq/G11-/-
G11-/-
Vhold = -28 mV
1000
2000
3000
Vhold = -28 mV
0.6
1000
2000
pir
10
P ir
musc /
pir
P i rMu sc
-50
-150
3000
4-DAMP
DA M P
musc /
4-DAMP
D A MP Mu sc
pir /
4-DAMP
-100
-50
V (mV)
time (s)
musc /
pir /
4-DAMP
P i rDA MP
P i rDA MP Mu sc
-10
in 4-DAMP
100
I (pA)
d
change in amplitude (%)
50
0.7
time (s)
G11-/-
I (pA)
Inor
Inor
0.8
100
Gq/G11-/-
1.0
0.9
in pirenzepine
muscarine (50 M)
1.1
1.0
0.6
4-DAMP (1 M)
G11-/-
50
Gq/G11-/-
0
Gq/G11-/-
-20
G11-/-
-50
-150
-30
-100
-50
V (mV)
.
=
tonic (fi =4922 Hz) firing in Gq G11 mice (n=3;
data not shown).
These data argue against any substantial compensatory
mechanisms in TC neurons of Gq =G=
mice at the level
11
of effector channels and mAChR and demonstrate that the
Gq-independent modulation of TASK channels is intact.
=
=
Discussion
The results of the present study can be summarized as
=
follows: (1) Forebrain-specific Gq =G11 -deficient mice
show a strong reduction of Gq/G11 protein levels in the
thalamus. Cre-mediated recombination is observed only in
TC neurons not in local GABAergic interneurons or neurons
of the nRET. (2) The actions of ACh in TC neurons depend
on intracellular signaling cascades mainly activated by
m1AChR and m3AChR coupled to Gq-proteins. The effects
of muscarine and OxoM under current clamp conditions
=
were significantly reduced in Gq =G11 . As a consequence, stimulation of mAChR could not induce a shift from
burst to tonic firing of action potentials. (3) The steady-state
parameters
Vrest and ISO amplitude were unchanged in
.
=
Gq G11 with no evidence for compensatory mechanisms on the level of effector channels. (4) These data point
1057
m1AChR
NeuN
merge
m3AChR
NeuN
merge
WT
Gq/G11-/-
WT
Gq/G11-/-
Gq/G11-/G11-/-
3
2
1
5
m
4
m
3
m
2
m
1
m
SK
1
TA
N4
SK
TA
HC
N3
HC
N2
HC
N1
HC
r2
.3
0
Ki
1058
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CELLULAR NEUROSCIENCE
Edited by:
Barry W. Connors, Brown University,
USA
Reviewed by:
Charles Cox, University of Illinois at
Urbana-Champiagn, USA
Barry W. Connors, Brown University,
USA
*Correspondence:
Thomas Budde, Institut fr Physiologie
I, Westflische Wilhelms-Universitt
Mnster, Robert-Koch-Street 27a,
48149 Mnster, Germany.
e-mail: tbudde@uni-muenster.de
Present address:
Tilman Broicher, Department of
Bioengineering, University of Utah, 20
South 2030 East, Salt Lake City, UT
84112, USA
Introduction
The thalamocortical (TC) network takes up two states of activity:
slow and highly synchronized oscillatory burst activity during slow
wave sleep, and tonic generation of action potentials alongside fast
oscillations during mental alertness and REM sleep. Slow oscillatory activity has a frequency of <15Hz, while fast oscillations
occur at 40 Hz (Steriade et al., 1997). Chemically-coded projections from the brainstem activate the forebrain by releasing
acetylcholine (ACh), noradrenalin (NA), and serotonin (5-HT).
These neurotransmitters mainly act on G-protein-coupled membrane receptors (McCormick, 1992a). In a similar way, the arousing
action of glutamate, released from corticothalamic axons, is mediated by metabotropic glutamate receptors (mGluR) (Salt, 2002).
A common action of these neurotransmitters is a depolarizing
shift of the membrane potential of TC neurons, causing rhythmic bursting to cease and tonic activity to commence. Membrane
depolarization is caused to a large part by the inhibition of a leak
K+ conductance (IKL), the molecular correlate of which are the
two pore-domain K+ (K2P) channels TASK-1 and TASK-3 (Meuth
etal., 2003, 2006). The activation of muscarinic ACh receptors
(mAChR) and mGluR1 coupled to Gq/G11 inhibits TASK-1 and
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Coulon etal.
