JUDY-FAY FERRON

ETUDE IN VIVO DU BURST-SUPPRESSION

Thèse présentée à la Faculté des études supérieures de l’Université Laval dans le cadre du programme de maîtrise en neurobiologie pour l’obtention du grade de maîtrise ès sciences (MSc)

FACULTÉ DE MÉDECINE

UNIVERSITÉ LAVAL QUÉBEC

2009 © Judy-Fay Ferron, 2009

Résumé

Cette étude résume certains concepts liés à l’anesthésie générale, détaille les mécanismes

d’action de l’isoflurane, un anesthésiant volatil, et aborde le phénomène du burst-

suppression. Elle vise principalement la compréhension de l’impact de l’isoflurane, à des

doses amenant le burst-suppression, sur l’inhibition dans le réseau thalamo-cortical. Nous

effectuons des enregistrements intracellulaires de neurones corticaux in vivo et de potentiels

de champs locaux à différentes doses d’anesthésiants chez le chat. Conjointement à ces

enregistrements, nous appliquons des drogues en iontophorèse en péri-synaptique des

neurones enregistrés et nous stimulons les noyaux thalamiques projetant dans les aires

corticales enregistrées. Nous suggérons que l’isoflurane amène une diminution de

l’inhibition corticale, via une plus grande recapture du glutamate par les glies, ce qui

diminue l’activation des interneurones corticaux.

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Abstract

This study summarizes some concepts about general anesthesia, details the mechanisms of

action of the volatile anesthetic isoflurane and describes the phenomenon of burst-

suppression. It aims at understanding the impact of isoflurane, under doses sufficient to

induce burst-suppression, on inhibition in the thalamo-cortical network. We performed

intracellular recordings of cortical neurons in vivo and local field potentials under different

doses of anesthesia in cats. Additionally, we applied drugs in iontophoresis in the

perisynaptic space of the recorded neurons and we stimulated thalamic nuclei projecting to

the areas where recordings were performed. We suggest that isoflurane diminishes the

cortical inhibition, by an increase of the glutamate uptake by glial cells leading to a

diminished activation of cortical interneurons.

Avant-propos

Le corps de ce mémoire débute par un court historique des débuts de l’utilisation de

l’anesthésie générale et des idéologies s y rattachant, de même que par des données

récentes sur les mécanismes d’action proposés pour sous-tendre cet état. Le mémoire

contient un chapitre présentant l’article soumis à la publication et une conclusion générale

résumant les principaux sujets abordés dans l’article.

L’article présenté dans ce mémoire est le résultat des expériences que j’ai effectuées et

analysées en totalité dans le laboratoire du Dr Florin Amzica. Je tiens sincèrement à

exprimer ma gratitude envers le Dr Amzica qui m’a les portes de son laboratoire et a fait

preuve de beaucoup de flexibilité afin de me permettre de continuer simultanément mes

études au doctorat en médecine. Il m’a également offert son expérience, son sourire et sa

disponibilité tout au long de ma maîtrise et de la rédaction de l’article soumis a xxxxxxx. Je

remercie également Pierre Giguère et tous mes collègues étudiants pour leur collaboration

professionnelle et morale. Je remercie également la direction de l’École des gradués pour

m’avoir permis d’intégrer l’article à mon mémoire.

Je dédie cette thèse a mes collègues Oana et Daniel qui ont fait preuve d une incroyable

bienveillance et amitié envers moi. Ils ont littéralement vécu avec moi les bons et moins

bons moments inhérents à ce mémoire et m’ont fait gagner en maturité. Une part est aussi dédiée à mon ami Louis-Charles pour

son constant soutien à distance

Table des matières Résumé.....................................................................................................................................i Abstract.................................................................................................................................. ii Avant-propos ........................................................................................................................ iii Table des matières ................................................................................................................ iii Liste des abréviations.............................................................................................................iv Liste des figures ......................................................................................................................v Introduction.............................................................................................................................1 Chapitre 1 :..............................................................................................................................2 Concepts concernant l’anesthésie générale.............................................................................2 Chapitre 2 :..............................................................................................................................5 Caractéristiques clinique de l’isoflurane.................................................................................5 Chapitre 4 :............................................................................................................................10

4.1 Évolution des idées et des techniques concernant les mécanismes fondamentaux de l’anesthésie générale ....................................................................................................11 4.2 Augmentation de l’inhibition................................................................................12

4.2.1 Inhibition synaptique ....................................................................................12 4.2.2 Inhibition intrinsèque (canaux K+)................................................................14

4.3 Diminution de l’excitation ....................................................................................14 4.3.1 Baisse de la relâche de neurotransmetteurs ..................................................14 4.3.2 Blocage de récepteurs post-synaptiques .......................................................15 4.3.3 Augmentation de la recapture du glutamate par les glies .............................15

4.4 Résumé..................................................................................................................17 Chapitre 5 :............................................................................................................................18 Le burst-suppression .............................................................................................................18 Chapitre 6 :............................................................................................................................20 Cortical inhibition during burst-suppression induced with isoflurane anesthesia ................20 Abstract.................................................................................................................................21

6.1 Introduction.................................................................................................................22 6.2 Material and Methods .................................................................................................24

6.2.1 Animal preparation ..............................................................................................24 6.2.3 Analysis ...............................................................................................................26

6.3 Results.........................................................................................................................27 6.3.1 Database...............................................................................................................27 6.3.2 Patterns of activity related to the induction of BS ...............................................27 6.3.3 Modification of inhibition during BS ..................................................................28 6.3.4 Pharmacological correlates of inhibition during BS............................................31

Discussion.............................................................................................................................34 Figures ..................................................................................................................................38 Conclusion ............................................................................................................................49 Bibliographie ........................................................................................................................51

iv

Liste des abréviations

AMPA acide α-amino-3-hydroxy-5-methyl-4-

isoxalepropionique

α-amino-3-hydroxy-5-methyl-4-

isoxalepropionique acid

BS Burst-suppression

Ca+2 Ion calcium

EAAT Excitatory amino acid transporter

EEG Électroencéphalogramme Electroencephalogram

DHK Acide dihydrokainique Dihydrokainic acid

GABA Acide gamma-aminobutyrique Gamma-aminobutyric acid

H+ Ion hydrogène

K+ Ion potassium

MAC Concentration alvéolaire minimale Minimal alveolar concentration

Na+ Ion sodium

NMDA Acide N-methyl-D-aspartique N-methyl-D-aspartic acid

PPSE Potentiel post-synaptique excitateur Excitatory post-synaptic potential

PPSI Potentiel post-synaptique inhibiteur Inhibitory post-synaptic potential

SNC Système nerveux central

Vm Potentiel membranaire

v

Liste des figures

Figure 1: Typical progression toward burst-suppression (BS) pattern. ...............................38 Figure 2: Comparison of slow oscillatory activity of intracellular potentials during sleep-like patterns and bursting during BS. ...................................................................................39 Figure 3: Typical responses of cortical neurons to thalamic (CL) electrical stimuli ...........40 Figure 4: Voltage dependence of cortical responses to thalamic (CL) electrical stimuli ....41 Figure 5: Unmasking of excitatory components during BS responses matching the timing of inhibitory components during control conditions. ...............................................................42 Figure 6: Extracellular Cl- concentration ([Cl-]o) during BS induction and recovery. .........43 Figure 7: Blockage of glial glutamate uptake restores inhibition during BS. ......................44 Figure 8: Effect of iontophoretic application of GABA on a cortical neuron from area 5. .45 Figure 9: Effect of iontophoretic application of GABA on cortical responsiveness to thalamic stimulation. ............................................................................................................46 Figure 10: Effect of isoflurane-induced BS on the neuronal input resistance. ....................47 Figure 11: Reduction of cortical inhibition under barbiturate (thiopental) anesthesia. .......48

Introduction

L’intérêt de ce mémoire a été de décrire l’effet d’une augmentation de la dose d’isoflurane,

et donc de la profondeur de l’anesthésie, menant a l’état de burst-suppression, sur

l’inhibition dans le réseau neuronal cortical. Malgré le fait que le burst-suppression a été

précédemment étudié, la plupart de ces études ont été faites sur des cellules en culture et/ou

in vitro, le comportement des neurones in vivo, en relation avec tous les circuits cérébraux,

ayant été peu étudié.

Les sections suivantes révisent sommairement certains concepts généraux concernant

l’anesthésie générale, les propriétés cliniques de l’isoflurane et les théories concernant ses

mécanismes d’action. L’accent a été mis plus particulièrement sur comment l’isoflurane

influence la balance excitation/inhibition dans le cortex et comment ceci se reflète au

niveau électrophysiologique. Le tout s’inscrit dans une perspective d’une meilleure

compréhension de l’anesthésie générale d’un point de vue clinique ainsi que des états

comateux.

Chapitre 1 :

Concepts concernant l’anesthésie générale

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Les débuts de l’utilisation de l’anesthésie générale sont riches en rebondissements. D’abord

employée par un modeste médecin de campagne vers 1840, Crawford Long, l’anesthésie

générale a commencé à être l’objet de démonstrations publiques avec Horace Wells, qui

utilisa de l’oxyde nitrique. La démonstration échoua en un certain sens, car bien que

l’oxyde nitrique réduise la douleur, il n’induisait pas nécessairement une totale immobilité,

ce qui laissa douter le public de l’efficacité de la méthode. L’idée fut ensuite reprise par

William Morton et Charles Jackson qui utilisèrent avec succès de l’éther, ce qui amena une

bataille concernant la primauté de la découverte de l’anesthésie générale

150 ans après cette découverte, il n’y a toujours pas de consensus sur la définition même de

l’anesthésie générale, car cette définition se fond en fait avec les finalités cliniques que l’on

souhaite atteindre grâce à l’anesthésie. Il s’agit d’un état induit pharmacologiquement dans

lequel les buts principaux de l’anesthésie générale sont atteints et l’agent anesthésiant est la

substance qui permet l’atteinte de ces buts (Franks, 2006 ; Antognini et al., 2002). Le

problème est donc déplacé sur l’identification des buts essentiels, qui varient selon le

groupe de personnes concerné. Les patients souhaiteront l’amnésie et l’inconscience

(l’analgésie pouvant être considérée implicite à l’inconscience), le chirurgien voudra un

patient immobile et le cardiologue la préservation de l’équilibre hémodynamique

(Antognini et al., 2002).

Il est fascinant de constater qu’une multitude de composés peuvent induire ces effets : des

gaz rares (xénon), des petites molécules organiques (oxyde nitrique), des hydrocarbures

halogénés (isoflurane) et d’autres à la structure plus complexes (barbituriques) (Morgan et

al., 2002). Mais rares sont les composés qui répondent à toutes les exigences mentionnées

lorsque utilisés seuls. Ils sont souvent combinés en clinique afin de couvrir le spectre de

ces actions tout en minimisant l’intensité (mais non la diversité) des effets secondaires.

Ceci a contribué à rendre l’anesthésie un acte médical sécuritaire, avec un décès sur 200

000 procédures aux États-Unis. Néanmoins, les anesthésistes veulent un contrôle toujours

plus serré des doses administrées afin de réduire les effets secondaires et sont constamment

à la recherche d’agents plus spécifiques (Urban et al., 2002). Malgré cela, les indicateurs

cliniques de l’anesthésie profonde se limitent encore traditionnellement aux réponses

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musculo-squelettique et autonomes aux stimuli per-opératoire alors qu’un meilleur

monitorage de l’activité cérébrale aiderait à un contrôle plus serré des doses (Hartikainen,

1995). Dans cette perspective, une meilleure compréhension de ses mécanismes d’action

des anesthésiants est donc pertinente. Mes travaux de recherche portent plus spécifiquement

sur un composé de la classe des anesthésiants volatiles, l’isoflurane, en raison de ses

caractéristiques cliniques décrites ci-dessous. Nous avons choisi cet anesthésiant largement

utilisé afin que nos résultats puissent servir éventuellement au point de vue clinique.

