Contrôle de la position des vibrisses chez le rat ...
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Contrôle de la position des vibrisses chez le rat : intégrations sensori-motrices sous-tendant le contrôle moteur volontaire : Polysémies vibrissales Thèse Michaël Elbaz Doctorat en neurosciences Philosophiæ doctor (Ph. D.) Québec, Canada © Michaël Elbaz, 2021
Transcript of Contrôle de la position des vibrisses chez le rat ...
Contrôle de la position des vibrisses chez le rat : intégrations sensori-motrices sous-tendant le
contrôle moteur volontaire : Polysémies vibrissales
Thèse
Michaël Elbaz
Doctorat en neurosciences
Philosophiæ doctor (Ph. D.)
Québec, Canada
© Michaël Elbaz, 2021
Contrôle de la position des vibrisses chez le rat : intégrations sensori-motrices sous-tendant le contrôle moteur volontaire
Polysémies vibrissales
Thèse Doctorat de neurosciences
Michaël ELBAZ
Sous la direction de :
Martin DESCHÊNES, directeur de recherche
Christian ETHIER, co-directeur de recherche
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Résumé
Que ce soit par nos yeux ou par nos mains, nous interagissons avec notre environnement
par l’intermédiaire de senseurs mobiles. L'utilisation efficace de tels organes, dits
« sensorimoteurs », implique de garder trace de leur position tandis que nous les bougeons.
Dans le cas contraire, la stabilité perceptive et la préhension dirigée seraient profondément
entravées. Cependant, les mécanismes neuronaux qui informent le système nerveux de la
position de ces organes restent largement inexplorés. Deux mécanismes impliquant une
information retour peuvent théoriquement être en jeu : la copie efférente et/ou le retour
sensoriel. Pour évaluer leur contribution, nous avons entraîné des rats à placer une vibrisse
dans une plage angulaire prédéterminée sans contact, une tâche dépendant de la
connaissance qu’ils ont de la position de leur vibrisse. Nous établissons que le retour
sensoriel n'est pas requis pour effectuer cette tâche. Le cortex moteur n'est pas non plus
requis, même en l'absence de retour sensoriel. Enfin, nous démontrons que le noyau rouge,
qui reçoit des entrées du cortex moteur, du cervelet, des afférences vibrissales, et qui
projette vers les motoneurones faciaux est impliqué de façon critique dans l'exécution de
cette tâche. Ces résultats démontrent l'existence d'un modèle interne, indépendant du
cortex moteur et suffisant au contrôle moteur volontaire. Ils suggèrent en outre que le
cervelet pourrait être impliqué en tant qu’élément efférent de ce modèle interne.
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Abstract
Whether through our eyes or our hands, we interact with our environment through mobile
sensors. The efficient use of these organs implies to keep track of their position over time.
Otherwise, perceptive stability and directed prehension would be profoundly impeded. Yet,
the neural mechanisms informing the nervous system about organs’ position remain largely
unexplored. Two feedback mechanisms may theoretically be at play: efference copy and/or
sensory feedback. To assess their contribution, we trained head-restrained rats to place
their vibrissae in a predetermined angular range without contact, a task that depends on the
knowledge of vibrissa position. We find that sensory feedback is not required to perform the
task. The motor cortex is neither required, even in the absence of sensory feedback. Finally,
we find that the red nucleus, which receives motor cortex, cerebellar and vibrissa inputs,
and projects to facial motoneurons is critically involved in the execution of the task. These
results demonstrate the existence of a motor cortex-independent internal model sufficient
for voluntary motor control. They further suggest that the cerebellum may be involved as an
efferent part of this internal model.
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Table des matières
Résumé ............................................................................................................................................................ ii
Abstract ............................................................................................................................................................ iii
Table des matières ....................................................................................................................................... iv
Liste des figures ............................................................................................................................................. v
Liste des acronymes ................................................................................................................................... vii
Remerciements .............................................................................................................................................. x
Avant-propos .................................................................................................................................................. xi
Introduction ..................................................................................................................................................... 1
Chapitre 1 A cortical-independent internal model for the control of voluntary vibrissa
movement ..................................................................................................................................................... 24
1.1 Résumé .............................................................................................................................................. 24
1.2 Abstract .............................................................................................................................................. 24
Chapitre 2 A Vibrissal Pathway that Activates the Limbic System .............................................. 48
2.1 Résumé .............................................................................................................................................. 48
2.2 Abstract .............................................................................................................................................. 48
Conclusion .................................................................................................................................................... 77
Bibliographie................................................................................................................................................. 79
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Liste des figures
Figures de l’introduction
Figure Introduction 1 – Photographie d’un rat à l’arborescence vibrissale manifeste (issu
de (Deschenes et al., 2012))
Figure Introduction 2 – Gros plan sur les macro-vibrisses mystaciales (issu de (Knutsen and
Ahissar, 2009))
Figure Introduction 3 - Mouvements de vibrisses et locomotions (selon (Sofroniew and
Svoboda, 2015))
Figure Introduction 4 - Un rat soumis au "gap crossing" (issu de (Carvell and Simons, 1990))
Figure Introduction 5 - Tâche de localisation d'objets réalisée par une vibrisse en
mouvement
Figure Introduction 6 - Complexe sinusal folliculaire d’où émane une vibrisse (selon (Rice et
al., 1993))
Figure Introduction 7 - Noyaux de la colonne trigéminale (d’après (Urbain and Deschênes,
2009))
Figure Introduction 8 - Voies trigémino-thalmiques vibrissales (d’après (Pouchelon et al.,
2012))
Figure Introduction 9 - Barrelettes vibrissales dans le PrV (selon (Bosman et al., 2011))
Figure Introduction 10 - Cortex somatosensoriel des rongeurs (adapté de (Maier et al.,
1999))
Figure Introduction 11 - Schéma des voies lemniscale 1 et paralemniscale (depuis (Frangeul
et al., 2014))
Figure Introduction 12 - Désinhibition de la voie paralemniscale (issu de (Urbain and
Deschênes, 2009))
Figure Introduction 13 - Muscles intrinsèques et extrinsèques (issu de (Hill et al., 2008))
Figure Introduction 14 - Cartographie des motoneurones des vibrisses au sein du noyau
facial ("ML", "NL", "DN" et "NLP" désignent des muscles extrinsèques ; issu de (Deschenes
et al., 2016))
Figure Introduction 15 - Boucles sensorimotrices vibrissales (depuis (Prescott et al., 2016))
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Figures du Chapitre 1
Figure 1 & Supplementary Figure 1: Intact animals can learn the vibrissa positioning task
Figure 2 & Supplementary Figure 2: Sensory feedback is neither required before, nor after
learning
Figure 3 & Supplementary Figure 3: Motor cortex is not required to learn the task
Figure 4 & Supplementary Figure 4: Inactivation of the rubro-facial pathway disrupts
performance
Figures du Chapitre 2
Figure 1. Axonal Projections of Interpolaris Cells that Give Rise to the Paralemniscal
Pathway
Figure 2. Vibrissa-Evoked Responses in KF and MdD
Figure 3. Axonal Projections of KF/PB Cells that Receive Interpolaris Input
Figure 4. Vibrissa-Evoked Responses in Central Amygdala
Figure 5. Separate Cellular Populations in KF/PB Project to the CeA and the Facial Nucleus
Figure 6. Transsynaptic Labeling of Brainstem Cells that Receive Input from the KF
Figure 7. Optogenetic Stimulation of KF Axons Inhibits Vibrissa-Evoked Responses in
Monovibrissa-Responsive Interpolaris Cells
Figure 8. Summary of the First-Order Axonal Projections of Interpolaris Cells, and the
Second-Order Projections that Derive from the KF/PB Complex
Figure S1. Interpolaris cells project to KF/PB and MdD (Supplementary information related
to Figure 1)
Figure S2. Additional projection sites of KF/PB cells that receive interpolaris input
(Supplementary information related to Figure 3)
Figure S3. Labeling of KF cells that project to lower brainstem (Supplementary information
related to Figure 4)
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Liste des acronymes
ION : infraorbital
KF : Kölliker-Fuse
LFP : Long Field Potential
M1 : cortex moteur primaire
MdD : dorsal medullary reticular nucleus
PB : parabrachial
PO : noyau postérieur du thalamus
PrV : Principal du V
S1 : cortex sensoriel primaire
S2 : cortex sensoriel secondaire
SpV : noyau spinal du V
SpVc : noyau spinal du V, caudalis
SpVi : noyau spinal du V, interpolaris
SpVo : noyau spinal du V, oralis
vIRT : formation réticulaire vibrissale
VPM : noyau ventral postéromedial
VPMdm : noyau ventral postéromédial dorsomédial
ZIv : zona incerta ventrale
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À mon Maître,
le Rav Pin'has haCohen Pachter זצ"ל.