Drugs
Results
5-HT receptor signaling and competition between different
receptor classes in Gq/G11/ mice
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Coulon etal.
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Coulon etal.
Figure2 | t-ACPD effect on firing properties. (A,B) Functional consequences of t-ACPD administration during current clamp recordings in a control mouse (A) and
in Gq/G11/ (B). (C) Mean voltage vs. time plot (black squares, control animal; gray circles, Gq/G11/). The horizontal bar indicates substance application. (D) Mean
bar graph representation of the depolarization induced by two different substance concentrations as indicated.
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Coulon etal.
To provide further evidence that Gq-coupled receptors are functionally expressed in TC neurons we performed immunohistochemical stainings by antibody labeling.
Application of 5-HT2AR-specific antibodies did not lead to
staining by binding of the secondary antibody (Figure4B, middle
image). Strong staining was observed upon application of 5-HT2CR(Figure 4C, middle image) and mGluR1-specific antibodies
(Figure4D, middle image). We co-stained slices with the neuronspecific nucleus marker NeuN (Figures4BD left images; merged
images to the right). This revealed that 5-HT2CR and mGluR1
were not somatically expressed in neurons of the LGN. We verified
this by examining slices that were co-stained with a marker for
microtubule associated protein 2 (MAP2) at a higher magnification. The images revealed that neither the staining for 5-HT2C (not
shown) nor for mGluR1 clearly marked the outline of the soma
(Figure S1 in Supplementary Material). We could not observe any
differences between stainings in the dLGN of control and Gq/
G11/ mice (Figure S2 in Supplementary Material).
The densitometric analysis of 5-HT2CR-specific fluorescence
revealed significant differences between control (mean fluorescence intensity=321a.u.; n=3 independent slices) and Gq/
G11/ mice (mean fluorescence intensity=411a.u.; n=3; data
not shown). No differences were found for mGluR1-specific
Figure3 | DHPG effect on firing properties. (A,B) Functional consequences of DHPG administration during current clamp recordings in a control mouse (A) and in
Gq/G11/ (B). (C) Mean voltage vs. time plot (black squares, control animal; gray circles, Gq/G11/). The horizontal bar indicates substance application. (D) Mean
bar graph representation of the DHPG-induced depolarization.
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Coulon etal.
Discussion
Waking, but not REM sleep, is accompanied by increased serotonergic activity in the thalamus (Steriade etal., 1997). The role of 5-HT
in the thalamus seems complex and is not yet fully understood:
Cellular effects may be direct or indirect and show regional differences. In vitro studies demonstrated that 5-HT causes a small
depolarization and a shift in voltage dependency of the hyperpolarization activated cation current, Ih. The latter is achieved via
GS-proteins and cAMP production (McCormick and Pape, 1990;
Lee and McCormick, 1996). Moreover, inhibition of an ISO component occurred, resembling the current mediated by TASK channels
(S. G. Meuth and T. Budde, unpublished results). In consequence,
oscillatory burst activity is suppressed. An indirect modulation of Ih
via Gq-proteins may include IP3-induced Ca2+ release from intracellular stores and subsequent activation of a Ca2+-dependent adenylyl
cyclase (Lthi and McCormick, 1998). The findings of the present
study indicate that a significant part of the 5-HT-induced depolarization is mediated by Gq/G11-coupled receptors. All receptors of
the 5-HT2 subclass are coupled to Gq/G11-proteins. These in turn,
activate PLC-. 5-HT2A and 5-HT2C are widely distributed in the
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Coulon etal.
brain and are present in the rodent dLGN (Li etal., 2004). Thus,
the strong reduction of the effect of -m-5-HT in Gq/G11/ is in
good agreement with a 5-HT2 expression in dLGN and is possibly
connected to the modulation of TASK channels.