Chapitre 2 :

Caractéristiques cliniques de l’isoflurane

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Nous avons porté notre attention sur l’isoflurane, car il constitue l’un des meilleurs choix

d’anesthésiants chez l’humain en raison de ses effets modérés sur la baisse de contractilité

et du rythme cardiaque et la baisse de tension artérielle, de ses propriétés anticonvulsivantes

et de sa non-toxicité hépatique et rénale (Eger, 1984 ). À l’instar d’autres composés

volatiles, tels le desflurane, il est reconnu pour ses effets neuroprotecteurs, (Hoffman,

1998 ; Doyle et al., 1999). que certains attribuent à la baisse de métabolisme cérébral qu’il

amène (Newberg et al., 1983 ; Doyle et al., 1999). Cette baisse de métabolisme se traduit

par une diminution de l’activité électrique des neurones, énergivore, qui préserve ainsi le

glucose et l’oxygène pour le métabolisme basal des cellules, leur permettant de mieux

résister à une éventuelle ischémie. Ceci justifie l’utilisation de l’isoflurane dans certaines

chirurgies où l’on induit l’hypoxie, comme celles à cœur ouvert (Pascoe et al., 1996) et

pour les anévrysmes intra-craniaux (Lavine et al., 1997). Il faut toutefois mentionner que le

sevoflurane et le desflurane sont souvent préférés à l’isoflurane, notamment à cause de la

grande rapidité d’élimination de ce dernier (Morgan et al., 2002).

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Chapitre 3 :

Corrélation entre les effets cliniques et lieux d’action de

l’isoflurane

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Il est reconnu qu’aux différents effets cliniques des anesthésiants correspondent différents

sites d’action dans le système nerveux (Grasshoff et al., 2005).

Par exemple, il est possible d’attribuer l’amnésie induite par l’isoflurane à une action au

niveau de l’hippocampe et de l’amygdale (Caraiscos et al., 1994; Ranft et al., 2004).

L’immobilité et une partie de l’analgésie résulteraient d’un blocage de la transmission au

niveau de la moelle épinière. Ceci prévient, d’une part, la génération de mouvements

réflexes et d’autre part, la réception des informations nociceptives par le cerveau

(Antognini et al., 2000).

La perte de conscience a été reliée à une altération de la communication dans le réseau

thalamo-cortical chez l’humain (White et al., 2003). D’autres expériences, menées à des

concentrations plus faibles d’isoflurane, mais suffisantes pour induire l’hypnose (absence

de réponse à des commandes verbales), soulignent l’importance du cortex cérébral, dans

lequel on observe une diminution de la décharge des neurones similaire malgré la

déconnection des structures sous-corticales (Hentschke et al., 2005 ). Mes travaux de

recherche se concentrent sur le rôle de l’isoflurane dans la boucle thalamo-corticale et plus

spécifiquement sur le comportement des neurones corticaux.

De la perte de conscience induite par l’anesthésie générale, il est possible de glisser vers

celle induite par le sommeil, avant de détailler davantage les mécanismes d’action

spécifiques à l’isoflurane. En effet, il est intéressant de dresser un parallèle entre la perte de

conscience induite dans ces deux états, les deux partageant une base commune. En effet,

l’altération du réseau thalamo-corticale mentionnée ci-dessus est aussi retrouvée pendant le

sommeil et est sous-tendue par l’hyperpolarisation des neurones thalamo-corticaux. Dans

les deux cas, cette hyperpolarisation amène un changement dans le patron de décharge de

ces neurones, qui passent d’un mode tonique à un mode en bouffées, ceci coïncidant avec

un changement dans le patron de l’EEG (oscillations rapides et de faible amplitude vers des

ondes plus lentes et amples) (Steriade, 1992 ; White et al., 2003). Ce changement dans le

patron de décharge des neurones entraîne une altération de l’information transitant par le

thalamus, déconnectant ainsi, d’un point de vue fonctionnel, le cortex des sensations

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extérieures. Alors que l’hyperpolarisation des neurones thalamo-corticaux peut être

attribuée à une diminution de l’excitation tonique des centres d’éveil du tronc cérébral

(Steriade, 1994) pendant le sommeil, elle pourrait aussi être causée pendant l’anesthésie,

par une action directe des anesthésiants sur le thalamus, en plus de celle sur les centres

d’éveil (White et al., 2003).

Néanmoins, il faut mentionner que l’anesthésie diffère du sommeil sur certains points. On

parle des stades du sommeil mais de la profondeur de l’anesthésie; cette dernière n’étant

pas un phénomène cyclique (Antognini et al., 2003). Aussi, un stimulus ne peut nous sortir

de l’état d’anesthésie et la température corporelle et le tonus musculaire y sont régulés

différemment (White et al., 2003).

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Chapitre 4 :

Mécanismes fondamentaux sous-tendant l’anesthésie

générale et l’action de l’isoflurane

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4.1 Évolution des idées et des techniques concernant les

mécanismes fondamentaux de l’anesthésie générale

L’une des plus célèbres théories concernant le mécanisme d’action des anesthésiants

généraux est celle de Meyer et Overton, dans laquelle le caractère lipophile des agents

volatils est mis en relief. Les deux scientifiques observèrent, de façon indépendante, que le

pouvoir anesthésiant d’un composé augmentait proportionnellement à sa solubilité dans les

lipides. Cette observation prit davantage de sens lorsque l’on découvrit le rôle des lipides

dans la structure membranaire; la bicouche lipidique fût perçue comme le site d’action des

anesthésiants, et l’on supposa que ces derniers altéraient son état normal. Cette théorie se

raffina et prévalu jusque dans les années 1980. Séduisante de par sa simplicité, elle ne

tenait pas compte cependant de la différence de potentiel entre les énantiomères d’un même

anesthésiant (molécule miroir) et n’expliquait pas comment la perturbation dans la

bicouche pouvait résulter en un dysfonctionnement des protéines membranaires qui

altérerait ultimement la transmission synaptique (Doyle, et al., 1999; Franks, 2006).

La maîtrise de nouvelles techniques permit de constater qu’il existe une diversité de cibles

potentielles pour les agents spécifiques et qu’il existe une certaine spécificité entre eux.

Ceci amena l’idée que les anesthésiants affectaient la transmission synaptique en se liant

directement aux récepteurs sur des sites précis. La perspective des mécanismes d’action des

anesthésiants est dans ce cas davantage agent-spécifique, où chaque agent affecte

différemment un ou des récepteurs en augmentant leur temps d’ouverture, leur fréquence

d’ouverture, en les bloquant, etc, par opposition à l’unification tentée par Meyer et Overton.

(Dilger, 2002 ; Franks, 2006). Plus récemment, les études génétiques, avec les techniques

de knock-out et knock-in, ont permis d’élaborer des liens puissants entre le niveau

moléculaire et systémique. Dans la première technique, la cible potentielle d’un

anesthésiant, souvent un canal ionique, est manquante, résultant en une baisse de

l’efficacité de l’agent. Dans la seconde, un canal ionique connu pour être modulé par un

anesthésiant est modifié au niveau d’un acide aminé, tout en laissant souvent la fonction

physiologique intacte, afin d’identifier plus précisément des sites de liaison ou de

modulation anesthésiant (Grasshoff et al., 2005).

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Les effets observés au niveau des réseaux neuronaux, thalamo-cortical, cortico-cortical,

etc., sont la sommation d’actions au niveau des neurones pris individuellement. Par

exemple, une altération de la communication dans un réseau pourrait être due à une

augmentation de l’inhibition et/ou une diminution de l’excitation, amenant un déséquilibre

dans la balance excitation/inhibition qui soutient l’éveil. Ces propos sont illustrés par des

travaux concernant l’altération, par des anesthésiants intraveineux (thiopentone et

propofol), de l’oscillation de circuits d’interneurones présents dans le néocortex et

l’hippocampe. Cette oscillation gamma (30-60 Hz), qui a été associée à des fonctions

cognitives, résulte de la coordination de courants GABAergiques (Whittington et al., 1996;

Bragin et al., 1995) et sera donc affectée par une prolongation de ces courants. Ceci montre

comment un changement au niveau moléculaire se répercute sur un réseau. Bien que ces

travaux ne portent pas directement sur l’isoflurane, on peut supposer que ses actions sur les

multiples cibles décrites ci-dessous influencent le fonctionnement global de réseaux

neuronaux.

Nous nous sommes davantage attardés à l’effet de l’isoflurane sur l’inhibition corticale

4.2 Augmentation de l’inhibition

4.2.1 Inhibition synaptique

Les anesthésiants généraux peuvent agir en augmentant l’inhibition au niveau synaptique

en potentialisant la signalisation inhibitrice déjà en place, i.e. en agissant comme agonistes

des récepteurs GABAergiques et glycinergiques (Dilger, 2002 ; Franks, 2006), court-

circuitant par conséquent les entrées excitatrices de plusieurs neurones simultanément et

modifiant leur patron de décharge.

4.2.1.1 Les récepteurs GABAA

Beaucoup d’attention a été portée aux liens unissant l’anesthésie et le récepteur GABAA, ce

dernier étant le canal ionique à ligand le plus abondant, chez le mammifère, pouvant

produire une inhibition rapide. Présent à travers le système nerveux, il est surtout situé en

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post-synaptique et parfois en péri-synaptique. Il est formé de 5 sous-unités, amenant une

diversité de sous-types de récepteurs dans le système nerveux (Franks, 2006).

Les récepteurs GABAA ont été identifiés comme la cible non équivoque des barbituriques

propofol et étomidate. Les étapes de cette découverte, qui débutent dans les années 80,

forment en quelque sorte un modèle; il a d’abord été observé que ces agents prolongeaient

les courants GABA dans les tranches d’hippocampe, puis que la différence de pouvoir

anesthésiant entre les énantiomères se reflétait par une différence dans leur pouvoir de

modulation du récepteur GABAA et finalement qu’une modification dans les sous-unités du

récepteur pouvait mener à une insensibilité à ces agents (Franks, 2006).

Des travaux ont montré que l’isoflurane aussi prolongeait les courants GABAA in vitro

(Nakahiro et al., 1989; Lin et al., 1992) et des sites de liaison sur des sous-unités du

récepteur ont même été identifiés (Mihic et al., 1997 ; Jenkins et al., 2001). Vahle-Hinz et

al (2001) ont aussi observé, in vivo, que l’administration de bicuculline, un antagoniste

GABAA, dans le thalamus, reversait l’effet suppressif de l’isoflurane sur les neurones

thalamo-corticaux et restaurait leur patron de décharge tonique, associé à la transmission de

l’information.

La feuille de route décrite ci-dessus n’a pu cependant être davantage suivie pour l’ensemble

des agents volatiles. En effet, la potentiation des récepteurs GABAA, bien qu’établie, est

habituellement moindre, à des concentrations d’anesthésiants équivalentes, que celle

observée par le propofol et l’etomidate, laissant supposer que les récepteurs GABAA ne

peuvent être la seule cible (Franks, 2006).

4.2.1.2 Les récepteurs glycinergiques

Les récepteurs glycinergiques, souvent colocalisés avec les récepteurs GABAA, sont aussi

considérés comme une cible plausible de l’isoflurane. Ils sont potentialisés par de faibles

concentrations de différents anesthésiants volatiles. (Harrison et al., 1993 ; Downie et al.,

1996). De par leur importance particulière dans la moelle épinière, ils seraient

particulièrement impliqués dans la perte de réponse aux stimuli douloureux (Sonne et al.,

2003). Cependant, comme les approches touchant la génétique de ces récepteurs ne sont pas

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encore concluantes, le rôle des récepteurs glycinergiques dans l’anesthésie générale ne peut

être considéré comme définitif (Franks, 2006).