Votre dévouement, votre intégrité,
votre aversion pour les mirages de ce monde,
les lueurs que vous m'avez si généreusement
permis d'entrevoir me guideront toujours.
Aucun remerciement ne rendra justice
à ce que je vous dois.
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הבן בחכמה וחכם בבינה ספר יצירה
« Quî enim posset fieri, si salus in promptu esset, et
sine magno labore reperiri posset, ut ab omnibus ferè
negligeretur ? Sed omnia praeclara tam difficilia,
quàm rara sunt. »
Ethica, Pars V, Propositio 42, Scholium
B. Spinoza
„Wir fühlen, daß selbst, wenn alle möglichen
wissenschaftlichen Fragen beantwortet sind, unsere
Lebensprobleme noch gar nicht berührt sind.“
Tractatus logico-philosophicus
L. Wittgenstein
« Objection contre la science :
Ce monde ne mérite pas d’être connu. »
Syllogismes de l’amertume
E. Cioran
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Remerciements
Voici donc venu le moment de boucler une petite folie entamée en septembre 2014. De la
définition d’un projet à sa laborieuse implémentation (3264 expériences comportementales
tout de même), ce doctorat m’aura permis d’entrevoir ce qu’être chercheur peut signifier.
Je le dois avant tout à Martin Deschênes dont la passion incarnée au quotidien a constitué,
pour moi, la plus belle illustration de ce que la recherche est magnifiée quand elle relève
d’une vocation. A rebours de cette « ère technologique » qui en puissance s’empare des
neurosciences, vous m’avez appris à d’abord envisager la pertinence des questions
scientifiques, pour seulement alors mettre les techniques appropriées au service de leur
élucidation. Ces instants dont j’ai parfois eu la chance d’être témoin à vos côtés où la refonte
des hypothèses s’impose, où les perspectives se renversent pour peu qu’on y soit prêt, ont
été les plus inattendus, les plus heureux et les plus instructifs de mon doctorat. Vous m’avez
permis en somme de soupçonner in situ qu’un cercle vertueux en recherche se fonde sur
un échange permanent entre intuition et analyse, gage d’une rigueur non bornée.
Christian Ethier est devenu co-superviseur de ce doctorat en mai 2016, pour guider ce qui
allait en constituer le projet principal. Nos échanges, toujours engagés, m’ont d’abord fait
réaliser la primauté des protocoles comportementaux, puisque leurs particularités
détermineront ni plus ni moins ce qui sera déductible de toute l’expérience. Je te dois de
m’avoir donné la possibilité de m’impliquer dans bien d’autres projets, dont l’un au cœur de
mon intérêt pour les neurosciences (des systèmes cognitives), et toucher à des méthodes
d’analyse contemporaines. Ton ouverture, ton écoute et ta patience démesurée m’ont été
très souvent essentielles.
Maxime Demers, je ne saurais trop te remercier pour ton aide primordiale et généreuse dans
la réalisation concrète des projets. Je me suis grâce à toi confronté à cette intelligence
pratique en action qui te semble si naturelle.
Merci à tous les membres des laboratoires, en particulier à Saravanan, Windsor (le meilleur
professeur d’anglais qui se pût concevoir), Sergiu, Maxime, Quentin, Marie-Andrée et
Alireza.
Au-delà du centre de recherche, ces personnes devenues des ami-es dont la présence a
éclairé mon expérience québécoise : l’extraordinaire Myriam, David qui a fini par s’auto-
proclamer « bouc émissaire de [m]a procrastination », Warren pour l’art et le génie
combinés, Jocelyne pour m’avoir fait chanter du Bach sans le regretter, Jonathan pour ta
confiance dans l’organisation de ces conférences aux thèmes m’échappant tout à fait.
Merci enfin à mes parents, à ma fratrie, à ma grand-mère et à mon oncle dont la présence
constante me permet coûte que coûte de braver ce drôle de monde.
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Avant-propos
Le premier chapitre de cette thèse correspond à un manuscrit non encore soumis ; il devrait
être soumis en juin 2021. L’article publié devrait correspondre à la version présentée dans
cette thèse. J’en suis le premier auteur. L’idée maîtresse de ce projet a été définie avec
Martin Deschênes, les arcanes du protocole expérimental établis avec Christian Ethier et
j’en ai réalisé expériences et analyses. Les co-auteurs en sont : Maxime Demers, Christian
Ethier et Martin Deschênes.
Le second chapitre est un manuscrit soumis en juin 2020, actuellement en révision. L’article
publié devrait correspondre à la version présentée dans cette thèse. J’en suis premier
auteur. Mon rôle a consisté à penser ce projet aux côtés de Martin Deschênes, à en réaliser
des expériences et analyses. Les co-auteurs en sont : Amalia Callado-Pérez, Maxime
Demers, David Kleinfeld et Martin Deschênes.
1
Introduction
Que cela soit par nos yeux ou par nos mains, nous nous représentons le monde qui nous
entoure par l’intermédiaire de senseurs mobiles, également appelés organes
sensorimoteurs. L’utilisation efficace de tels organes suppose de garder trace de leur
position tandis que nous les bougeons. Considérons à titre d’illustration un objet posé sur
une table que nous tenterions de saisir : sans connaissance de la position de notre main en
mouvement, il n’y aurait d’adéquation possible (autre qu’aléatoire) entre la position visée et
la position de notre main, et nos tentatives s’avéreraient le plus souvent infécondes.
Comment la connaissance de la position d’organes en mouvement est-elle rendue
possible ? Les vibrisses offrent l’opportunité d’explorer les fondements neurophysiologiques
de ce défi, commun à tous les organes sensorimoteurs.
Les vibrisses sont des moustaches sensibles dont disposent de nombreuses espèces de
mammifères (Ahl, 1986; Brecht et al., 1997), hautement mobiles chez certains rongeurs.
Rats et souris sont en effet capables de palper activement leurs environs via des
mouvements de vibrisse rythmiques, mouvements appelés « whisking » (Grant et al., 2011).
Ils peuvent localiser précisément (à 6° près) des objets touchés par leur intermédiaire
(O'Connor et al., 2010), impliquant qu’ils en « connaissent » la position alors que celles-ci
bougent par rapport à leur museau ; les vibrisses exemplifient donc le défi sensorimoteur.
Les vibrisses offrent de surcroît d’importants avantages expérimentaux : leurs mouvements
relativement simples (pour l’essentiel définis par un unique degré de liberté) sont très
précisément quantifiables et perturbables, tout comme les phénomènes
neurophysiologiques associés.
En guise d’introduction aux deux projets détaillés dans cette thèse, nous commencerons
par un tour d’horizon de la riche éthologie vibrissale. En un second temps, nous décrirons
les principaux acquis de la neurophysiologie vibrissale autant sensorielle que motrice. Enfin,
nous montrerons en quoi un fossé demeure ici entre éthologie et neurophysiologie ; nos
projets ont été pensés afin de contribuer à le réduire.
1. Ethologie vibrissale
Les souris et les rats sont pour la plupart des espèces qui les composent des animaux
nocturnes (Hartmann, 2011; Mrosovsky and Hattar, 2005; Pisano and Storer, 1948),
pourvus d’une faible acuité visuelle (Birch and Jacobs, 1979; Prusky et al., 2002). Le sens
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du toucher médié par leurs vibrisses leur est à ce titre crucial pour explorer leur
environnement.