In the course of this study, we made the interesting observation that muscarinic and serotonergic receptors seem to compete
for the same Gq-protein-coupled signaling pathway. The effect of
activation of one receptor class is strongly and negatively correlated to the strength of prior activation of the other receptor class,
thereby suggesting convergence onto the same limited pool of
Gq-protein-coupled signaling pathways. This view is supported by
results obtained from cat and guinea pig TC neurons that suggested
acetylcholine- and noradrenalin-induced slow depolarizations to
occur through the activation of the same second-messenger system
(McCormick, 1992b).
mGluR-dependent signaling
Group I mGluRs consist of mGluR1 and mGluR5 that are positively coupled to PLC-. Several types of mGluRs are expressed in
the dLGN of different species, with retinal (mGluR5) and cortical (mGluR1) inputs accessing specific subtypes (Godwin etal.,
1996b; Lourenco Neto etal., 2000). Application of t-ACPD and
selective mGluR1 agonists depolarizes TC neurons and switches
their activity mode from burst to tonic firing, thereby mediating
TC transmission (McCormick and von Krosigk, 1992; Godwin
etal., 1996a; Salt, 2002). The results of the present study are in line
with these findings. However, the remaining t-ACPD effect in Gq/
G11/ indicates that mGluRs not coupled to Gq/G11 contribute to
the response. This conclusion is corroborated by the finding that
mGluR3, mGluR4, and mGluR7 are expressed in rodent dLGN
(Lourenco Neto et al., 2000). During postnatal development,
specific changes in the subcellular location of mGluRs have been
observed (Liu etal., 1998) which are the basis for the topographical association to different input systems (Godwin etal., 1996b;
Turner and Salt, 2000).
Residual effects of neurotransmitters in Gq/G11/ mice
Immunohistochemical stainings provided evidence that Gqcoupled receptors are functionally expressed in TC neurons.
However, the application of 5-HT2AR-specific antibodies failed to
stain the tissue. Why this is the case remains unclear. The strong
staining that was observed upon application of 5-HT2CR- and
mGluR1-specific antibodies was not reduced in Gq/G11/ mice,
suggesting that the deletion of Gq does not lead to a down regulation of its upstream receptors (compare Figure4 and Figure S2
in Supplementary Material). Co-staining slices with the neuronspecific nucleus marker NeuN revealed that 5-HT2CR and mGluR1
were not somatically expressed in neurons of the LGN. This was
confirmed in a co-staining with MAP2.
The finding that a specific agonist for 5-HT2C receptors had the
same effect on the membrane potential in current clamp recordings
than -m-5-HT in control mice suggests that 5-HT2C receptors in
the dLGN could be the functionally dominant isoform.
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Coulon etal.
References
Broicher, T., Kanyshkova, T., Meuth, P.,
Pape, H. C., and Budde, T. (2008a).
Correlation of T-channel coding gene
expression, IT, and the low threshold
Ca2+ spike in the thalamus of a rat
model of absence epilepsy. Mol. Cell.
Neurosci. 39, 384399.
Broicher, T., Wettschureck, N., Munsch, T.,
Coulon, P., Meuth, S. G., Kanyshkova,
T., Seidenbecher, T., Offermanns, S.,
Pape, H. C., and Budde, T. (2008b).
Muscarinic ACh receptor-mediated
control of thalamic activity via G(q)/
G(11)-family G-proteins. Pflugers
Arch. 456, 10491060.
Chemin, J., Girard, C., Duprat, F., Lesage,
F., Romey, G., and Lazdunski, M.
(2003). Mechanisms underlying excitatory effects of group I metabotropic
glutamate receptors via inhibition of
2P domain K+ channels. EMBO J. 22,
54035411.
Chen, X., Talley, E. M., Patel, N., Gomis,
A., McIntire, W. E., Dong, B., Viana,
F., Garrison, J. C., and Bayliss, D. A.
(2006). Inhibition of a background
potassium channel by Gq protein
{alpha}-subunits. Proc. Natl. Acad.
Sci. U. S.A.103, 34223427.
Exton, J. H. (1996). Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other
agonists linked to G proteins. Annu.
Rev. Pharmacol. Toxicol. 36, 481509.
Godwin, D. W., Vaughan, J. W., and
Sherman, S. M. (1996a). Metabotropic
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Godwin, D. W., Van Horn, S. C., Eriir, A.,
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inputs access different metabotropic
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geniculate nucleus. J. Neurosci. 16,
81818192.