4.2.2 Inhibition intrinsèque (canaux K+)

Il est aussi possible de moduler l’inhibition neuronale en modifiant les propriétés

intrinsèques des neurones, qui ne dépendent pas de la liaison directe avec un

neurotransmetteur. Par exemple, il a été rapporté que l’isoflurane augmentait des courants

K+ de fuite, i.e. qui sont médiés par des canaux dont l’ouverture n’est pas contrôlée par un

récepteur. Cette ouverture amène une baisse de la résistance membranaire du neurone et

court-circuite ainsi les conductances Na+ et Ca+2 excitatrices. (Ries et al., 1999). Plus

récemment, les canaux K+ two-pore-domain, se sont imposés comme une autre cible

potentielle (Patel et al., 2001). Ils sont vus comme des régulateurs de l’excitabilité

membranaire et sont soumis à une grande modulation (Franks, 2006). Leur distribution

dans le SNC est complexe et ils sont situés en pré et post-synaptique. Heurteaux, et al.,

(2004) ont montré que des souris n’exprimant pas TREK-1, un membre de cette famille de

canaux, étaient insensibles à l’action de plusieurs agents volatiles, dont l’isoflurane.

4.3 Diminution de l’excitation

Les anesthésiant généraux peuvent aussi agir en diminuant la transmission excitatrice et il

semblerait que l’isoflurane puisse y parvenir de façon pré ou post-synaptique.

4.3.1 Baisse de la relâche de neurotransmetteurs

Une diminution de la relâche des neurotransmetteurs, et plus particulièrement du glutamate,

a été envisagée comme un mécanisme pré-synaptique pour diminuer l’excitation

(Westphalen et al., 2003 ; Wu et al., 2004). La cause de cette diminution de la relâche a été

située, selon les auteurs, au niveau de l’entrée de Ca+2 dans le bouton terminal (MacIver et

al., 1996), de la machinerie même de l’exocytose (Hemmings et al., 2005) ou de

l’amplitude du potentiel d’action (Wu et al., 2004), dont la diminution pourrait être due à

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une action sur des canaux Na+ (Rehberg et al., 1996) ou K+ (Ries et al., 1999; Patel et al.,

2001). Bien qu’aucun de ces auteurs n’expliquent précisément comment l’isoflurane inhibe

davantage la libération de glutamate que celle de GABA, afin de produire un effet

globalement dépresseur, Westphalen donne quelques pistes en suggérant notamment une

différence physiologique entre les boutons terminaux GABAergiques et glutamatergiques.

Il envisage aussi une baisse de libération de glutamate et de GABA mais compensée par

une potentiation des récepteurs GABAA.

4.3.2 Blocage de récepteurs post-synaptiques

La transmission glutamatergique peut aussi être réduite par un blocage ses récepteurs post-

synaptiques. On reconnaît à l’isoflurane le potentiel de bloquer les récepteurs AMPA et

NMDA (Carla et al., 1992 ; Martin et al., 1995), quoique la littérature ne soit pas très

abondante sur cet aspect. Par contre, l’isoflurane pourrait diminuer l’efficacité des NMDA

via l’hyperpolarisation neuronale qu’elle amène via l’augmentation des conductances K+

(Ries et al., 1999).

Il est aussi question que l’isoflurane puisse inhiber des courants Ca2+ voltage-dépendants

sur des cellules chromaffines (Pancrazio et al., 1993) et dans l’hippocampe (Study, 1994).

4.3.3 Augmentation de la recapture du glutamate par les glies

En plus des mécanismes d’action pré et post-synaptique de l’isoflurane pourrait en exister

un autre dépendant des cellules gliales. Davantage qu’une simple matrice cellulaire servant

de support, les glies jouent un rôle important dans la régulation des fonctions neuronales,

incluant la transmission glutamatergique. Une fois libéré, le glutamate est rapidement

recapturé, car il peut être neurotoxique à haute dose, amenant un influx massif de Ca2+ via

les récepteurs NMDA. Cette recapture se fait au niveau des boutons terminaux des

neurones, certes, mais surtout par les processus des astrocytes qui entourent les synapses.

La recapture s’effectue par des transporteurs spécifiques au glutamate qui utilisent les

gradients électrochimiques du Na+, K+ et H+. Les astrocytes convertissent le glutamate en

16

glutamine qui est retournée aux neurones. Ces derniers peuvent éventuellement la

retransformer en glutamate au niveau du bouton terminal et l’emmagasiner à nouveau dans

des vésicules synaptiques (Shigeri et al., 2003).

Plus précisément, cinq transporteurs de glutamate ont été caractérisés jusqu’à maintenant

chez l’humain (EAAT 1 à 5). Les glies expriment surtout EAAT1 et EAAT2 et ce dernier

est considéré comme le transporteur glial type, étant exprimé de façon prédominante par

celles-ci. EAAT2 est responsable de plus de 90% de la recapture du glutamate et il est

exprimé dans l’ensemble des régions cérébrales (Shigeri et al., 2003 ; Su et al., 2003).

Il a été observé, sur des cultures gliales, que l’isoflurane pouvait augmenter la recapture du

glutamate via ces transporteurs (Zuo, 2001; Miyazaki et al., 1997 ; Huang et al., 2003). Le

même phénomène a été noté chez d’autres anesthésiants volatiles – halothane, enflurane,

sevoflurane - mais non avec les anesthésiants intraveineux tels le pentobarbital et la

kétamine. L’augmentation de la recapture causerait une diminution rapide du glutamate

dans la synapse et diminuerait ainsi son action sur les récepteurs post-synaptiques. Même si

ceci ne serait pas suffisant pour bloquer complètement la transmission d’une synapse prise

isolément, il est probable que cette diminution de transmission ait des répercussions au

niveau du réseau neuronal, où la multitude de signaux s’unissent et s’amplifient (Miyazaki

et al., 1997 ). Cette diminution du glutamate synaptique pourrait aussi être un mécanisme

de la neuroprotection que procurent les agents volatiles. Il a d’ailleurs été observé que

l’inhibition des EAATs augmentaient l’amplitude du potentiel post-synaptique excitateur

(PPSE) in vitro (Tong et al., 1994). et augmentait le MAC (concentration alvéolaire

minimale; i.e. la concentration d’anesthésiant où 50% des patients n’ont pas de réaction lors

d’une incision chirurgicale) pour l’isoflurane in vivo (Cechova et al., 2006). Cette dernière

observation signifiant qu’il faut davantage d’isoflurane pour obtenir un effet anesthésiant

lorsqu’il y a davantage de glutamate dans les synapses.

17

4.4 Résumé

La majorité des auteurs cités ci-dessus s’entendent pour attribuer plus d’un mécanisme

d’action à l’isoflurane mais leurs avis diffèrent concernant l’importance relative de chacun.

Certains attribuent davantage d’importance à l’augmentation de l’inhibition par rapport à la

diminution de l’excitation ou à l’action pré-synaptique plutôt que post-synaptique de

l’isoflurane. Vahle-Hinz et ses collègues, par exemple, ont montré que le blocage des

informations du thalamus pouvait à la fois être restauré par des agonistes AMPA/NMDA

ou des antagonistes GABAA, illustrant la contribution relative des mécanismes inhibiteurs

et suppresseurs (Vahle-Hinz et al., 2007). Et bien que j’aie tenté de présenter ces

mécanismes de façon distincte pour des fins pédagogiques, il m’apparaît clair qu’ils sont en

interactions les uns avec les autres dans les modèles in vivo; une diminution de l’excitation

devrait influencer les neurones inhibiteurs qui eux-mêmes modulent les neurones qui les

excitent.

Beaucoup des conclusions des travaux cités ci-dessus doivent être regardées d’un œil

critique. Les énoncés du type : «À des concentrations cliniques d’un anesthésiant x, les

récepteurs GABAA sont affectés par x anesthésiant alors que les canaux Na+ ne le sont

pas » sont communs dans les publications. Il ne faut pas perdre de vue qu’il y a plus d’un

type de récepteur GABAA et de canaux Na+. Une myriade de facteurs peut influencer la

réponse à un anesthésiant et par conséquent la concentration de l’anesthésiant nécessaire

pour obtenir certains effets; or, on attribue souvent beaucoup d’importance à se situer dans

une fourchette de concentration pertinente cliniquement. Ceci est susceptible d’être modulé

par l’environnement cellulaire, les cascades de seconds messagers et les connections dans

les réseaux étudiés (Urban et al., 2002).

Finalement, mentionnons qu’en marge de tous ces mécanismes électrophysiologiques, il a

été découvert plus récemment que l’isoflurane peut affecter l’expression des gènes,

dessinant une piste pour l’explication de certains effets secondaires post-opératoires

(Rampil, et al., 2006).

18

Chapitre 5 :

Le burst-suppression

19

Mes travaux de recherche portent donc sur l’action de l’isoflurane dans le cortex, en

relation avec le thalamus, à des doses qui induisent un patron d’EEG nommé burst-

suppression (BS). Il peut être observé chez l’humain à des niveaux modérés et profonds

d’anesthésie et il peut être induit par l’isoflurane, mais aussi par des composés appartenant

à d’autres classes d’anesthésiants, tels les barbituriques. Ce patron d’EEG montre une

alternance entre des bouffées d’activité et des lignes isoélectriques. (Hartikainen et al.

1995; Steriade et al. 1994). En plus de pouvoir être induit pendant certaines chirurgies, ce

patron d’EEG est aussi visible dans certains comas dus à une intoxication de drogues ou de

l’hypoxie suivant un arrêt cardio-respiratoire, par exemple. L’état de burst-suppression,

dans ce cas-là, est souvent signe de mauvais pronostic (Brenner, 2005). Les mécanismes

sous-tendant le BS sont peu connus, mais on peut espérer qu’une meilleure compréhension

de ceux-ci pourrait conduire vers des gestes thérapeutiques améliorant le pronostic de ces

patients.

La première étude du BS au niveau cellulaire, bien que non sous isoflurane, a été faite par

Stériade et al (1994). Ils ont montré que l’activité intracellulaire des neurones néocorticaux

et thalamiques as correspondait à celle observée au niveau de l’EEG. Ils ont aussi montré

que 30 à 40 % des neurones thalamiques déchargeaient encore pendant les suppressions,

mais de moins en moins avec la profondeur de l’anesthésie, et que des stimulations

thalamiques pouvaient restaurer l’activité de l’EEG. Cette dernière observation a

également été faite chez l’humain pendant le BS induit par l’isoflurane, suggérant que la

circuiterie corticale est encore fonctionnelle (Hartikainen, et al., 1995). Mes travaux de

recherche se sont effectués à des doses d’anesthésiant atteignant le burst-suppression, afin

d’étudier plus en profondeur le comportement des neurones corticaux pendant cet état et de

le comparer avec celui sous de plus faibles doses d’anesthésiant.

20

Chapitre 6 :

Cortical inhibition during burst-suppression induced with isoflurane anesthesia

Judy-Fay Ferron, Daniel Kroeger, Oana Chever and Florin Amzica*

21

Abstract

Isoflurane is a widely used anesthetic which safely and reversibly induces deep coma and

associated burst-suppression (BS) electroencephalographic patterns. Here we investigate

possible underlying causes for the state of cortical hyperexcitability which was recently

shown to be one of the characteristics of BS. Our hypothesis was that cortical inhibition is

diminished during isoflurane-induced BS. Experiments were performed in vivo employing

intracellular recordings of cortical neurons to assess their responsiveness to stimulations of

connected thalamic nuclei. We demonstrate that during BS excitatory post-synaptic

potentials were diminished by 44%, whilst inhibitory potentials were completely

suppressed. This finding was supported by additional results indicating that a decrease in

neuronal input resistance normally found during inhibitory responses under low isoflurane

conditions was abolished in the BS condition. Moreover, removal of inhibition occasionally

revealed excitatory components which were absent during control recordings prior to the

induction of BS. We also show that the absence of inhibition during BS is not caused by a

blockage of GABA receptors, since iontophoretically applied GABA shows receptor

availability. Moreover, the concentration of extracellular chloride was increased during BS,

as would be expected after reduced flow of chloride through GABAA receptors. Also

inhibitory responses were re-instated by selective blockage of glial glutamate transporters

with dihydrokainate. These results suggest that the lack of inhibition during BS is caused

by reduced excitation, probably resulting from increased glial uptake of glutamate

stimulated by isoflurane, which creates a diminished activation of cortical interneurons.