Description des vibrisses
De part et d’autre du museau des rats se trouvent une trentaine de macro-vibrisses mobiles,
longues de 2 à 5 cm (ce sont les vibrisses dites « mystaciales »), ainsi qu’une centaine de
micro-vibrisses immobiles d’environ 7 mm (Brecht et al., 1997; Grant et al., 2011; Vincent,
1913) (Figure Introduction 1).
Figure Introduction 1
Photographie d’un rat à l’arborescence vibrissale manifeste (issu de (Deschenes et al., 2012))
Les macro-vibrisses mystaciales sont organisées en une matrice de cinq lignes (A à E dans
la direction dorso-ventrale) et de sept colonnes (1 à 7 dans la direction caudo-rostrale)
(Figure Introduction 2) ; quatre vibrisses, désignées par des lettres grecques (alpha, bêta,
gamma et delta) forment un arc additionnel caudalement à ladite matrice. L’étendue de
déflection maximale des vibrisses est bien supérieure dans l’axe rostro-caudal que dans
l’axe dorso-ventral.
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Figure Introduction 2 – Gros plan sur les macro-vibrisses mystaciales (issu de (Knutsen and Ahissar, 2009))
Outre les macro-vibrisses mystaciales existent des macro-vibrisses supraorbitales, génales
et infra-mandibulaires (Chorev et al., 2016; Severson et al., 2019). À moins de précision
contraire, nous nous référerons aux vibrisses mystaciales par l’emploi du seul mot
« vibrisses ».
Utilisation qu’un rat fait de ses vibrisses dans divers contextes
Lorsqu’un rat rencontre des congénères (Ebbesen et al., 2017), explore son environnement
(Ebbesen et al., 2017) ou attend une récompense (Dominiak et al., 2019), ses vibrisses
décriront des mouvements typiques rythmiques rapides (5-10 Hz) de protractions-
rétractions : le « whisking ». Le whisking (verbe apparié : « whisker ») peut être décomposé
en trois sous-mouvements : la phase, l’amplitude et le « setpoint », soit l’angle moyen autour
duquel les mouvements rapides surviennent (Hill et al., 2011).
L’apparition du whisking en des contextes comportementaux différents atteste de ce que
l’utilisation des vibrisses répond chez les rongeurs à des fonctions multiples. Distinguer ces
fonctions s’impose afin de saisir la « valence » distincte des informations sensorielles
récoltées, impliquant des mécanismes neurophysiologiques possiblement distincts.
Explorer son environnement
Se mouvoir : marcher, courir, nager
Le whisking a été comparé aux tâtonnements par lesquels, avec sa canne, un aveugle
sonde activement ses environs (Kleinfeld and Deschenes, 2011). Et pour cause, durant la
marche, les pattes d’un rat iront sur la surface que ses vibrisses auront préalablement
palpée par le whisking (Grant et al., 2018). Lorsqu’ils courent par contre, les rats
maintiennent leurs vibrisses protractée sans mouvement rythmique, une stratégie semblant
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suggérer qu’ils sont alors moins intéressés par explorer leur enrivonrment que par éviter
des obstacles (Arkley et al., 2014). Des rats privés de vision ont été ainsi entraînés à courir
au travers d’un couloir pour y trouver de la nourriture : ceux qui devaient s’attendre à ce que
des obstacles soient disposés à des endroits indéterminés de leur parcours avaient
tendance à y courir moins vite et à maintenir leurs vibrisses plus protractées,
comparativement à ceux qui s’attendaient à un couloir vide d’obstacles (Arkley et al., 2014).
En matière de marche comme de course, tout se passe donc comme si les vibrisses
« ouvraient la voie » (Figure Introduction 3).
Figure Introduction 3 - Mouvements de vibrisses et locomotions (selon (Sofroniew and Svoboda, 2015))
Aussi, une asymétrie de mouvements vibrissaux (côtés gauche vs. droite de la face)
précède les mouvements de tête (Grant et al., 2012; Towal and Hartmann, 2006), les
guidant possiblement. Tailler toutes les vibrisses d’un côté induit un « thigmotactisme »
(Meyer and Meyer, 1992), soit une propension à garder les vibrisses non taillées en contact
permanent avec une paroi extérieure tel un mur. Couper les vibrisses de rats nageurs altère
profondément leur nage, entraînant une inclinaison du corps et/ou une plongée du nez dans
l'eau ; leurs vibrisses contribuent donc à leur permettre de maintenir la tête hors de l’eau
(Ahl, 1982).
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Détection et localisation d’objets
Les vibrisses contribuent largement à la détection et à la localisation d’objets et ce dès les
premiers jours post-partum des rats : couper les vibrisses de tous jeunes rats leur fera
manquer le mamelon qu’ils visent (Sullivan et al., 2003), ce qui résulte en une moindre
croissance (Arakawa and Erzurumlu, 2015). Cette implication des vibrisses dans le fait de
se nourrir se retrouvera, chez les adultes, jusque dans la détection des courants d’air
(l’« anémotactisme »), dont les effluves peuvent mener vers des cibles d’intérêt comme de
la nourriture (Yu et al., 2016).
Afin de mieux quantifier les capacités de détection d’objets en laboratoire, deux tâches
comportementales bien connues des vibrissologues ont été amplement utilisées : le
franchissement d’un fossé (« gap crossing ») et la localisation fine d’objets.
Gap crossing – Un rat placé sur une plateforme évaluera, par l’intermédiaire de ses
vibrisses, la présence d’une autre plateforme espacée de celle sur laquelle il se trouve par
un fossé. Sur la seule base des informations recueillies par ses vibrisses, le rat décidera de
se rendre sur l’autre plateforme (Carvell and Simons, 1990). Moyennant un entraînement
suffisant, les rats sont capables d’accomplir cette tâche avec une seule vibrisse (Hutson and
Masterton, 1986), quoique plus lentement que lorsqu’ils en ont plusieurs (Celikel and
Sakmann, 2007).
Figure Introduction 4 - Un rat soumis au "gap crossing" (issu de (Carvell and Simons, 1990))
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Localisation d’objets – Chez des rats libres de leurs mouvements, la localisation d’objets
est ralentie lorsque les macro-vibrisses sont coupées (Brecht et al., 1997; Sellien et al.,
2005). À la détection d’un objet par les vibrisses suit un mouvement d’orientation de la tête
(réduit si les vibrisses sont coupées (Symons and Tees, 1990)), de sorte à ce que le museau
se trouve symétriquement en regard de l’objet détecté, avec une palpation impliquant alors
les vibrisses des deux côtés du museau (Grant et al., 2009). S’ensuivra un contrôle de la
protraction des vibrisses, maximisant le nombre de contacts avec l’objet (Mitchinson and
Prescott, 2013) : un type de whisking qualifié de « fovéal » (Berg and Kleinfeld, 2003;
Carvell and Simons, 1990). Une expérience très contrôlée visant à estimer les capacités de
localisation fine d’objets consiste à entraîner des animaux libres de leurs mouvements, ou
bien maintenus tête fixe, à comparer deux positions distinctes (Cheung et al., 2019; Knutsen
et al., 2006; Mehta et al., 2007; O'Connor et al., 2010; O'Connor et al., 2013) (Figure
Introduction 5). Ces expériences ont démontré une capacité maximale de discrimination de
deux positions de l’ordre de 6° tandis que seul l’un des deux objets est présent, une capacité
sous-tendue donc par la mémoire des deux emplacements (O'Connor et al., 2010).