Greif, G. J., Sodickson, D. L., Bean, B.
P., Neer, E. J., and Mende, U. (2000).
Altered regulation of potassium and
calcium channels by GABAB and
Acknowledgments
Supported by the German Research Foundation (DFG): FOR1086,
TP2 to Thomas Budde and Sven G. Meuth. Supported by the
Interdisziplinres Zentrum fr Klinische Forschung Mnster,
Bud3/010/10 to Thomas Budde. The authors wish to thank A. Jahn, E.
Nass, A. Markovic, and R. Ziegler for excellent technical assistance.
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Coulon etal.
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Coulon etal.
Supplementary material
Figure S1 | Immunohistochemical staining of mGluR1 and MAP2. Neurons of the dLGN were stained using specific antibodies against microtubule
associated protein 2 (MAP2) and against mGluR1 conjugated to Cy2 and Cy3, respectively. While MAP2 was clearly present in the soma, mGluR1 appears to be
absent there. Scale bars indicate 10 m.
Figure S2 | Immunohistochemical detection of mGluR and 5-HTR subtypes in dLGN of G11/ mice. Specific antibodies and secondary antibodies were the
same as in Figure4. 5-HT2AR-specific antibodies again did not cause staining (A). 5-HT2CR- (B) and mGluR1-specific antibodies (C) caused strong staining as in the
dLGN of Gq/G11/ mice. Scale bars indicate 100 m.
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INVITED REVIEW
Received: 26 May 2011 / Revised: 8 August 2011 / Accepted: 11 August 2011 / Published online: 13 September 2011
# Springer-Verlag 2011
Introduction
Sleep occupies roughly one third of our lifetime, and yet,
sleep research is unable to fully identify, let alone explain,
the nature, mechanisms, and functions of sleep. Reducing
sleep time is a common route in a notoriously busy society,
but prolonged sleep loss will cause (among other defects)
impaired metabolism, impaired immune function, impaired
cognitive function, and will, ultimately, lead to death [149].
Many pathophysiological states, such as lethargic encephalitis [190], insomnia [47], narcolepsy [114], apnoea [186],
cataplexy [126], or epilepsy [104], are related to disturbances in the mechanisms of sleep induction or maintenance.
Despite these valuable findings, the fundamental question of sleep research remains largely unanswered: Why do
we need to sleep? One of the main functions of sleep was
proposed to be memory consolidation [46, 102], but as
fundamental as this is, loss of memory consolidation cannot
explain the grave effects of sleep loss or disturbed sleep,
nor can the reduced immune and endocrine functions [120,
121], or impaired thermal regulation [180] that are hallmark
symptoms of sleep deprivation.
A key to sleep function could be to determine how and
when sleep is induced. The proposed mechanisms of sleep
54
by an intrinsic mechanism (the brainstem reticular activating concept) and (b) sensory input from the periphery is
needed for the maintenance of wakefulness (maintenance of
cerebral tone by sensory pathway activity, see [176]).
The main argument against theories of passive sleep has
been that sleep or sleep-like states can be induced by
stimulation either of sensory receptors or of central
structures [176]. The theories of active sleep have evolved
around the fact that peripheral or central low-frequency
stimulation causes sleep or EEG synchronization (see
[118]). Moreover, afferent volleys from the vagus nerve
are relevant for producing sleep with a synchronized EEG
pattern [147] and earlier findings showed that repetitive
visual stimulation evoked sleep-like behavioural signs with
matching EEG patterns in cats and humans [38, 58, 98].
However, the sites that cause EEG synchrony after
stimulation are numerous and applying synchronous stimuli
was considered nonsensical for the study of normal brain
activity, since they disturb natural patterns of neuronal
discharges and thus cannot be used to induce natural states
of sleep (see [176]). Lesion and pharmacological studies
have provided evidence that two major cerebral sites are
implicated in active sleep generation: The nucleus of the
solitary tract of the medulla [8, 96] and the preoptic area of
the basal forebrain ([112, 124, 156], see [176]). However,
the idea of active sleep induction and an active hypnogenic
focus also requires data at the cellular level. Neurons
promoting sleep were thought to at least require projections
toward the cholinergic and noradrenergic neurons in the
upper brainstem core [176]. This could provide a disfacilitation of brainstem to thalamus excitatory pathways,
supporting thalamic synchronization [97].