Thus cortical hyperexcitability during BS is favored by suppressed inhibition.

22

6.1 Introduction

It is commonly believed that general anesthetics act by shifting the excitatory-inhibitory

balance of brain signaling towards inhibition. This assumption is mainly supported by

investigations into molecular mechanism of anesthesia suggesting that most of these agents

act on GABA receptors (for review see Franks, 2006). Within the large variety of

anesthetics, inhalation agents such as isoflurane are widely used in clinical practice.

Isoflurane appears to target several membrane proteins: glycine and GABAA receptors

(Harrison et al., 1993; Nishikawa et al., 2002) , as well as two-pore-domain K+ channels

(Franks and Lieb, 1988; Patel et al., 1999). In addition, isoflurane has been shown to

stimulate the activity of the GLT1/EAAT2 glial glutamate transporter (Larsen et al., 1997;

Zuo, 2001).

A great wealth of data has been produced relating to the targets and mechanisms of

anesthesia in general, and of isoflurane in particular. Interestingly, most of these studies

were conducted in vitro or in cultures and despite the simplified preparation most still did

not lead to definitive conclusions. The few in vivo studies (Vahle-Hinz et al., 2001; Detsch

et al., 2002; Vahle-Hinz et al., 2007) used extracellular recordings which render the

interpretation of excitatory vs. inhibitory activities uncertain. In slices, isoflurane reduced

excitatory synaptic transmission in the hippocampus (Berg-Johnsen and Langmoen, 1986;

Miu and Puil, 1989) and amygdala (Ranft et al., 2004). Moreover, at concentrations of

about 0.2-0.3 mM isoflurane increased the duration of GABAA inhibitory synaptic currents

without affecting their amplitude (Ranft et al., 2004). This result contrasts somewhat with a

series of studies showing that similar concentrations of volatile anesthetics decreased the

current-amplitude of GABAA IPSCs in hippocampal neurons (Jones and Harrison, 1993;

Pearce, 1996; Banks and Pearce, 1999) as well as in cerebellar neurons (Antkowiak and

Heck, 1997) , while confirming the prolonged decay time. These effects are believed to

result from postsynaptic binding of the agent (Banks and Pearce, 1999; Westphalen and

Hemmings, 2003).

Recently, we have employed a variety of anesthetic agents to demonstrate that a

deep level of anesthesia, producing burst-suppression (BS), generates a state of unstable

cortical hyperexcitability (Kroeger and Amzica, 2007). This finding prompted us to

23

question the strength of the associated inhibitory mechanisms within states of anesthesia-

induced unconsciousness.

BS is an electroencephalographic (EEG) pattern consisting of an alternating pattern

of (a) epochs with silenced cortical activity and associated EEG isoelectric line, and (b)

periods displaying high-amplitude slow and sharp waves (Swank and Watson, 1949). BS is

generally developed during coma (Brenner, 1985) with various etiologies ranging from

anesthesia (safe, fully-controlled and reversible) to hypoxia (Silverman, 1975), drug-related

intoxications (Weissenborn et al., 1991; Ostermann et al., 2000; De Rubeis and Young,

2001) and hypothermia (Pagni and Courjon, 1964; Michenfelder and Milde, 1991;

Nakashima et al., 1995). The common EEG patterns of BS, regardless of the underlying

etiology (Chatrian, 1990), suggest common cellular mechanisms. For instance, the

hyperexcitable responses evoked during BS were elicited employing various anesthetic

agents (Kroeger and Amzica, 2007).

For our present investigation we began with the assumption that the

hyperexcitability seen during BS results from diminished or blocked inhibition.

24

6.2 Material and Methods

6.2.1 Animal preparation

All experimental procedures were performed in acute preparations (cats) and were

approved by the committee for animal care of Laval University. They fully comply with the

principles of responsible conduct toward animals of the NIH. Experiments started with an

initial i.m. application of ketamine-xylazine anesthesia (10 mg/kg and 2 mg/kg,

respectively). Once a painless state was achieved (verified through absence of withdrawal

reflex), all future pressure and incision points were infiltrated with lidocaine and the animal

was tracheotomized, artificially ventilated (respiratory rate around 30 strokes/min) and

paralyzed with gallamine triethiodide (20 mg/kg) through a cannula inserted in the cephalic

vein. These procedures lasted for about 15 min. From this point on, the animal was

continuously monitored in order to maintain vital signs within a physiological range. Body

temperature was controlled through a heating pad (38 ± 1oC), end-tidal CO2 concentrations

were kept at 3.7% (±0.2%) by adjusting the O2 in the inhaled airflow. Deep anesthesia was

verified by continuous patterns of slow and ample EEG waves. We also monitored the heart

rate throughout the experiments (below 110 beats/min). Before the onset of surgical

procedures we gradually switched anesthesia to isoflurane (1-1.5%) in order to compensate

for the vanishing initial dose of ketamine-xylazine. From that point onwards the animal was

kept only under isoflurane anesthesia. Following the isoflurane induction, the animal

underwent craniotomy exposing the suprasylvian gyrus. First we lowered bipolar tungsten

stimulation electrodes into the centro-lateral (CL) and latero-posterior (LP) nuclei of the

thalamus (respective sterotaxic coordinates: anterior 10 mm, lateral 3.5 mm and vertical 4

mm above the ear bar plane; and anterior 10 mm, lateral 6 mm and vertical 4 mm). Next we

placed intracellular microelectrodes as well as field potential electrodes and Cl--sensitive

electrodes into the cortex. The stability of the recordings was enhanced by bilateral

pneumothorax, hip suspension, drainage of the atlanto-occipital space and filling of the

hole in the calvarium with a 4% agar solution. Fluid loss during the experiment was

compensated for by intravenous injections of saline (20-30 ml/experiment). Unless

25

otherwise indicated, all chemicals used in these experiments were purchased from Sigma.

At the end of the experiments, the animals received a lethal dose of sodium pentobarbital.

6.2.2 Electrode preparation and recordings

Intracellular recordings were obtained from the association areas 5 and 7 of the

suprasylvian gyrus using glass micropipettes (tip diameter <0.5 μm) filled with a 3 M

solution of potassium acetate (in situ impedance 30-50 MΩ). Field potential electrodes

were similarly shaped, except that the tip was widened under the microscope to achieve in

situ impedance of around 10 MΩ. These field potential electrodes were filled with a 0.2 M

NaCl solution. The EEG was recorded monopolarly with stainless steel screws implanted

into the skull in the immediate vicinity (~2 mm) of the cellular recordings. All electrodes

shared a chlorided silver reference placed in the paralyzed neck muscles. The EEG

potentials were bandpass filtered between 0.3 Hz and 1 kHz. Thalamic stimulation was

elicited with current impulses of 0.1-0.2 ms duration and 0.05-0.8 mA intensity. Under BS

conditions, the electrical stimuli were generally delivered during the suppression periods in

order to avoid the interference of spontaneous (bursting) activities.

Our Cl--sensitive microelectrodes were manufactured according to procedures

described in other studies (Massimini and Amzica, 2001). We employed double-barrel

pipettes in which the Cl--sensitive electrode was pretreated with dimethylchlorosilane, dried

at 120oC for 2 hours, and the tip filled with the Cl- ionophore I-cocktail A (Fluka). This

provides a rapid electrode (see below) and logarithmic selectivity against bicarbonate of 1.5

(Kondo et al., 1989) corresponding to a 3% selectivity.The rest of the barrel was loaded

with NaCl (0.1 M), while the other barrel contained NaCl (0.2 M). The Cl--sensitive

electrodes were calibrated in two solutions containing respectively: 1) NaCl 100 mM, and

2) NaCl 10 mM and NaGlu 90 mM (pH 7.4). In order to ascertain the relationship between

concentration and voltage we employed the Nicolsky-Eisenmann equation (Ammann,

1986).

We measured the time course of the response of Cl--sensitive microelectrodes by

stepping the electrodes through 2 drops containing different Cl- concentrations (10 and 100

mM). The drops were held at close distance by silver rings, which were individually

grounded. Only electrodes reaching 90% of the response in less than 20 ms were selected

26

for recording, ensuring that the electrode response was far faster than the phenomena under

investigation. To preclude the possible contamination of ion potentials through capacitive

coupling by field potentials, the latter were measured with the pair electrode and subtracted

from the former. The resulting signal was linearized and transposed into concentration

values using the parameters extracted from the logarithmic fitting of the calibration points.

The headstage amplifier for Cl--sensitive electrodes was modified with an ultra ultra low

input current (<25 fA) amplifier (National Semiconductor). Intracellular, ion-sensitive and

field potential signals were passed through a high-impedance amplifier with active bridge

circuitry (Neurodata). All signals were digitally converted (20 kHz sampling rate) and

stored on a computer for offline analysis.

Iontophoretic application of GABA was performed by means of double barrel glass

electrodes which were broken to a diameter of 7 to 15 µm. One of the barrels was loaded

with GABA (0.25 M in distilled water, pH 4), whilst the other barrel contained NaCl (0.2

M) and served as a blank electrode. The double barrel electrode was then glued to an

intracellular microelectrode whose recording tip protruded by 40-60 µm beyond the

iontophoretic tip. GABA was retained in its barrel by negative current (8-10 nA) and was

ejected by positive current (3-70 nA) by means of an electrophoresis device (Dagan,

Minneapolis, USA).

Dihydrokainate is an antagonist of the glutamate transporter GLT1/EAAT2. In our

experiments a 9 mM solution of dihydrokainate in lactate ringer was applied topically using

a Hamilton syringe on a small filter paper (3x3 mm) placed over the suprasylvian gyrus.

The paper had a central hole through which the recording intracellular microelectrode was

lowered into the cortex.

6.2.3 Analysis

All of our analysis was performed with software from WaveMetrics Inc., and relies on time

relationships between the recorded voltage (concentration) time series. Most of the results

are presented as stimulus-triggered averages (evoked potentials). These were obtained

through extracting sweeps of the intracellular or extracellular field/EEG potentials and

synchronization with the triggering stimulus. Statistical analysis was carried out with

XLSTAT.

27

6.3 Results

6.3.1 Database

Fifty-six neurons were recorded intracellularly in 24 cats. Only recordings satisfying high

quality criteria were retained for this study: stable membrane potentials more negative than

-60 mV without holding current and overshooting action potentials. The average resting

membrane potential recorded under low isoflurane conditions, immediately following

impaling and after stabilization of the membrane potential was -72.4 ± 3.2 mV (average ±

SD). We also recorded the cell’s input resistance: 23.6 ± 9.7 MΩ (tested with 0.5 nA

negative current pulses). These parameters were modulated during the induction of BS (see

below). All recorded neurons displayed regular discharge patterns according to the

established criteria (Connors et al., 1982; Nuñez et al., 1993). All intraneuronal recordings

were held for more than 30 min in order to include at least one recording of a change of

state (e.g. control to BS) - although some recordings lasted for up to 4 such cycles.

6.3.2 Patterns of activity related to the induction of BS

All recording sessions started under 1.5% isoflurane anesthesia, which produced activity

patterns comparable to those recorded during slow-wave sleep (SWS) and under ketamine-

xylazine anesthesia (Fig. 1A) (for further examples, see Amzica and Steriade, 1995;

Contreras and Steriade, 1995). Briefly, these patterns consist of a quasi regular slow

oscillation with a frequency range just below 1 Hz. The oscillation is the result of

membrane potential alternations in the recorded neurons between depolarized (“up”) states

and hyperpolarized (“down”) states. This oscillation is reflected in an in-phase lock of EEG

and field potential signals. The up-states correspond to field negative potentials, whilst

down-states match field positive potentials (Contreras and Steriade, 1995).