Figure Introduction 5 - Tâche de localisation d'objets réalisée par une vibrisse en mouvement
Discrimination de formes et de textures
Les rats sont capables de discriminer avec leurs vibrisses des objets (Brecht et al., 1997) et
même des textures (Guic-Robles et al., 1989), dans ce dernier cas avec un degré de
précision comparable aux humains utilisant leurs doigts (Carvell and Simons, 1990). Si les
rats mettent plus de temps à localiser des objets lorsqu’ils sont dépourvus de leurs macro-
vibrisses mystaciales, ils mettent plus de temps à discriminer des textures s’ils s’ont
dépourvus de micro-vibrisses (Brecht et al., 1997; Kuruppath et al., 2014), suggérant une
implication éthologique des macro-vibrisses dans la localisation et des micro-vibrisses dans
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la discrimination fine. Ainsi typiquement, un rat muni de toutes ses vibrisses, ayant détecté
la présence d’un objet par l’intermédiaire de ses macro-vibrisses, en approchera alors son
museau pour le palper par l’intermédiaire de ses micro-vibrisses. Cette séquence
éthologique n’empêche pas pour autant les rats de discriminer des objets par l’intermédiaire
de leurs macro-vibrisses, lorsque l’expérimentateur les y conduit (von Heimendahl et al.,
2007).
Enjeux sociaux
Les vibrisses ne servent pas qu’à l’exploration d’objets, mais aussi à celle de congénères
(Bobrov et al., 2014; Wolfe et al., 2011). Les souris, tout comme probablement les rats, les
utilisent de surcroît corrélativement à l’instauration de hiérarchies sociales (les souris
dominantes taillant les vibrisses des dominées) (Long, 1972). Enfin, comme précédemment
évoqué, les rats whiskent lorsqu’ils s’attendent à recevoir une récompense (Dominiak et al.,
2019), ce qui pourrait jouer un rôle social autant avec les congénères qu’avec
l’expérimentateur se sentant désiré.
Nous concluons ainsi ce tour d’horizon de ce en quoi les vibrisses contribuent à l’éthologie
des rats, éthologie dont nous retiendrons l’hétérogénéité. Nous reviendrons sur certaines
des facultés tout juste évoquées en troisième partie, après avoir planté le décor
neurophysiologique de base en deuxième.
2. Neuroanatomie et neurophysiologie vibrissales
Suivant les distinctions communes, nous allons dans un premier temps tenter de distinguer
les physiologies sensorielle et motrice, pour ensuite en évoquer les interactions. Notons
d’emblée cependant que, dès le tronc cérébral, processus sensoriels et moteurs
interagissent largement (puisque certains des premiers relais sensoriels sont aussi des
prémotoneurones).
Voies sensorielles
Mécanorécepteurs
Chaque vibrisse se trouve logée dans un complexe sinusal folliculaire, constitué d’un
follicule encapsulé dans un sinus vasculaire (Rice et al., 1986) (Figure Introduction 6).
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Figure Introduction 6 - Complexe sinusal folliculaire d’où émane une vibrisse (selon (Rice et al., 1993))
Au moins huit types de mécanorécepteurs innervent le follicule (Dorfl, 1985; Ebara et al.,
2002; Rice et al., 1993; Rice et al., 1986), se distinguant notamment selon qu’ils s’adaptent
rapidement à des déflections vibrissales et qu’ils sont sensibles à de plus ou moins fortes
amplitudes de déflections (Stuttgen et al., 2006). Chaque follicule est innervé par 100 à 200
afférences (Rice et al., 1986), lesquelles cheminent, pour ce qui est des vibrisses
mystaciales, vers la branche infraorbitale du nerf trijumeau. En cas de toucher, les moments
de forces s’exerçant à la base de la vibrisse varient selon la courbature de la vibrisse, la
position radiale du contact et l’orientation de la vibrisse (Quist and Hartmann, 2012; Quist
et al., 2014).
9
Ganglions
Chaque afférence n’innerve qu’un mécanorécepteur (Zucker and Welker, 1969). Les
neurones ganglionnaires encodent de façon très robuste les déflections vibrissales (Jones
et al., 2004) vis-à-vis desquelles ils présentent une forte préférence angulaire (Lichtenstein
et al., 1990). Outre le toucher, les mêmes neurones encodent de façon fiable des signaux
ré-afférents, du moins durant le whisking, c’est-à-dire un retour sensoriel des mouvements
de vibrisses effectués par l’animal (Severson et al., 2017; Severson et al., 2019).
Les neurones ganglionnaires projettent vers les différents noyaux de la colonne du trijumeau
(ou colonne trigéminale), lesquels donneront lieu à de nombreuses voies parallèles que voici
décrites.
Voies trigémino-thalamo-corticales
Les vois trigémino-thalamiques vibrissales émanent des différents noyaux de la colonne
trigéminale soit le noyau principal (PrV), le noyau spinal (SpV) qui consiste en l’oralis
(SpVo), en l’interpolaris (SpVi) et en le caudalis (SpVc) (Figure Introduction 7).
Figure Introduction 7 - Noyaux de la colonne trigéminale (d’après (Urbain and Deschênes, 2009))
Cinq voies trigémino-thalamiques vibrissales ont été anatomiquement caractérisées (Figure
Introduction 8) (Urbain and Deschênes, 2009) : quatre qui communiquent avec différentes
sous-régions du noyau ventral postéromédial thalamique (VPM) et une qui projette vers le
noyau postérieur thalamique (PO – sous-région tapissant la partie dorsomédiale du VPM).
10
Figure Introduction 8 - Voies trigémino-thalmiques vibrissales (d’après (Pouchelon et al., 2012))
Voie lemniscale 1
La première voie lemniscale est de loin la plus étudiée, peut-être du fait de ce qu’elle est
quasi-parfaitement discrétisée du tronc cérébral, au cortex, en passant par le thalamus : les
neurones de cette voie ont une préférence de réponse marquée pour une vibrisse
spécifique. Cette discrétisation est d’ordre anatomique, visible en cytochrome oxydase : à
chaque vibrisse correspond une « barrelette » trigéminale, un « barréloïde » thalamique et
11
un « barrel » cortical. Cette voie connecte le PrV au cœur du VPM et se termine dans la
couche 4 du cortex somatosensoriel (le fameux « barrel cortex » ou « cortex à tonneaux »).
Tronc cérébral : PrV
Le PrV est localisé à l’extrémité antérieur de la colonne trigéminale. Les barrelettes qu’on
y trouve (Ma and Woolsey, 1984) suivent la disposition des vibrisses (Figure Introduction
9).
Figure Introduction 9 - Barrelettes vibrissales dans le PrV (selon (Bosman et al., 2011))
Les neurones peuplant les barrelettes présentent donc une activité électrophysiologique
directement influencée par une seule vibrisse, quoique 90% des neurones répondent, à plus
forte latence, à d’autres vibrisses, du fait de connexions inter-barelettes. Ils projettent
seulement vers le VPM dorsomédial (VPMdm) contralatéral (Minnery and Simons, 2003).
Thalamus : cœur du VPMdm
Le VPMdm n’est constitué que de neurones de relai formant les barréloïdes (Van Der Loos,
1976), dont chacun reçoit des projections d’un à deux neurones du PrV (Castro-Alamancos,
2002). Tout comme le PrV, les neurones du VPMdm répondent à faible latence (4-6 ms)
surtout à une vibrisse en particulier, pour laquelle ils présentent en outre une sélectivité
directionnelle (Minnery et al., 2003). Ces neurones projettent vers le noyau réticulaire
12
thalamique (Harris, 1987) duquel ils reçoivent en retour des projections inhibitrices
(Peschanski et al., 1983; Varga et al., 2002), unique source locale d’inhibition (Barbaresi et
al., 1986).
Cortex : cortex somatosensoriel primaire
Les axones barréloïdeux projettent vers le cortex somatosensoriel primaire (S1), en
particulier vers ses couches 3 et 4 (laquelle couche 4 constitue le « cortex à tonneaux »
(Woolsey and Van der Loos, 1970)) et dans une moindre mesure vers sa couche 6 (Arnold
et al., 2001; Petersen, 2007). À l’instar des autres aires corticales primaires sensorielles, les
neurones de la couche 4 projettent vers les couches 2/3, lesquelles projettent vers la couche
5, laquelle établit des sorties sous-corticales (Lubke and Feldmeyer, 2007). Ainsi, lorsqu’une
vibrisse est défléchie, une excitation rapide se met en branle de la barrelette, au barréloïde
au tonneau correspondants (Armstrong-James et al., 1992; Lefort et al., 2009).