Hypothalamus
The ventro-lateral preoptic nucleus was suggested to initiate
sleep onset by several mechanisms of reciprocal inhibition
of the following [131]: (a) the cholinergic, noradrenergic,
and serotonergic arousal systems in the brainstem; (b)
histaminergic systems of the tuberomammillary nucleus in
the posterior hypothalamus; and (c) cholinergic systems of
the basal forebrain. All of these are in addition modulated
by the orexinergic arousal system of the lateral hypothalamus [64]. When active, these systems promote wakefulness, and accordingly, their inhibition promotes sleep. In
this scenario, the ventro-lateral preoptic nucleus is a switch
to initiate sleep: With increasing time of neuronal activity
during wakefulness, sleep regulatory substances accumulate
and eventually trigger the ventro-lateral preoptic nucleus
[131] in conjunction with circadian input from the suprachiasmatic nucleus [2] in the anterior hypothalamus, the
circadian rhythm of which is controlled by a series of genes
[87]. Sleep regulatory substances include adenosine in the
55
56
57
ITASK and Ih [93, 110]. Both reciprocally interact as major determinants of the resting membrane potential [113]. The brainstem transmitters inactivate ITASK and shift the voltage dependency of activation
of Ih to more depolarised potentials. Consequently, the membrane
depolarises and the input resistance increases, thereby favouring the
tonic firing mode. Orexins, histamine, and nitric oxide (NO) exert a
similar influence, while adenosine (but also volatile anaesthetics such
as halothane) suppresses tonic firing by a local modulation of these
ion channels. GABAergic input from the NRT or local interneurons
provide a hyperpolarisation that allows a de-inactivation of T-type Ca2
+
channels and activation of Ih, which then allows a cyclic interaction
that generates rhythmic burst activity (see Fig. 3 for details)
neurons, whose activity generalizes by progressive recruitment of neurons in the thalamocortical network. This
recruitment is thought to underlie the waxing phase of a
sleep spindle wave [26, 83]. The rhythm itself is thought to
base upon the reciprocal interactions between NRT neurons
and interconnected TC neurons, orchestrated in their
spatiotemporal pattern through corticofugal influences (see
Fig. 1) [25]: Individual TC neurons follow network
oscillations due to a fine tuned interplay between
GABAergic inhibitory postsynaptic potentials (IPSPs)
that are received from NRT neurons, and two types of
voltage-gated membrane conductances, which are as follows: the T-type Ca2+ current (IT) and the hyperpolarisationactivated cation current (Ih; see Figs. 2 and 3) [107, 110,
177].
Additionally, a long-lasting potassium-dependent IPSP
mediated by GABAB receptors primes TC neurons for burst
firing by activating low-threshold calcium potentials [32].
During spindling, NRT neurons rhythmically convey
GABAergic IPSPs onto TC neurons at 714 Hz. The
membrane hyperpolarisation provided by the IPSPs causes
a de-inactivation of IT, paralleled by an activation of Ih. The
repolarisation then activates IT again, triggering a so-called
rebound burst, representing the already mentioned LTS
which is crowned by several fast Na+/K+ APs. The
58
issue [33, 34]. Many more studies show that besides being
a charge carrier in rhythmic burst discharges during slowwave sleep, Ca 2+ is also crucially involved in the
generation and maintenance of sleep-related activity (for
review see [138, 175]):
The hyperpolarisation-activated cyclic nucleotide-gated cation (HCN) channels give rise to Ih. A regulation of Ih by
intracellular Ca2+ has been shown to be particularly
important during sleep spindling and slow-wave sleep [43,
44, 181]. In an attempt to provide a basis for the waxing and
waning behaviour of sleep spindles, a mathematical model of
synchronized oscillations in thalamic networks has implemented an activity-dependent up-regulation of Ih by intracellular Ca2+ (entering via T-type Ca2+ channels) in TC
neurons [43]. Here, a Ca2+ influx through the T-Type Ca2+
channels and the resulting positive shift of the activation
curve of Ih will increase the available Ih-mediated conduc-
59
60
Fig. 4 Ca2+ in the nucleus reticularis thalami. Ca2+ influx through Ttype Ca2+ channels in dendrites of NRT neurons was shown to activate
Ca2+-activated K+ channels of the SK2 type to regulate the strength of
the endogenous NRT oscillation [35]: depolarisations by Ca2+ influx
are followed by hyperpolarisations due to K+ efflux through SK2
channels. The hyperpolarisation de-inactivates T-type Ca2+ channels
and the cycle starts again. Sarco-(endo-)plasmic reticulum Ca2+
ATPase (SERCA) competes with SK2 channels for cytosolic Ca2+,
which interrupts the cyclic activity. Ca2+-induced Ca2+ release (CICR)
61
62
63
64
regulation. As such, adenosine can be regarded a sleepinducing factor [7, 145]. For instance, A1 adenosine
receptors decreased excitatory postsynaptic currents elicited
by stimulation of the ventrobasal thalamus in both
inhibitory and excitatory neurons in the barrel cortex [51].