BS was induced by raising isoflurane in the inhaled airflow to 2.5%. The first

change in activity patterns was characterized by an accelerated and spikier EEG as well as

by a hyperpolarization of the neuronal membrane (Fig. 1B). With a delay of about 5 min

(for details, see Tétrault et al., 2008) the first episodes of isoelectric lines (asterisks in Fig.

1B) appeared and soon afterwards a stable pattern of BS was established (Fig. 1C). The

28

EEG patterns during BS consisted of periods of bursting and isoelectric lines. During the

latter, all recorded cortical neurons displayed a hyperpolarized membrane potential devoid

of any synaptic activity, while bursts consisted of depolarizing triangular-shaped potentials,

synchronous with corresponding field and EEG waves.

The distribution of membrane potential values differed between low isoflurane

conditions and BS (Fig. 2). SWS patterns always produced a bimodal distribution

illustrating the preference for either the “up” state or the “down” state (Fig. 2A left). In

contrast, the bursting activity displayed a unimodal distribution (Fig. 2A right) even though

this activity was also characterized by depolarizing events - albeit exhibiting prevalence for

hyperpolarized values and an absence of peaks in the depolarizing range. This aspect

reflects the short duration of the depolarizing potentials during the bursting phase of BS.

Under low isoflurane anesthesia depolarizing phases of the slow oscillation also reflect the

activity of cortical interneurons, thus the above observation is a first indication of the

possible reduction of inhibitory activity during BS (see Discussion).

6.3.3 Modification of inhibition during BS

Under low isoflurane conditions cortical neurons respond to volleys from functionally

related thalamic nuclei with an initial, short-latency EPSP (Fig. 3A), followed by an

inhibitory potential (a), a long-lasting hyperpolarization (b), and a rebound depolarization

(c). At hyperpolarized membrane potentials, such as during spontaneously occurring

“down” states, the initial IPSP (a) appeared reverted and added on top of the initial EPSP.

On the other hand, the secondary hyperpolarization reached a similar membrane potential

(2nd trace, Fig. 3A). As proposed in other reports with similar responses (Connors et al.,

1988; Berman et al., 1991; Contreras et al., 1997), we suggest that the initial

hyperpolarization might represent a GABAA IPSP, while the second one could be GABAB

mediated. During the low isoflurane condition we observed no membrane potential

dependence of the rebound effect in all tested neurons (n = 37). However, after the

induction of BS (3rd trace) the rebound excitation (c) was virtually abolished, despite the

more hyperpolarized membrane potential. In addition, the reverted (a) component was

decreased. The comparison of the amplitudes of the initial EPSPs between low isoflurane

29

conditions and BS shows a reduction of the EPSP by 44% (n = 12). This comparison was

performed with each neuron kept at the same membrane potential (± 0.5 mV) during the

two conditions (this implied depolarization of the membrane through intracellular current

injection during BS). No analysis of the duration the EPSPs was made, due to the

variability in resting membrane potential among the recorded population of neurons, which

could have been responsible for voltage-dependent components such as NMDA, INa(p), etc.

Additionally, BS unmasked occasionally excitatory components (see below) that would

have rendered the task unrealistic. Furthermore, no inhibitory responses were observed

during BS even when depolarizing the neuronal membrane at values close to firing

threshold (-60 mV in Fig. 3B).

We suggest that the (a) component in the response represents an active inhibition.

Therefore we measured the input resistance of the neuron during the respective time frame

(Fig. 3B). Under low isoflurane conditions the input resistance was decreased during the

period following the initial EPSP, as compared to periods without electrical stimulation

(compare black traces in Fig. 3B1 and 2), as expected if an IPSP would have occurred. In

the depicted neuron, the initial IPSP was associated with a 23% drop in resistance, while at

the extent of the complete population of tested neurons (n = 37) the resistance drop was of

27%. During BS on the other hand, a similar comparison, at the same membrane potential,

showed no overt modification of the input resistance and indeed only displayed the overall

increased resistance during BS unaffected by the presence or absence of preceding

stimulation (red traces in Fig. 3C). The lack of resistance modification during the time

frame of the IPSP under BS conditions was a consistent finding in all tested neurons (n =

37).

Furthermore, we could demonstrate that late inhibitory potentials evoked by

thalamic electrical stimulation during low isoflurane conditions were voltage-dependent, as

would be expected by a phenomenon which opens membrane conductances. Thus, long-

lasting inhibition was enhanced by depolarization under low isoflurane conditions (Fig. 4A,

black trace) as compared to those at resting membrane potential (Fig. 4B). In contrast,

responses to the same stimuli were markedly reduced by steady hyperpolarization of the

membrane potential (Fig. 4C). The fact that this component was absent at a very

hyperpolarized level (-107 mV in this case) suggests that it might be generated by K+

30

currents. This hyperpolarizing component was canceled at a membrane potential of -93 mV

(± 2.1 mV; n = 16). We were, however, unable to reverse this putative GABAB IPSP in

order to provide a clear reversal potential. During BS overt inhibitory potentials were

completely abolished at any levels of membrane polarization (Fig. 4, red traces).

In several neurons (n = 14, 25% of the recorded neurons) the thalamic response

during BS consisted of the initial EPSP and a secondary excitation. They occurred with

variable delay within the time frame of the low isoflurane inhibition (Fig. 5). In these cases,

control responses exhibited the usual excitation-inhibition-rebound sequences (Fig. 5A1).

The BS responses, however, were devoid of the rebound phase, in a similar way as in the

overall majority of the recorded cells. Moreover, we observed that the secondary excitation

displayed a clear voltage-dependence (Fig. 5A2), but found it unlikely that this may

represent a reversal of the IPSP because this response was also present at rather depolarized

membrane potentials (e.g. -65 mV in Fig. 5A2). In addition, the latency of this component

was variable and was expressed at varying points during the inhibitory window (Fig. 5B).

We suggest that the observed excitatory potential may represent a secondary synaptic

excitatory event unmasked by the absence of inhibition during BS. Under low isoflurane

conditions, this response is shunted by inhibitory potentials.

Since Cl- inhibition is achieved by opening of the GABAA receptors and entrance of

Cl- ions in the cell, we investigated whether the observed absence of inhibition during BS

might have consequences on the extracellular distribution of various ions, particularly those

involved in inhibitory processes (Cl- and K+). Our results suggest that during low isoflurane

conditions ongoing inhibitory activities establish a given extracellular Cl- concentration

([Cl-]o). Application of high levels of isoflurane (3%) caused an increase of [Cl-]o in parallel

with the onset of BS. The extracellular concentration returned to control values after the

supply of isoflurane was discontinued. We observed an average increase in [Cl-]o by 27.3

mM (± 7.4, n =6) with an onset latency of 112 s (± 21). Considering that the average [Cl-]o

is around 150 mM (Dietzel et al., 1982), our recorded difference thus represents a 18%

increase. Within seconds of the onset of this increase we recorded the appearance of the

first isoelectric episode (asterisk in the inset in Fig. 6, marking the onset of BS), suggesting

that the reduction of the inhibitory activity in the cortex might create a surplus of Cl- ions in

the extracellular space.

31

6.3.4 Pharmacological correlates of inhibition during BS

Thus far, our data suggest that inhibition is strongly impaired during isoflurane-induced

BS. One reason for this impairment may be the action of isoflurane on an enhancement of

glutamate uptake by specific glial GLT1/EAAT2 transporters (Larsen et al., 1997; Zuo,

2001) and the consequent reduction of excitatory neuronal inputs to inhibitory interneurons.

The idea behind this reasoning is that a reduction in excitation would result in a diminished

inhibitory drive. If this was the case, then blockage of glial glutamate transporters should

reinstate inhibition even during BS.

To test this hypothesis we applied dihydrokainate (9 mM) topically onto the

neocortex during isoflurane-induced BS and performed intracellular recordings from

neurons situated in the dihydrokainate affected area (Fig. 7). In all cases (n= 12), we

observed an overall depolarized membrane potential in these neurons as compared to

recordings without dihydrokainate. The average membrane potential was –62.3 mV (± 3.7;

statistically different from controls, p = 0.02, signed-rank Wilcoxon test). We observed a

continuation of neuronal bursting activity in synchrony with the EEG. However, neurons

exhibited continuous synaptic potentials during the contralaterally recorded EEG isoelectric

line (Fig. 7A). These potentials might in part also be the result of a direct enhancement of

the excitatory activity by dihydrokainate. The amplitude of this activity during isoelectric

episodes was clearly increased as compared to recordings without dihydrokainate (Fig. 7B).

The width of the Gaussian fit of the membrane potential distribution measured at half

amplitude was 3.3 mV (± 1.2), and was statistically different (p<0.001, signed-rank

Wilcoxon test) from the corresponding values of neurons without dihydrokainate (0.4 mV ±

0.09). By itself, this result provides evidence that our topical application of dihydrokainate

was indeed effective.

Moreover, dihydrokainate application during BS re-instated the inhibitory responses

to thalamic stimulations, which were in fact comparable to those obtained in the same

neuron before the induction of BS (Fig. 7C). This suggests that the diminished inhibition

observed during isoflurane-induced BS mainly relies on a reduction of glutamate

availability within cortical networks.

Further investigation into the status of interneurons focused on verifying the

availability of GABAergic receptors for ligand binding during BS. We therefore employed

32

an iontophoretic application of GABA directly onto intracellularly recorded neurons. The

effectiveness of this method of GABA application was confirmed by an observed

amplitude reduction of synaptic intracellular activity (Fig. 8). Assessment of the

distribution of membrane potential values shows a progressive reduction of the width of the

Gaussian fit measured at half amplitude by 42% (n = 7) and a return to control values after

the discontinuation of iontophoresis. Similarly, GABA application also reduced the

amplitude of cortical responses to thalamic stimuli. During low isoflurane conditions the

amplitude of EPSPs was reduced by 66% whilst IPSP amplitudes were reduced by 82%

(Fig. 9A1). During BS on the other hand no inhibitory component could be elicited and the

observed EPSPs were reduced by 59% (Fig. 9A2). Additionally, we recorded a reduction in

input resistance of neurons undergoing GABA iontophoresis (Fig. 9B), a finding that

meshes well with the known GABA shunting of neuronal membranes. During low

isoflurane conditions and during BS input resistance values dropped by 70% and 68%,

respectively. These virtually equal values imply that the availability of the GABA receptors

was similar in the two states.

We also investigated the effect of increasing doses of isoflurane on neuronal

membranes during BS induction. As described above, we measured the input resistance of

neurons with intracellularly injected current pulses. A minimum of 5 pulses were applied

during each state (low isoflurane and BS) and we averaged the ensuing responses. Overall,

neurons (n = 35 tested) displayed a slight increase in resistance from 23.6 MΩ (± 9.7) to

26.8 MΩ (± 11.7), representing a 14% increase (Fig. 10A1). This increase was, however,

not statistically significant (signed-pair Wilcoxon test). On the other hand, we noticed a

clear increase of input resistance in a subset of neurons (n = 24), while others displayed a

decrease (n = 11). This suggests the presence of two populations which are modulated

differently by isoflurane application (Fig. 10A2). Thus, we recalculated the response

averages for each group: neurons with increased input resistance displayed an increase of

32% (from 20.2 MΩ to 26.7 MΩ), while the other group displayed a drop of 16% (from

27.9 MΩ to 23.3 MΩ). The resistance changes within these separated groups were indeed

statistically significant (p < 0.01, signed-pair Wilcoxon test). We could not, however,

correlate these to neuronal populations with any particular location within the suprasylvian

gyrus or with a particular recording depth.

33

Nevertheless, since the majority of the recorded neurons displayed a clearly

increased input resistance during BS induced by isoflurane, our results are at least partially

in agreement with the reduced inhibition measured in this study and with the hypothesized

effect of isoflurane on the increased transglial transport of glutamate. In order to further

clarify this point we monitored the dynamic evolution of the input resistance during the

induction of BS in several neurons (n = 5) (Fig. 10C). During these procedures we ensured

constant membrane potential values with current injection in order to avoid the contribution

of voltage-dependent currents. We observed a clear increase of input resistance with each

induction of BS. Occasionally we applied thalamic stimuli to assess the amplitude of the

initial EPSP. Our results suggest a clear reduction in EPSP amplitudes during BS (Fig.