Le cortex à tonneaux occupe une part majoritaire du cortex somatosensoriel des rongeurs
(Figure Introduction 10, (Prescott et al., 2016)), et le ratio entre le volume du cortex
somatosensoriel primaire et le volume du cortex visuel primaire est supérieur chez les
rongeurs nocturnes que diurnes (Campi et al., 2011; Campi and Krubitzer, 2010).
Figure Introduction 10 - Cortex somatosensoriel des rongeurs (adapté de (Maier et al., 1999))
Chaque tonneau contient, chez le rat, environ 20.000 neurones (Meyer et al., 2010). Bien
que depuis sa découverte en 1970, les descriptions du cortex à tonneaux abondent dans la
13
littérature, notamment quant aux méandres de son organisation neuronale (Feldmeyer et
al., 2013; Petersen, 2007; Petersen, 2019; Stuttgen and Schwarz, 2018), notons que le rôle
de cette organisation demeure incertain, s’agissant d’un cas relativement particulier dans le
monde animal. L’hypothèse qu’il s’agisse d’une « trompe » évolutive (spandrel) a été
avancée (Horton and Adams, 2005).
S1 a été très étudié pour ses propriétés plastiques, tant durant le développement (Van der
Loos and Woolsey, 1973) qu’à l’âge adulte (Polley et al., 2004), ouvrant la voie à des
comparaisons (souvent à des analogies) avec les cortex visuel et auditif (Feldman, 2009;
Fox and Wong, 2005). En bref, contrairement au thalamus, le cortex somatosensoriel
connaît une période critique durant laquelle, à défaut d’informations vibrissales, il
dégénèrera (Fox et al., 2002; Glazewski and Fox, 1996).
Le cortex somatosensoriel primaire projette vers le cortex somatosensoriel secondaire (S2)
et vice versa (Aronoff et al., 2010; Chakrabarti and Alloway, 2006). Il a été démontré que
les réponses de S2 à la déflection des vibrisses dépendaient davantage que S1 des
corrélations de déflections entre vibrisses, et étaient sujettes à une plus longue fenêtre
d’intégration que S1 (Goldin et al., 2018). De là à envisager que S2 serait davantage
impliqué dans la mémoire à court-terme…
Voie lemniscale 2
Tronc cérébral : PrV
La voie lemniscale 2 naît d’une population du PrV répondant identiquement à plusieurs
vibrisses, même chez le rat anesthésié (Urbain and Deschenes, 2007b; Veinante and
Deschenes, 1999).
Thalamus : tête du VPM dorsomédial
Le PrV multi-vibrissal projette à la lisière dorsale du VPM, en bordure du PO, au sein d’une
fine couche cellulaire. Celle-ci présente, contrairement aux corps des barréloïdes, des
réponses multi-vibrissales (Urbain and Deschenes, 2007b).
Cortex : S1
La tête du VPMdm projette vers les régions septales de la couche 4 de S1, c’est-à-dire entre
les tonneaux (Furuta et al., 2009), ainsi que vers sa couche 6.
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Voie extralemniscale
Tronc cérébral : SpVi caudal
La voie extralemniscale naît d’une population répondant identiquement à plusieurs vibrisses
du SpVi caudal (Veinante et al., 2000).
Thalamus : VPM ventrolatéral
La voie extralemniscale chemine vers le VPM ventrolatéral (c’est-à-dire vers la queue des
barréloïdes). Une population spécifique du réticulaire thalamique y projette (Bokor et al.,
2008).
Cortex : S1 et S2
Le VPM ventrolatéral projette vers les couches 6 et 4, où il pourrait contribuer aux activités
septales (Urbain and Deschênes, 2009).
Voie paralemniscale
Figure Introduction 11 - Schéma des voies lemniscale 1 et paralemniscale (depuis (Frangeul et al., 2014))
Tronc cérébral : SpVi rostral
La partie rostrale de l’interpolaire contient des cellules multi-vibrissales (Williams et al.,
1994), ayant majoritairement une préférence directionnelle (du bas vers le haut) (Furuta et
al., 2006). Ces neurones montrent une arborescence très riches, pourvus de collatérales
entre autres au sein du PO, de la zone incerta ventrale (ZIv), du collicule supérieur, de l’olive
inférieure (Veinante et al., 2000), du Kölliker-Fuse (KF) (Elbaz et al., 2021).
15
Thalamus : PO
Suivant la déflection de vibrisses, des réponses à fortes latences (de l’ordre de 15 ms) sont
enregistrables dans le PO (Diamond et al., 1992; Sosnik et al., 2001), et abolies lorsque S1
est inhibé (Diamond et al., 1992). Il a été établi qu’une inhibition exercée par la ZIv sur le
PO était responsable de l’inhibition des réponses ascendantes émanant du SpVi rostral
(Bartho et al., 2002; Lavallee et al., 2005). L’inactivation de la ZIv ré-établit en effet la
transmission de réponses à faible latence du SpVi rostrale vers le PO (Trageser and Keller,
2004). Il a par conséquent été proposé qu’un mécanisme de désinhibition (inhibition de
l’inhibition incerto-thalamique) permettait d’ouvrir la voie lemniscale. Cette possibilité a été
corroborée par la découverte de ce que le cortex moteur est capable d’inhiber les réponses
vibrissales au sein de la ZIv, une inhibition médiée par un réseau inhibiteur intra-incertal
(Figure Introduction 12) (Urbain and Deschenes, 2007a).
Figure Introduction 12 - Désinhibition de la voie paralemniscale (issu de (Urbain and Deschênes, 2009))
Le défi principal concernant la voie paralemniscale reste de déterminer les contextes
comportementaux durant lesquels elle est désinhibée (soit ceux durant lesquels la ZIv
projetant au PO est inhibée), permettant un encodage par le PO des informations
sensorielles émanant du SpVI rostral. De nombreux éléments laissent à penser que la voie
paralemniscale serait désinhibée dans un contexte aversif (cf. chapitre 2 de cette thèse).
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Cortex
Le PO paralemniscal projette vers de nombreuses régions corticales (incluant S1, S2, cortex
périrhinal, insulaire, moteur) (Urbain and Deschênes, 2009).
Voie issue du SpVo
Une population neuronale multi-vibrissale située dans le SpVo (Jacquin and Rhoades, 1990)
projette vers la partie la plus postérieure du VPM et du PO, devant le prétectum, lequel
projette pour sa part vers le cortex périrhinal, le striatum et l’amygdale.
Interactions entre voies trigémino-thalamo-corticales
Lorsqu’un traceur rétrograde est injecté au sein de n’importe lequel des noyaux de la
colonne trigéminale, la vaste majorité des cellules rétrogradement marquées apparaissent
dans d’autres noyaux de la colonnes trigéminale, dans les aires corticales
somatosensorielles et dans le noyau cholinergique pédonculopontin (Urbain and
Deschênes, 2009). Ainsi par exemple, le PrV reçoit des projections inhibitrices depuis le
SpVi caudal et excitatrices depuis le SpVc (Furuta et al., 2008). La projection depuis S1 est
massive et topographiquement organisée, connectant des tonneaux aux barrelettes
correspondantes (Urbain and Deschênes, 2009).
Voies trigémino-colliculaire et cortico-colliculaire
Les couches intermédiaires du collicule supérieur reçoivent des informations vibrissales
sensorielles autant de de la colonne trigéminale que du cortex à tonneaux (Bruce et al.,
1987; Huerta et al., 1983; Veinante and Deschenes, 1999). Du fait des projections
trigéminales, les neurones colliculaires répondent de façons robuste et rapide aux
déflections vibrissales (Hemelt and Keller, 2007). Du fait des projections corticales, les
neurones colliculaires répondent par un second pic d’activité aux déflections vibrissales
(Cohen et al., 2008).