Agonists for adenosine receptors A1 and A2A applied to the
prefrontal cortex modulate cortical ACh release, behavioural arousal, EEG delta power, and sleep. Additionally,
cortical adenosine agonist or antagonist application significantly altered ACh release in the pontine reticular
formation [185]. Obviously, the prefrontal cortex is able to
modulate behavioural arousal via descending inputs to the
pontine brainstem. The A1 adenosine receptors within the
prefrontal cortex may comprise part of a descending system
that can inhibit wakefulness.
Brainstem and hypothalamus
Adenosine can regulate sleep-related activity in the pons and
the hypothalamus (see [146]). In the hypothalamus, adenosine promotes NREM sleep by inhibiting the histaminergic
neurons in the tuberomammillary nucleus [130]. In the pons,
an adenosine A1 receptor agonist or an inhibitor of the
adenylyl cyclase increase REM sleep in rats, while an A2A
agonist increased ACh release in the pontine reticular
formation and both REM and NREM sleep [23, 101], the
latter probably via GABA-mediated inhibition of arousal
promoting neurons in the pontine reticular formation.
In a simplified view, the duration spent in the state of
wakefulness is inversely proportional to arousal activity.
This is reflected in diminishing discharge activity of
mesopontine cholinergic neurons [148] that play a major
role during arousal [177]. During wakefulness, when the
activity of these neurons is high, increased metabolic
activity may cause an increase in both intracellular and
extracellular adenosine. Whole cell patch clamp and
extracellular recordings in acute brainstem slice preparations
have shown that mesopontine cholinergic neurons are under
tonic inhibitory control of such endogenous adenosine. This is
mediated by increasing an inwardly rectifying K+ current and
a reduction of Ih [148]. Both alterations may act in concert in
mesopontine cholinergic neurons to reduce excitability and
increase the tendency of thalamic neurons to burst.
Thalamus
In the thalamus, adenosine hyperpolarises TC neurons and
reduces the apparent input resistance due to an A1-receptormediated increase in membrane K+ conductance [132].
Additionally, Ih is reduced in TC neurons in the presence of
an A1 receptor agonist, possibly due to an inhibition of
adenylyl cyclase activity. As described in detail above, Ih
has been proposed to be particularly important during
65
Fig. 6 From molecules to networks: thalamic mediators of sleeprelated activity. The hypothetical homeostat of NREM sleep is made
up by molecular networks of several sleep regulatory substances,
including tumour necrosis factor- (TNF-), interleukin-1 (IL-1),
growth hormone-releasing hormone (GHRH), prostaglandin D2 (PG),
brain-derived neurotrophic factor (BDNF), and adenosine. On a
cellular level, many studies show that differential regulation of
intrinsic cellular properties support or suspend thalamic sleep-related
activity. Finally, neuronal networks, e.g. the functional syncytium of
electrically coupled NRT neurons or the intra- and intercellular
networks between NRT neurons, TC neurons, and thalamic local
interneurons impose, support, or maintain sleep-related activity during
whole organism sleep. It is unknown whether individual neuronal
sleep-like states as described for cortical neurons also exist in thalamic
66
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