10C).

Lastly, we studied the modulation of inhibitory processes during BS induced with a

short-acting barbiturate (thiopental, 32 mg/kg applied systemically). Since it is known that

the primary action of barbiturates is an enhancement of GABAA receptor activity, we

expected that control values of inhibition would be maintained or even increased during

barbiturate-induced BS. However, all recorded neurons (n = 5) displayed a reduction of

inhibitory potentials during BS, although some inhibitory components were maintained

(Fig. 11). Therefore our results raise the possibility that the reduced inhibition reported in

this study might not result from a specific anesthetic action on GABAergic receptors, but

rather from a more general mechanism particular to the state of BS. Two facts support this

idea: (a) the periodic variations of extracellular Ca2+ and its overall reduced availability,

especially during the suppression phase, decreases the synaptic efficacy in cortical

networks (Kroeger and Amzica, 2007), and (b) thalamic neurons display rhythmic bursting

activities during the suppression phase of the BS (Steriade et al., 1994) creating an

excitatory pressure on cortical elements.

34

Discussion

The results presented above show a significant reduction of cortical inhibition during

isoflurane-induced BS and thus support earlier reports of a hyperexcitable state during this

level of anesthesia-induced coma (Kroeger and Amzica, 2007). Several facts highlight this

conclusion:

(1) The composition and timing of our recorded responses to thalamic stimulation

during low isoflurane conditions were similar to responses observed in other studies

(Connors et al., 1988; Berman et al., 1991; Contreras et al., 1997). Both inhibitory

components were absent during isoflurane induced BS.

(2) The decreased input resistance measured during inhibitory responses under low

isoflurane conditions is abolished under BS conditions. Moreover, as observed in some

neurons, excitatory potentials replaced inhibitory responses during stimulation, which is

probably the result of a lifted shunting effect.

(3) During BS only the initial excitatory response to thalamic stimuli was preserved.

All subsequent components did not display voltage dependence, as would be expected if

receptor channels were open.

(4) We observed a steady and significant rise in the extracellular Cl- concentration

during BS induction emphasizing the possibility that inhibitory activity through GABAA

receptors may have been absent.

At a first glance, it may surprise to learn that inhibition is abolished during a state

that is associated with deep coma (Brenner, 1985) , especially if the coma is provoked by a

drug that a) occupies a specific binding site on GABAA receptors (Mihic et al., 1997;

Jenkins et al., 2001) and b) enhances GABAergic transmission (Harrison et al., 1993;

Banks and Pearce, 1999; Nishikawa et al., 2002). However, a blocking effect of volatile

anesthetics on GABAA receptors has been described in vitro by Banks and Pears (1999),

who found that this block was most prominent using enflurane. However, isoflurane only

produced a 20% reduction in the permeability of the receptor at aqueous concentrations of

0.6 mM (Fig. 3 in Banks and Pearce, 1999). This concentration corresponds to about 2

MAC and is thus within the range employed in the present experiments Moreover, in our

experiments GABA application still elicited an effect on neuronal membranes even under

35

high isoflurane concentration. This result was probably mediated by extrasynaptic receptors

(Salin and Prince, 1996) and possibly reflecting a shunting effect (reduced amplitude of the

synaptic potentials and increased input conductance). Therefore, the direct effect of

isoflurane on GABA receptors might represent only a part of the mechanisms of isoflurane

action.

Further membrane targets of isoflurane are two-pore-domain K+ channels (Franks

and Lieb, 1988; Patel et al., 1999). These channels have been shown to underlie isoflurane

resistance in transgenic mice with deficient TREK-1 proteins (Heurteaux et al., 2004).

However, isoflurane action on these channels cannot fully account for the reduced

inhibition seen in our study, but could possibly explain the decreased input resistance of a

subpopulation of our recorded neurons. On the other hand, results from the neuron

population displaying increased resistance upon isoflurane application support the

suggestion that reduced GABAergic influence could cause the loss of inhibition during BS.

In our view, the main effect of isoflurane in these experiments was a reduction in

excitation, which in turn resulted in a reduced activation of interneurons, and thus

diminished inhibition. This result may be explained by modulation of: 1) neurotransmitter

release, 2) blockage of postsynaptic targets, and/or 3) glutamate uptake by glial cells. The

first explanation receives support from findings indicating a reduced synaptic release of

glutamate under isoflurane due to various presynaptic mechanisms (MacIver et al., 1996;

Westphalen and Hemmings, Jr., 2003; Wu et al., 2004). The most appealing target of

presynaptic depression, found in the calyx of Held, appears to be a reduction in amplitude

of action potentials invading the axon terminal, resulting in a 50% reduction of glutamate

release (Wu et al., 2004). This value compares well with our 44% reduction in the

amplitude of the thalamically evoked EPSPs.

A similar presynaptic effect might exist also at the level of GABA release, even

though experimental evidence is so far lacking. Isoflurane has little effect at the

glutamatergic postsynaptic site (Perouansky and Antognini, 2003), although a moderate

inhibition of AMPA receptors has been reported at concentrations of 2 MAC (Dildy-

Mayfield et al., 1996), which are sufficient to induce BS.

Glial cells play an important role in regulating the neuronal excitability by several

mechanisms, e.g. glutamate uptake via specific transporters. This process is enhanced by

36

volatile anesthetics such as isoflurane (Larsen et al., 1997; Zuo, 2001; Huang and Zuo,

2003) and halothane (Miyazaki et al., 1997). Blockage of glutamate uptake leads to an

increased demand of anesthesia (Cechova and Zuo, 2006), which is employed to counteract

increasing excitatory signaling. Therefore, the net result of isoflurane is a diminished

availability of glutamate in the synaptic cleft, as glia glutamate uptake is enhanced.

Moreover, the isoflurane-induced reduction in glutamatergic signaling reduces the

activation of inhibitory cells, and thus inhibition declines. With this scenario in mind, we

investigated the effect of a blockage of the glial transporters. Our results indicate that

blocking glial transporters does indeed restore inhibitory responses. However, since

dihydrokainate also acts directly on glutamatergic receptors, it may be conceivable that this

result could be obtained by dihydrokainate itself. Indeed, during the EEG isoelectric line

the membrane potential displayed increased synaptic noise suggesting local excitatory

activity. On the other hand, dihydrokainate restored phasic inhibition related to the

stimulation of ascending excitatory pathways. This suggests an increased availability of

glutamate in the synapse which could then have produced this response.

Increasing doses of isoflurane also result in a diminished duration of the

depolarizing phases during spontaneous bursting phases of BS with respect to the slow

oscillations expressed under low isoflurane anesthesia. Indeed, all excitatory and inhibitory

cortical neurons discharge during the depolarizing phase (“up-state”) of the control slow

(<1 Hz) sleep-like oscillation (Steriade et al., 1993). The measured reduction in duration of

the “up state” during BS could be caused by increased inhibitory activity. However,

considering that inhibitory responses are absent during BS, we conclude that the shape of

the recurrent depolarizations during bursting activity would rather lack a contribution of

local inhibitory interneurons.

A further deepening of the comatose state with higher doses of isoflurane results in

progressively shorter EEG bursts and longer isoelectric episodes until a constant cortical

silence is achieved. During this state cortical excitability is probably suppressed altogether.

From the above-discussed mechanisms it appears that both excitatory and inhibitory

pathways are affected by isoflurane - at least during concentrations eliciting BS. Inhibition

is more drastically affected, which in turn leads to a shift in the excitation/inhibition

balance towards excitation, even though excitation is reduced. The abolishing of inhibition

37

leads to a hyperexcitable state permitting the genesis of EEG bursts occasionally

interrupting the cortical silence of the EEG isoelectric line (Kroeger and Amzica, 2007).

EEG bursts may then be elicited either by subliminal stimuli (Kroeger and Amzica, 2007)

or by ongoing subcortical activities: thalamic (Steriade et al., 1994) or hippocampal (D.

Kroeger and F. Amzica, in preparation).

In clinical settings, this hyperexcitability, and the associated bursting activity, may

occasionally be mistaken for symptoms of epileptic fits (Dan and Boyd, 2006). This idea

receives further support from the abolishment of inhibition shown in the present study.

Both results are surprising findings especially since isoflurane is frequently used for the

treatment of status epilepticus at concentrations which induce BS (Kofke et al., 1985;

Kofke et al., 1989; Meeke et al., 1989). This therapeutic effect probably relies on the ability

of isoflurane to block gap junctions (Mantz et al., 1993), a primary instrument in the

generalization of spike-wave seizures (Amzica et al., 2002). The above described paradox

underlines the complexity, and probably the heterogeneity, of the epileptic syndromes.

38

Figures

Fig. 1 Typical progression toward burst-suppression (BS) pattern. Recorded at the EEG and field potential level as well as intracellularly in a cortical neuron in the suprasylvian gyrus (areas 5-7). (A) Under light anesthesia (1.5% isoflurane) neuronal activity is mainly characterized by slow-wave sleep-like (SWS) oscillation dominated by a quasi regular slow rhythm of <1 Hz. (B) Application of higher concentrations of isoflurane (2.5%) hyperpolarizes the neuronal membrane potential and accelerates cortical oscillations. Occasionally, this transitory activity is interrupted by short periods of flat EEG (*) which increase in duration and become more frequent. (C) The transition period is followed by a stable pattern of BS, which is characterized by long silent periods of flat EEG and occasional bursts of activity. In this and following figures, signals are presented with positive polarity upwards.

39

Fig. 2. Comparison of slow oscillatory activity of intracellular potentials during sleep-like patterns and bursting during BS. (A1) Intracellular activity of the same neuron recorded under lighter (1%) isoflurane anesthesia (left) and during BS (right). The latter trace was obtained by artificially eliminating all isoelectric phases, which served the purpose of producing an oscillatory period of equivalent duration to the trace depicting activity under light anesthesia. During the low isoflurane recording the neuron was injected with hyperpolarizing current (-0.5 nA) to prevent firing of action potentials and to reach comparative membrane potentials as during BS. (A2) Histograms of membrane potentials during recording periods displayed above (20 s) gave raise to different patterns: sleep-like activity under light anesthesia resulted in a biphasic oscillatory pattern with clear up-and-down states, whilst BS activity produced a unimodal distribution reflecting a prevalence of the down state and a shortened up state. The ordinate represents the incidence of a particular membrane potential value during the analyzed period. (B) Pairs of histograms (gray for light anesthesia, white for BS) from 2 other neurons recorded under the same conditions as in A, showing consistently similar distributions of the membrane potentials.

40

Fig. 3 Typical responses of cortical neurons to thalamic (CL) electrical stimuli. (A) Responses of a cortical neuron (area 5) to electrical stimuli delivered to the thalamic LP nucleus. During spontaneous depolarized membrane potentials of the “up-state” of the slow oscillation (upper trace) under low isoflurane anesthesia we observed the following response sequence: an initial EPSP, a fast IPSP (a), a long lasting hyperpolarization (b), and a sustained rebound depolarization (c). The same stimulus was applied during the hyperpolarized “down-state” (middle trace) and we recorded a very similar response with the exception of a modified short IPSP (a). After BS induction with 3% IF, the initial excitatory response diminished in amplitude despite a further hyperpolarized membrane potential. Moreover, the rebound component (c) appeared mostly abolished (lower trace). (B) Absence of inhibitory components in the response of another cortical neuron to thalamic stimulation even under steady depolarization through current injection (+1 nA) to bring the membrane potential at -60 mV. Average of 20 responses. (C) Assessment of input resistance: responses under low isoflurane in black, BS responses in red recorded 9 minutes later (average traces from the same neuron, n = 25). Note the overall increase in resistance during BS (panel 2). Resistance tests during the time frame of the inhibitory responses after stimulation displayed a lack of resistance modification under BS and an 18% reduced resistance during the low isoflurane condition (panel 1).