Voie trigémino-cérébelleuse
Le cervelet reçoit des informations vibrissales par deux voies impliquant des fibres
moussues. D’une part, directement depuis des cellules de la colonne trigéminale, dont des
cellules du PrV et du SpVi rostral (Watson and Switzer, 1978) ne correspondant pas à celles
projetant au PO (Woolston et al., 1982). D’autre part, par l’intermédiaire du noyau pontin,
17
lequel reçoit lui-même des projections de la colonne trigéminale (Mihailoff et al., 1989;
Swenson et al., 1984).
Le cervelet reçoit également des projections de l’olive inférieure (fibres grimpantes), à
laquelle projette le SpVi (Molinari et al., 1996; Yatim et al., 1996).
Enfin, le cervelet reçoit des projections de S1 et du cortex moteur vibrissal (Swenson et al.,
1989).
Les cellules de Purkinje répondant aux déflections vibrissales se trouvent surtout dans les
lobules corticaux CRUS1 et CRUS2 (Bosman et al., 2010).
Voies motrices
La grande majorité des études ayant porté sur la motilité des vibrisses se sont concentrées
sur le whisking et sur les macro-vibrisses mystaciales, tandis que les vibrisses
supraorbitales et génales sont également mobiles (Severson et al., 2019).
Musculature des follicules
L’innervation des vibrisses mystaciales est assurée par le nerf facial (Dorfl, 1982, 1985),
plus spécifiquement par ses branches buccales et mandibulaires (Semba and Egger, 1986).
Les muscles dits intrinsèques sont protracteurs des vibrisses, tandis que les muscles dits
extrinsèques sont protracteurs, rétracteurs et déflecteurs verticaux des coussinets sur
lesquels les vibrisses sont incrustées (Figure Introduction 13) (Haidarliu et al., 2015).
Figure Introduction 13 - Muscles intrinsèques et extrinsèques (issu de (Hill et al., 2008))
18
Les muscles extrinsèques et intrinsèques sont striés, de trois types pour les extrinsèques :
rapides, intermédiaires et lents (White and Vaughan, 1991). Les muscles intrinsèques
correspondent essentiellement à des fibres rapides (Haidarliu et al., 2010; Jin et al., 2004).
La protraction des vibrisses est surtout régie par les muscles intrinsèques, tandis que la
rétraction des vibrisses est largement passive (Berg and Kleinfeld, 2003). Les muscles
extrinsèques contrôlent davantage l’orientation et le gonflement du pad que la protraction
des vibrisses.
Motoneurones
Les motoneurones des vibrisses se trouvent dans les parties latérale et intermédiaire du
noyau facial. Les motoneurones des muscles intrinsèques se logent dans sa portion
ventrale, ceux des muscles extrinsèques dans sa portion dorsale (Figure Introduction 14).
Figure Introduction 14 - Cartographie des motoneurones des vibrisses au sein du noyau facial ("ML", "NL",
"DN" et "NLP" désignent des muscles extrinsèques ; issu de (Deschenes et al., 2016))
Prémotoneurones
Les régions prémotoneuronales contrôlant les motoneurones des vibrisses ont été
déterminées par des études transynaptiques sur lesquelles se fonde cette partie
(Sreenivasan et al., 2015; Takatoh et al., 2013). Ces régions prémotoneuronales sont
légion, en voici un florilège.
19
Noyaux trigéminaux
L’interpolaire rostral et l’oralis projettent au noyau facial, médiant un réflexe de rétraction
suivant le toucher (Bellavance et al., 2017).
Formation réticulaire intermédiaire ventrale
La formation réticulaire intermédiaire ventrale contient les prémotoneurones dont l’activité
rhythmiques est à l’origine du whisking (Deschenes et al., 2016; Moore et al., 2013).
Cortex moteur
D’éparses projections cortico-faciales existent mais jouent probablement un rôle secondaire
par rapport à celles, bien plus massives, connectant le cortex moteur et les ganglions de la
base ou le colliculus supérieur. Comme nous le verrons dans la partie 3 (et dans le chapitre
1), le rôle du cortex moteur demeure très nébuleux.
Noyau rouge parvocellulaire
Le noyau rouge parvocellulaire donne lieu à la voie rubro-faciale. Il reçoit des projections du
cervelet, du cortex moteur et des noyaux trigéminaux (Godefroy et al., 1998; Pacheco-
Calderon et al., 2012). Cette voie médie l’exécution du réflexe de clignement des paupières,
suivant l’exposition à un stimulus conditionnel (tel qu’une déflection vibrissale) annonçant
l’occurrence d’un stimulus inconditionnel (typiquement, un jet d’air visant les yeux) (Freeman
and Steinmetz, 2011; Pacheco-Calderon et al., 2012).
Kölliker-Fuse
Le Kölliker-Fuse (KF) envoie des projections glutamatergiques vers la région du facial
contrôlant les muscles intrinsèques (Elbaz et al., 2021). Une autre population GABAergique
du KF inhibe, possiblement par un mécanisme d’inhibition présynaptique, les noyaux de la
colonne trigéminale (Chamberlin, 2004; Elbaz et al., 2021).
Collicule supérieur
Chaque colliculus supérieur envoie de denses projections bilatérales aux noyaux faciaux
(Hattox et al., 2002; Miyashita and Mori, 1995).
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Voies sensorimotrices
Signaux moteurs et sensoriels interagissent dès le tronc cérébral (Bellavance et al., 2017),
au niveau du thalamus paralemiscal (PO), entre les cortex sensoriel et moteur, des
ganglions de la base, du cervelet, du colliculus supérieur, et du noyau rouge (Figure
Introduction 15).
Figure Introduction 15 - Boucles sensorimotrices vibrissales (depuis (Prescott et al., 2016))
Ganglions de la base
Le striatum dosrsolatéral reçoit des projections du PO, mais aussi de S1, S2, du cortex
pariétal postérieur et du cortex moteur (Smith et al., 2012). Une convergence de projections
depuis S1 et le cortex moteur sur de mêmes neurones du striatum dorsolatéral a été décrite
(Charpier et al., 2020), avec cependant une asymétrie dans leur physiologie (Lee et al.,
2019).
Le striatum peut contrôler la position des vibrisses d’au moins deux façons : via la boucle
ganglions de la base-thalamo-corticale, ou bien via ses projections vers le colliculus
supérieur (lequel projette également vers le noyau facial) (Kaneda et al., 2008).
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Cervelet
Comme précédemment évoqué, le cervelet répond au toucher vibrissal, mais son activité
corrélée avec les mouvements des vibrisses durant le whisking précède lesdits mouvements
(Chen et al., 2016). Le cervelet est donc le siège d’informations vibrissales autant
sensorielles qu’efférentes (relèveraient-elles d’une copie efférente).
Collicule supérieur
En plus de recevoir des projections de la colonne trigéminale et de projeter au noyau facial,
le collicule supérieur reçoit ipsilatéralement du cortex moteur (Miyashita et al., 1994), du
cortex somatensoriel (Wise and Jones, 1977) et du cervelet (May, 2006).
3. Neuroéthologie
Nous avons jusque-là évoqué d’une part des comportements divers impliquent les vibrisses,
d’autre part des fondements anatomo-physiologiques du « cerveau vibrissal ». Cependant,
nous nous tenons encore loin d’une explication de ces premiers par ces seconds. Nous
tenterons toutefois ci-dessous de faire un panorama des données neuroéthologiques
connues.
Mouvements spontanés (whisking)
Comme mentionné précédemment, le whisking est le fait d’un oscillateur qui projette
directement vers les motoneurones des vibrisses. Cependant, ce qui initie le whisking, soit
ce qui active l’oscillateur en question, demeure énigmatique. Si le cortex moteur des
vibrisses peut influencer l’amplitude du whisking (Ebbesen et al., 2017), les rats peuvent
tout aussi bien l’initier, avec ou sans cortex (Gao et al., 2003). Se pose donc la question du
rôle des activités, au sein du cortex moteur, depuis lesquelles le whisking (incluant son
initiation) peut être décodé (Hill et al., 2011).
Des activités ré-afférentes sont retrouvées, lorsqu’un rat whiske, autant au niveau des
mécanorécepteurs, du tronc cérébral, que dans le thalamus (Moore et al., 2015b; Severson
et al., 2019) et le cortex (Gutnisky et al., 2017), mais nous ignorons si ces activités jouent
un rôle proprioceptif, s’avérant peut-être utiles pour permettre aux rats adultes de savoir
« où sont leurs vibrisses ». Le whisking persiste cependant après déafférentation (Welker,
1964) et ablation corticale (Semba and Komisaruk, 1984).
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Explorer son environnement
Locomotion
Les liens entre informations vibrissales et locomotion, que les observations
comportementales permettaient déjà de soupçonner, ont été corroborés par une étude qui
a établi que l’activité neuronale, dans S1, associée aux vibrisses les plus proches du sol (3
vibrisses sous le menton) encodent la vitesse de la marche. La microstimulation de ces
neurones a pour effet de modifier la vitesse de la marche (Chorev et al., 2016).
Détection et localisation d’objets
La lésion de S1 rend impossible la réalisation du « gap crossing » (Hutson and Masterton,
1986). Cependant, S1 n’est pas requis pour la simple détection d’objets (Hong et al., 2018).
Nous en déduisons qu’outre la simple détection d’objets, l’estimation de distances pourrait
requérir S1. D’ailleurs, S1 est requis pour discriminer des positions distinctes (O'Connor et
al., 2010).
La détection d’une forte déflection vibrissale annonçant un choc électrique n’est détectable
que si ou le collicule supérieur ou le thalamus sensoriel est actif, mais requiert ces deux
régions si la déflection vibrissale est de faible amplitude (Cohen and Castro-Alamancos,
2010) ; intéressant cas de redondance et de synergie entre deux régions cérébrales.
Discrimination de textures
S1 est requis pour discriminer des textures (preprint « Deep and superficial layers of the
primary somatosensory cortex are critical for whisker-based texture discrimination in mice »)
mais l’étude en question n’a pas permis aux sujets expérimentaux l’utilisation de leurs
microvibrisses, pourtant utilisées dans un cadre éthologique pour discriminer des textures.
Questions ouvertes
Deux questions principales se dégagent : 1) Quels contextes comportementaux particuliers
implique telle voie sensorielle ou motrice ? 2) Quelle est la portée éthologique des signaux
corrélés avec la position des vibrisses et des signaux efférents retrouvés au sein du cortex
et du cervelet ?
Nous avons tâché de nous avancer sur chacune de ces questions, avec deux projets dont
les approches sont complémentaires. Le premier projet, partant d’un comportement, vise à
déterminer le circuit minimal par lequel un rat peut finement contrôler la position de ses
vibrisses, capacité dépendant de sa connaissance de leur position. Le second projet, partant
23
de données anatomo-physiologiques, établit l’existence d’une nouvelle voie vibrissale
sensori-motrice, qui pourrait être impliquée dans l’expression d’émotions.
24
Chapitre 1 A cortical-independent internal model
for the control of voluntary vibrissa movement
1.1 Résumé
Que ce soit par nos yeux ou par nos mains, nous interagissons avec notre environnement
par l’intermédiaire de senseurs mobiles. L'utilisation efficace de tels organes, dits
« sensorimoteurs », implique de garder trace de leur position tandis que nous les bougeons.
Dans le cas contraire, la stabilité perceptive et la préhension dirigée seraient profondément
entravées. Cependant, les mécanismes neuronaux qui informent le système nerveux de la
position de ces organes restent largement inexplorés. Deux mécanismes impliquant une
information retour peuvent théoriquement être en jeu : la copie efférente et/ou le retour
sensoriel. Pour évaluer leur contribution, nous avons entraîné des rats à placer une vibrisse
dans une plage angulaire prédéterminée sans contact, une tâche dépendant de la
connaissance qu’ils ont de la position de leur vibrisse. Nous établissons que le retour
sensoriel n'est pas requis pour effectuer cette tâche. Le cortex moteur n'est pas non plus
requis, même en l'absence de retour sensoriel. Enfin, nous démontrons que le noyau rouge,
qui reçoit des entrées du cortex moteur, du cervelet, des afférences vibrissales, et qui
projette vers les motoneurones faciaux est impliqué de façon critique dans l'exécution de
cette tâche. Ces résultats démontrent l'existence d'un modèle interne, indépendant du
cortex moteur et suffisant au contrôle moteur volontaire. Ils suggèrent en outre que le
cervelet pourrait être impliqué en tant qu’élément efférent de ce modèle interne.
1.2 Abstract
Whether through our eyes or our hands, we interact with our environment through mobile
sensors. The efficient use of these organs implies to keep track of their position over time.
Otherwise, perceptive stability and directed prehension would be profoundly impeded. Yet,
the neural mechanisms informing the nervous system about organs’ position remain largely
unexplored. Two feedback mechanisms may theoretically be at play: efference copy and/or
sensory feedback. To assess their contribution, we trained head-restrained rats to place
their vibrissae in a predetermined angular range without contact, a task that depends on the
knowledge of vibrissa position. We find that sensory feedback is not required to perform the
task. The motor cortex is neither required, even in the absence of sensory feedback. Finally,
we find that the red nucleus, which receives motor cortex, cerebellar and vibrissa inputs,
and projects to facial motoneurons is critically involved in the execution of the task. These
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results demonstrate the existence of a motor cortex-independent internal model sufficient
for voluntary motor control. They further suggest that the cerebellum may play a role as an
efferent part of this internal model.
Introduction
Decoding information gathered through moving sensors – the hallmark of “active sensing” –
requires keeping track of the sensors’ position (Kleinfeld and Deschenes, 2011; Wolpert and
Ghahramani, 2000). Haptic exploration in rodents instantiates this faculty: mice and rats can
report the location of an object in their vibrissa field with great precision (Knutsen et al.,
2006; Mehta et al., 2007; O'Connor et al., 2010), which implies that they know the position
of their vibrissae with respect to their own body (Cheung et al., 2019). As facial muscles are
devoid of proprioceptors, two non-exclusive feedback mechanisms may account for the
knowledge of vibrissa position (Fee et al., 1997; Wolpert and Ghahramani, 2000; Wolpert et
al., 1995): efference copy (internal feedback) and/or reafferent signals (external, sensory
feedback). With efference copy, internal brain signals are transmitted from motor regions
responsible for the movement to other regions, allowing the brain to keep track of the
consequences of motor actions. With reafferent signals, sensory receptors encode the
position of the vibrissae or the kinematics of the ongoing movement. Although previous
studies established that both mechanisms are plausible at the level of the vibrissa system
(Chen et al., 2016; Hill et al., 2011; Moore et al., 2015b; Severson et al., 2017; Severson et
al., 2019), their ethological value remains unknown. We designed a behavioral task which
allowed to untangle the contribution of these two mechanisms.
Vibrissa tasks involving touch are not well-suited for distinguishing the role of efference copy
and reafferent signals. First, primary vibrissa afferents multiplex exafferent (touch) and
reafferent (self-motion) signals (Gutnisky et al., 2017; Moore et al., 2015b; Severson et al.,
2017; Severson et al., 2019), which makes it virtually impossible to manipulate reafferent
signals without interfering with exafferent signals. Second, in the somatosensory cortex, a
region involved and required for localizing objects with vibrissae (Guo et al., 2014; O'Connor
et al., 2010), sensory and efference copy signals interact via sensorimotor loops (Mao et al.,
2011; Petreanu et al., 2012; Veinante and Deschenes, 2003), blurring their respective
contribution. To circumvent these limitations, we designed a vibrissa positioning task which
is carried out in the dark and does not involve touch, so that the sensory information at play
26
consists of reafferent signals only. We reasoned that if rats can finely control the position of
their vibrissae, they know their location.