41

Fig. 4 Voltage dependence of cortical responses to thalamic (CL) electrical stimuli. Intraneuronal recording in cortical area 5 at three levels of polarization: under depolarizing current injection of 1 nA (A), rest (B) and under hyperpolarizing current application of -1.5 nA (C). Note: light anesthesia (black traces) and BS (red traces). Each trace is the result of an average of 10 individual responses, which were selected among those occurring at comparable membrane potentials between the 2 states. Depicted above are the averaged traces of A with 2 examples for individual responses from each state. Note the presence of an inhibitory response after the initial excitation with a consecutive rebound excitation during light anesthesia, as well as the absence of the inhibition and rebound during BS.

42

Fig. 5 Unmasking of excitatory components during BS responses matching the timing of inhibitory components during low isoflurane conditions. Averaged evoked responses of a cortical neuron (area 5) to thalamic (LP) stimulation (n = 25). (A1) Responses of the same neuron: before BS induction (under low isoflurane), during BS (high isoflurane), and after recovery from BS (return to low isoflurane). During the BS period a depolarizing potential replaces the inhibitory component of the control period. This change is also apparent in the nearby recorded field potential (FP). All stimuli were applied during the suppression period. (A2) The BS-related depolarizing event displayed voltage dependence and increased with membrane hyperpolarization. (B) Similar patterns in another neuron exhibiting an excitatory BS component expressed early after the initial excitatory response.

43

Fig. 6 Extracellular Cl- concentration ([Cl-]o) during BS induction and recovery. Note progressively increased Cl- values after application of increased (3%) isoflurane (high IF) and return to control values after the withdrawal of the additional dose. The dotted horizontal line indicates the average low isoflurane Cl- concentration, whilst the continuous horizontal line represents the summation of the average and standard deviation values (SD). The time delay of the Cl- increase was determined as the first value crossing the SD line without subsequently returning (vertical arrow). The EEG period within the square is expanded below to show the appearance of the first isoelectric episode within seconds after the onset of the Cl- increase (vertical arrow).

44

Fig. 7 Blockage of glial glutamate uptake restores inhibition during BS. (A) Intracellular recording in the suprasylvian gyrus during topical application of dihydrokainate. Neurons discharged synchronously during the bursting phase of the BS pattern. However, the membrane potential displayed an unusually increased background activity during the isoelectric line (see inset). (B) Histograms of membrane potentials of 2 neurons during 20 s periods of isoelectric activity. The upper histogram corresponds to a neuron recorded under low isoflurane conditions, whilst the lower histogram reflects the neuron at left. (C) Neuronal response to CL thalamic stimulation during BS shows a similar excitatory-inhibitory pattern as during light anesthesia. Averaged responses (n = 15) are shown on the left, whilst individual responses during BS (top) and during low isoflurane conditions (bottom) are displayed on the right.

45

Fig. 8 Effect of iontophoretic application of GABA on a cortical neuron from area 5. (A) Spontaneous activity under light (1%) isoflurane anesthesia displaying a slow oscillatory activity. GABA application reduced the amplitude of this oscillation, which subsequent recovered following the discontinuation of GABA. (B) We quantified the amplitude of membrane potential variations of the five 17 s epochs indicated in A. The histograms (bars) were fitted with Gaussian functions (lines) and indicate the narrowing of amplitude ranges during the GABA application, suggesting a decrease in amplitude variations. The histogram widths (at half amplitude) for the five selected epochs are: 19, 12.8, 11.1, 11.5, and 18.3 mV, respectively.

46

Fig. 9 Effect of iontophoretic application of GABA on cortical responsiveness to thalamic stimulation. (A1) Averaged (n = 15) evoked membrane potentials during low isoflurane conditions (light 1% isoflurane anesthesia), before and after the application of GABA (black traces), and during GABA iontophoresis (red traces). Note the diminished amplitude of both excitatory and inhibitory components. (A2) Similar sequence of procedures in the same neuron recorded during BS. The application of GABA resulted in diminished excitatory responses, whilst inhibitory components were completely absent. (B) Effect of GABA application on membrane input resistance during low isoflurane conditions (1) and during BS (2). In both cases GABA reduced the input resistance of the neuron.

47

Fig. 10 Effect of isoflurane-induced BS on the neuronal input resistance. (A1) Global variation of the input resistance between low isoflurane conditions (hatched surface) and BS (black surface) displaying slight increase of the resistance during BS. The difference between the two conditions is not statistically significant (signed-rank Wilcoxon test). (A2) Separate populations of neurons show either increased input resistance during BS (top panel) or a resistance decrease (bottom panel). Differences in both groups were significant (signed-rank Wilcoxon test). (B) Hyperpolarizing pulses during low isoflurane (1) and BS (2) as employed in C, demonstrating increased resistance during BS. (C) Input resistance test (above) and EPSP amplitudes during 3 successive inductions of BS (periods marked with continuous lines), by high levels of isoflurane (3%) (periods indicated by dotted lines). Note the systematically increased resistance and diminished EPSP amplitude during BS.

48

Fig. 11 Reduction of cortical inhibition under barbiturate (thiopental) anesthesia. (A) Control conditions under ketamine-xylazine anesthesia with slow sleep-like oscillations in the EEG (left panel) and voltage-dependent responses to thalamic stimulations (right panel; average of 15 sweeps). Note enhanced EPSPs and reduced inhibition with hyperpolarization. (B) Systemic application of thiopental (32 mg/kg) induces BS in EEG signals (left) and decreasing amplitudes and durations of inhibitory responses (right).

49

Conclusion

Des expériences faites chez l’animal ont montré une baisse de la réactivité neuronale sous

anesthésie, souvent attribuée à une diminution de l’excitation ou une augmentation de

l’inhibition au niveau du cortex. Toutefois, ces expériences ont été principalement

effectuées sur des cellules en culture ou in vitro, ce qui ne permet pas d’étudier le

comportement des neurones et leurs complexes interactions avec le réseau cortical. Ainsi,

sous isoflurane et in vivo, des stimulations thalamiques produisant dans le cortex, de façon

classique, un PPSE suivi d’un PPSI, montrent une diminution du PPSE et une disparition

du PPSI avec l’augmentation de la concentration d’isoflurane et donc du niveau

d’anesthésie. La disparition du PPSI est maintenue malgré les changements de Vm induits

par injection de courant et elle s’accompagne d’une augmentation de la résistance.

L’injection iontophorétique de GABA et d’acide dihydrokainique (DHK) en périsynaptique

ont montré d’une part que le premier n’amenait pas une restauration du PPSI alors que l’on

pouvait observer du PPSI sous DHK.

Ceci montre qu’une augmentation du glutamate synaptique peut augmenter l’inhibition

exercée sur un neurone cortical, probablement par excitation des interneurones connectés à

celui-ci. Puisqu’un excès de glutamate peu provenir du blocage de sa recapture par les

glies, ceci nous amène à suggérer que les glies pourraient jouer un rôle dans cette

diminution de l’inhibition, par la diminution de la recapture du glutamate lors de

l’augmentation des doses d’isoflurane. Ceci génère une perspective nouvelle dans la

compréhension des mécanismes de l’anesthésie, car la plupart des modèles actuels se

concentrent sur les récepteurs GABA et ce qui se passe dans les domaines pré- et post-

synaptique des circuits exclusivement neuronaux, sans tenir compte de l influence des glies

sur l’activité neuronale.

D’un point de vue plus global, cette diminution de l’inhibition pendant l’augmentation du

niveau d’anesthésie et surtout pendant l’état de burst-suppression se rattache à l’idée qu’il

pourrait exister une fenêtre dans laquelle le cortex serait hyperexcitable (Kroeger et

50

Amzica, 2007). Ceci pourrait amener des indications dans le futur sur le traitement des

patients dans un état comateux présentant un patron d’EEG de burst-suppression.

Bibliographie Ammann D (1986) Ion sensitive microelectrodes. Berlin: Springer. Amzica F, Massimini M, Manfridi A (2002) Spatial buffering during slow and paroxysmal sleep oscillations in cortical networks of glial cells in vivo. J Neurosci. Vol. 22:1042-1053. Amzica F, Steriade M (1995) Short- and long-range neuronal synchronization of the slow (< 1 Hz) cortical oscillation. J Neurophysiol. Vol. 73:20-38. Antkowiak B, Heck D (1997) Effects of the volatile anesthetic enflurane on spontaneous discharge rate and GABA(A)-mediated inhibition of Purkinje cells in rat cerebellar slices. J Neurophysiol. Vol. 77:2525-2538. Arriza JL et al. (1994) Funtional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J Neurosci 14:5559-5569. Antognini JF, Carstens E et Raines DE Neural Mechanisms of Anesthesia, Totowa, NJ, Humana Press, 2003. Antognini JF et al. (2000) Isoflurane depresses electroencephalographic and medial thalamic responses to noxious stimulation via an indirect spinal action. Anesth Analg. Vol. 91(5):1282-1288. Antognini JF et Carsten E (2002) In vivo characterization of clinical anaesthesia and its components. Br J Anaest Vol. 89(1):156-166. Banks MI, Pearce RA (1999) Dual actions of volatile anesthetics on GABA(A) IPSCs: dissociation of blocking and prolonging effects. Anesthesiology 90:120-134. Berg-Johnsen J, Langmoen IA (1986) Isoflurane effects in rat hippocampal cortex: a quantitative evaluation of different cellular sites of action. Acta Physiol Scand. Vol. 128:613-618. Berman NJ et al. (1991) Mechanisms of inhibition in cat visual cortex. J Physiol. Vol. 440:697-722. Bragin A et al. (1995) Gamma (40-100 Hz) oscillation in the hippocampus of the behaving rat. J Neurosci Vol. 15(1 Pt 1):47-60. Brenner RP (1985) The electroencephalogram in altered states of consciousness. Neurol Clin. Vol. 3:615-631. Brenner RP (2005) The interpretation of the EEG in stupor and coma. Neurologist. Vol. 11(5):271-84. Caraiscos VB et al. (1994) Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane.. J Psychiatry Neurosci. Vol. 19(5):354-358. Carla V et Moroni F (1992) General anaesthetics inhibit the responses induced by glutamate receptor agonists in the mouse cortex. Neurosci Lett. Vol. 146(1):21-24. Cechova S et Zuo Z (2006) Inhibition of glutamate transporters increases the minimum alveolar concentration for isoflurane in rats. Br J Anaesth. Vol. 97(2):192-195. Chatrian GE (1990) Coma, other states of altered responsiveness, and brain death. In: Current Practice in Clinical Electroencephalography (Daly DD, Pedley TA, eds), pp 425-487. New York: Raven Press. Connors BW, Gutnick MJ, Prince DA (1982) Electrophysiological properties of neocortical neurons in vitro. J Neurophysiol. Vol. 48:1302-1320.

52 Connors BW, Malenka RC, Silva LR (1988) Two inhibitory postsynaptic potentials, and GABAA and GABAB receptor-mediated responses in neocortex of rat and cat. J Physiol 406:443-468. Contreras D, Durmüller N, Steriade M (1997) Absence of a prevalent laminar distribution of IPSPs in association cortical neurons of cat. J Neurophysiol. Vol. 78:2742-2753. Contreras D, Steriade M (1995) Cellular basis of EEG slow rhythms: a study of dynamic corticothalamic relationships. J Neurosci. Vol. 15:604-622. Dan B, Boyd SG (2006) Stimulus-sensitive burst-spiking in burst-suppression in children: implications for management of refractory status epilepticus. Epileptic Disord. Vol 8:143-150. De Rubeis DA, Young GB (2001) Continuous EEG monitoring in a patient with massive carbamazepine overdose. J Clin Neurophysiol. Vol 18:166-168. Detsch O et al. (2002) Differential effects of isoflurane on excitatory and inhibitory synaptic inputs to thalamic neurones in vivo. Br J Anaesth. Vol. 89:294-300. Dildy-Mayfield JE, Eger EI, Harris RA (1996) Anesthetics produce subunit-selective actions on glutamate receptors. J Pharmacol Exp Ther. Vol. 276:1058-1065. Dilger JP (2002) The effects of general anaesthetics on ligand-gated ion channels. Br J Anaesth. Vol. 89(1):41-51. Downie DL et al. (1996) Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes. Br J Pharmacol. Vol. 118(3):493-502. Doyle PW et Matta BF (1999) Burst suppression or isoelectric encephalogram for cerebral protection: evidence from metabolic suppression studies. Br J Anaesth- Vol. 83(4):580-584. Eger EI (1984) The pharmacology of isoflurane. Br J Anaesth. Vol. 56 Suppl 1:71S-99S. Franks NP (2006) Molecular targets underlying general anaesthesia. Br J Pharmacol- Vol. 147 Suppl 1:S72-S81. Franks NP, Lieb WR (1988) Volatile general anaesthetics activate a novel neuronal K+ current. Nature Vol. 333:662-664. Grasshoff C, Rudolph U et Antkowiak B (2005) Molecular and systemic mechanisms of general anaesthesia: the 'multi-site and multiple mechanisms' concept. Curr Opin Anaesthesiol. Vol. 18(4):386-391. Harrison NL et al. (1993) Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane. Mol Pharmacol.Vol. 44(3):628-632. Hartikainen KM et al. (1995) Cortical reactivity during isoflurane burst-suppression anesthesia. Anesth Analg. Vol. 81(6):1223-1228. Hemmings HC Jr anesthesia. EMBO J. Vol. 23(13):2684-2695. Hentschke H, Schwarz C et Antkowiak B (2005) Neocortex is the major target of sedative concentrations of volatile anaesthetics: strong depression of firing rates and increase of GABAA receptor-mediated inhibition. Eur J Neurosci. Vol. 21(1):93-102. Heurteaux C et al. (2004) TREK-1, a K+ channel involved in neuroprotection and general anesthesia. EMBO J. Vol 23:2684-2695. Hoffman WE et al. (1998) Thiopental and desflurane treatment for brain protection. Neurosurgery. Vol. 43(5):1050-1053. Huang Y et Zuo Z (2003) Isoflurane enhances the expression and activity of glutamate transporter type 3 in C6 glioma cells. Anesthesiology. Vol. 99(6):1346-1353.

53 Jenkins A et al. (2001) Evidence for a common binding cavity for three general anesthetics within the GABAA receptor. J Neurosci. Vol. 21(6):RC136. Jones MV, Harrison NL (1993) Effects of volatile anesthetics on the kinetics of inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurophysiol 70:1339-1349. Kofke WA et al. (1985) Prolonged low flow isoflurane anesthesia for status epilepticus. Anesthesiology Vol. 62:653-656. Kofke WA et al.. (1989) Isoflurane for refractory status epilepticus: a clinical series. Anesthesiology 71:653-659. Kroeger D et Amzica F (2007) Hypersensitivity of the anesthesia-induced comatose brain. J Neurosci. Vol. 27(39):10597-607. Larsen M et al. (1997) Isoflurane increases the uptake of glutamate in synaptosomes from rat cerebral cortex. Br J Anaesth 78:55-59. Lavine SD et al. (1997) Temporary occlusion of the middle cerebral artery in intracranial aneurysm surgery: time limitation and advantage of brain protection. J Neurosurg. Vol. 87(6):817-824. Lin LH et al. (1992) General anesthetics potentiate gamma-aminobutyric acid actions on gamma-aminobutyric acidA receptors expressed by Xenopus oocytes: lack of involvement of intracellular calcium. J Pharmacol Exp Ther. Vol. 263(2):569-578. Maclver MB et al. (1996) Volatile anesthetics depress glutamate transmission via presynaptic actions. Anesthesiology. Vol. 85(4):823-834. Mantz J, Cordier J, Giaume C (1993) Effects of general anesthetics on intercellular communications mediated by gap junctions between astrocytes in primary culture. Anesthesiology Vol. 78:892-901. Martin DC et al. (1995) Volatile anesthetics and glutamate activation of N-methyl-D-aspartate receptors. Biochem Pharmacol. Vol. 49(6):809-817. Massimini M, Amzica F (2001) Extracellular calcium fluctuations and intracellular potentials in the cortex during the slow sleep oscillation. J Neurophysiol. Vol 85:1346-1350. Meeke RI, Soifer BE, Gelb AW (1989) Isoflurane for the management of status epilepticus. DICP Vol. 23:579-581. Michenfelder JD, Milde JH (1991) The relationship among canine brain temperature, metabolism, and function during hypothermia. Anesthesiology Vol. 75:130-136. Mihic SJ et al. (1997) Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature- Vol. 389(6649):385-389. Miu P, Puil E (1989) Isoflurane-induced impairment of synaptic transmission in hippocampal neurons. Exp Brain Res. Vol 75:354-360. Miyazaki H et al. (1997) Increase of glutamate uptake in astrocytes: a possible mechanism of action of volatile anesthetics. Anesthesiology. Vol. 86(6):1359-1366. Morgan GE, Mikhail MS et Murray MJ Clinical Anesthesiology, Lange Medical Books/McGraw-Hill, New York, 2002. Nakahiro M et al. (1989) General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons. FASEB J. Vol. 3(7):1850-1854. Nakashima K, Todd MM, Warner DS (1995) The relation between cerebral metabolic rate and ischemic depolarization. A comparison of the effects of hypothermia, pentobarbital, and isoflurane. Anesthesiology Vol. 82:1199-1208.

54 Newberg LA, Milde JH et Michenfelder JD (1983) The cerebral metabolic effects of isoflurane at and above concentrations that suppress cortical electrical activity. Anesthesiology. Vol. 59(1):23-28. Nishikawa K et al. (2002) Volatile anesthetic actions on the GABAA receptors: contrasting effects of alpha 1(S270) and beta 2(N265) point mutations. Neuropharmacology Vol. 42:337-345. Nuñez A, Amzica F, Steriade M (1993) Electrophysiology of association cortical cells in vivo: intrinsic properties and synaptic responses. J Neurophysiol. Vol. 70:418-430. Ostermann ME et al. (2000) Coma mimicking brain death following baclofen overdose. Intensive Care Med. Vol. 26:1144-1146. Pagni CA, Courjon J (1964) Electroencephalographic modifications induced by moderate and deep hypothermia in man. Acta Neurochir (Wien) Suppl. Vol.14:35-49. Pancrazio JJ, Park WK et Lynch C (1993) Inhalational anesthetic actions on voltage-gated ion currents of bovine adrenal chromaffin cells. Mol Pharmacol- Vol. 43(5):783-794. Pascoe EA et al. (1996) High-dose thiopentone for open-chamber cardiac surgery: a retrospective review. Can J Anaesth. Vol. 43(6):575-579. Patel AJ et al. (1999) Inhalational anesthetics activate two-pore-domain background K+ channels. Nat Neurosci. Vol. 2:422-426. Patel AJ et Honore E (2001) Anesthetic-sensitive 2P domain K+ channels. Anesthesiology. Vol. 95(4):1013-1021. Pearce RA (1996) Volatile anaesthetic enhancement of paired-pulse depression investigated in the rat hippocampus in vitro. J Physiol. Vol. 492 ( Pt 3):823-840. Perouansky M, Antognini JF (2003) Glutamate receptors. In: Neural Mechanisms of Anesthesia (Antognini JF, Carstens EE, Raines DE, eds), pp 319-332. Totowa,NJ: Humana Press. Rampil IJ, Moller DH et Bell AH (2006) Isoflurane modulates genomic expression in rat amygdala. Anesth Analg. Vol. 102(5):1431-1438. Ranft A et al. (2004) Isoflurane modulates glutamatergic and GABAergic neurotransmission in the amygdala. Eur J Neurosci. Vol. 20(5):1276-1280. Rehberg B, Xiao YH et Duch DS (1996) Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. Anesthesiology. Vol. 84(5):1223-1233. Ries CR et Puil E (1999) Mechanism of anesthesia revealed by shunting actions of isoflurane on thalamocortical neurons. J Neurophysiol. Vol. 81(4):1795-801. Ries CR et Puil E (1999) Mechanism of anesthesia revealed by shunting actions of isoflurane on thalamocortical neurons. J Neurophysiol. Vol. 81(4):1795-1801. Salin PA, Prince DA (1996) Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol. Vol. 75:1573-1588. Shigeri Y et Shimamoto K (2003) Pharmacology of excitatory amino acid transporters (EAATs and VGLUTs). Nippon Yakurigaku Zasshi. Vol. 122(3):253-264. Silverman D (1975) The electroencephalogram in anoxic coma. In: Handbook of electroencephalography and clinical neurophysiology (Rémond A, ed), pp 81-94. Amsterdam: Elsevier. Sonne JM et al. (2003) Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg. Vol. 97(3):718-740. Steriade M (1992) Basic mechanisms of sleep generation. Neurology. Vol. 42(7 Suppl 6):9-17.

55

Steriade M, Nuñez A, Amzica F (1993) A novel slow (< 1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components. J Neurosci 13:3252-3265. Steriade M (1994) Sleep oscillations and their blockage by activating systems. J Psychiatry Neurosci. Vol. 19(5):354-358. Steriade M, Amzica F et Contreras D (1994) Cortical and thalamic cellular correlates of electroencephalographic burst-suppression. Electroencephalogr Clin Neurophysiol. Vol. 90(1):1-16. Study RE (1994) Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons. Anesthesiology. Vol. 81(1):104-116. Su ZZ et al. Insights into g(2003) Glutamate transport regulation in human astrocytes: cloning of the promoter for excitatory amino acid transporter 2 (EAAT2). Proc Natl Acad Sci U S A. Vol. 100(4):1955-1960. Swank RL, Watson CW (1949) Effects of barbiturates and ether on spontaneous electrical activity of dog brain. J Neurophysiol. Vol. 12:160. Tétrault S, Chever O, Sik A, Amzica F (2008) Opening of the blood-brain barrier during isoflurane anaesthesia. Eur J Neurosci. Vol. 28:1330-1341. Tong G et Jahr CE (1994) Block of glutamate transporters potentiates postsynaptic excitation. Neuron.- Vol. 13(5):1195-1203. Urban BW et Bleckwenn M (2002) Concepts and correlations relevant to general anaesthesia. Br J Anaesth. Vol. 89(1):3-16. Vahle-Hinz C et al. (2001) Local GABA(A) receptor blockade reverses isoflurane's suppressive effects on thalamic neurons in vivo. Anesth Analg. Vol. 92(6):1578-1584. Vahle-Hinz C et al. (2007) Contributions of GABAergic and glutamatergic mechanisms to isoflurane-induced suppression of thalamic somatosensory information transfer.. Exp Brain Res. Vol. 176(1):159-72. Weissenborn K et al. (1991) Burst suppression EEG with baclofen overdose. Clin Neurol Neurosurg. Vol. 93:77-80. Westphalen RI et Hemmings HC (2003) Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals. J Pharmacol Exp Ther. Vol. 304(3):1188-1196. Westphalen RI, Hemmings HCJr (2003) Effects of isoflurane and propofol on glutamate and GABA transporters in isolated cortical nerve terminals. Anesthesiology Vol. 98:364-372. White NS et Alkire MT (2003) Impaired thalamocortical connectivity in humans during general-anesthetic-induced unconsciousness. Neuroimage. Vol. 19(2 Pt 1):402-411. Whittington MA, Jefferys JG et Traub RD (1996) Effects of intravenous anaesthetic agents on fast inhibitory oscillations in the rat hippocampus in vitro. Br J Pharmacol- Vol. 118(8):1977-1986. Wu XS et al. (2004) Isoflurane inhibits transmitter release and the presynaptic action potential. Anesthesiology. Vol. 100(3):663-670. Zuo Z (2001) Isoflurane enhances glutamate uptake via glutamate transporters in rat glial cells. Neuroreport. Vol. 12(5):1077-1080.