Results
The vibrissa positioning task
Head-restrained rats were trained to move their left C1 vibrissa (Brecht et al., 1997) from a
retracted position, the “go zone”, to a protracted “reward zone”. Once rats self-initiated trials
by moving their vibrissa in the “go zone”, they were allowed a maximum of 10 seconds to
reach and hold their vibrissa within the “reward zone” for a given duration (figure 1A). The
required hold time in the reward zone adaptatively increased over learning, from 10 ms
initially to 1 s at the performance level (Online Methods). Once rats reached the 1 s-
performance criterion, the hold-time was fixed and the effect of permanent and/or transient
experimental manipulations was assessed.
Intact rats can learn the vibrissa positioning task
Water-deprived rats quickly learnt to reliably lick upon reward delivery (supplementary figure
1A). Given the short hold time initially required (10 ms), naïve rats were able to obtain
rewards through spontaneous vibrissa movements, such as upward twitches (figure 1B, left).
Over training, normal rats (n=10) tended to reach and maintain their vibrissa in the reward
zone more swiftly following trial onset (figure 1C; mixed-effect linear regression p<0.01).
Reaching the 1-sec hold time criterion required on average 6000 trials (4 weeks of training).
Expert animals exerted sustained protractions during the 1-sec successful hold times (figure
1B), without high-frequency (“whisking”) movements in most cases (93±1% of successful
hold time periods). As reward expectation is normally associated with whisking (Dominiak
et al., 2019), the task requires rats to curb their natural proclivity to whisk. An increasing
trend to protract was also apparent during intertrials (figure 1D); as a result, expert animals
started most trials via backwards twitches (supplementary figure 1B). This suggests either
that permanent sustained protraction is an effective strategy to succeed as learning
progresses, or that rats did not distinguish between trials and intertrials. We invalidate the
latter hypothesis by showing that, in expert animals, hold times preceding inopportune (false
alarms) licks are shorter during trials than during intertrials, all other things being equal
(supplementary figure 1C).
In summary, expert rats’ strategy consists in maintaining their vibrissa close to or within the
reward zone and briefly retracting the vibrissae to initiate a new trial.
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Sensory feedback is neither required before nor after learning
Do rats require sensory feedback to finely move their vibrissae? This is physiologically
plausible since reafferent signals are widespread throughout the rodent’s brain (encoded by
mechanoreceptors’ afferents (Severson et al., 2017; Severson et al., 2019), brainstem
(Moore et al., 2015b; Zucker and Welker, 1969), thalamus (Gutnisky et al., 2017; Moore et
al., 2015b), cortex (Berg and Kleinfeld, 2003; Cheung et al., 2020; Fee et al., 1997) and
cerebellum (Chen et al., 2016)). To test the requirement of sensory feedback during our
task, we first deafferented two expert rats once their steady-state level was measured (figure
2A; Online methods). They recovered their success rate pre-lesion level after some (5 and
6, respectively) experimental sessions. To assess whether deafferentation altered whiskers
motor control, we compared their movement parameters across conditions. Deafferentation
did not alter the whisker mean angle during trials (figure 2B; mixed-effect linear regression,
p>.05), nor the distribution of maximum hold times in the reward zone (figure 2C; KS test,
p>.05), nor the occurrence of 10°-wide holds in the reward zone (figure 2D; permutation test,
p>.05). During the 1-sec successful hold times, the mean vibrissa position remained
unchanged (figure; 2E mixed-effect linear regression, p>.05) as well as the inter-trial
variability (figure 2F, permutation test, p>.05). However, deafferentation diminished the
prevalence of high-frequency movements (whisking and discrete twitches) (supplementary
figure 2B, mixed-effect linear regression, p<.05).
The dispensability of a brain region in the execution of a behavior, once learnt, does not
exclude its requirement over learning (Kawai et al., 2015). To test the potential requirement
of sensory feedback for learning the task, we deafferented three rats before their first training
session. These animals displayed the same signs of learning as intact animals, reaching at
least similar performance level (supplementary figures 2C-F).
These results indicate that sensory feedback is required neither for learning nor for
executing the task; in other terms, rats can finely control their vibrissae’ position via an
internal model.
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Motor cortex is not required to learn the task
Since the vibrissa position can be decoded from the vibrissa motor cortex (Ebbesen et al.,
2017; Friedman et al., 2012; Hill et al., 2011; Sreenivasan et al., 2016) both in the presence
and in the absence of sensory feedback (Hill et al., 2011), we tested its requirement during
our task, before and after deafferentation. We bilaterally ablated the motor cortex
(supplementary figure 3A, Online methods) of two naïve rats and exposed them to the task.
These rats learned the task (supplementary figures 3B-D) and reached a steady-state
performance level at least similar to that of intact rats (figure 3A). This learning ability was
also verified in five rats whose motor cortex was unilaterally ablated, contralaterally to the
tracked vibrissa (data not shown). Thus, the motor cortex is neither necessary for learning
nor for executing our task in the presence of sensory feedback.
In the absence of motor cortex, sensory feedback is required for vibrissa
stability
To test the requirement of motor cortex in the absence of sensory feedback, we deafferented
the bilaterally decorticated expert rats. Both rats recovered their pre-deafferentation level in
terms of occurrence of holds in the reward zone and success rate (figure 3A; logistic mixed-
effect regression, p>.05). Yet, as in corticated rats, deafferentation diminished the
prevalence of fast oscillations during hold times (mixed-effect linear regression, p<.05).
Surprisingly, deafferentation in decorticated rats led to changes that did not occur in intact
rats. First, following deafferentation, the variance of the vibrissa angle increased during trials
(test permutation, p<.05) while the mean angle did not change (linear mixed-effect, p>.05).
Second, the inter-trial variability over the 1-sec successful hold time fragments was
increased (figure 3C-D, stats). Taken together, these results indicate that, in the absence of
motor cortex, sensory feedback plays a non-compensable role in motor precision and the
ability to maintain vibrissae still. Nevertheless, in the absence of motor cortex and sensory
feedback, the overall ability to voluntarily protract vibrissae is preserved, suggesting the
existence of an internal model independent from the vibrissa motor cortex and afferents.
Inactivation of the rubro-facial pathway disrupts performance
Which brain pathway might sustain the ability to do the task without requiring the motor
cortex, but involving it at least in case of deafferentation? Amid the numerous premotor
nuclei controlling vibrissa motoneurons (Hattox et al., 2002; Isokawa-Akesson and
Komisaruk, 1987; Sreenivasan et al., 2015; Takatoh et al., 2013), the parvocellular part of
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the red nucleus seems of particular interest: first, it receives both cortical and cerebellar
inputs (Daniel et al., 1987; Hattox et al., 2002; Pacheco-Calderon et al., 2012); second, it
has access to vibrissae’ sensory information via direct projections from the primary sensory
relay (Elbaz et al., 2021; Godefroy et al., 1998); third, Purkinje cells’ activity anticipates
vibrissa position, even when the motor cortex is inactivated (Chen et al., 2016).
We examined the involvement of the rubrofacial pathway by expressing inhibitory DREADDs
(Zhu and Roth, 2014) in neurons projecting from the red nucleus to the facial nucleus (Online
methods; figure 4A and supplementary figure 4A). This expression allowed for the
conditional inactivation of rubrofacial neurons during the entire duration of chosen
experimental sessions, following intraperitoneal injection of Clozapine N-oxide (CNO; Online
Methods). To account for potential endogenous effects of CNO (Gomez et al., 2017; Mahler
and Aston-Jones, 2018; Manvich et al., 2018), the effects of CNO were compared between
rats expressing DREADDs (n=3) and rats not expressing DREADDs (n=3).
Inactivation of the red nucleus dramatically impaired rats’ ability to maintain their vibrissa in
the reward zone (figure 4B and supplementary figure 4B; logistic mixed-effect regression on
success rates, interaction effect group:CNO, p<0.05). The vibrissa position was more
retracted during trials (figure 4C), including during whisking periods (regression mixed-effect
regressions on whisking setpoint, interaction effect group:CNO, p<.05). Finally, the duration
of holds within the reward zone prior to “false alarm” licks were shorter (supplementary figure
4C; linear mixed-effect regression, interaction effect group:CNO, p<.05), suggesting an
impeded motor perception. Rubrofacial neuron inactivation led to the same deficits in
deafferented animals (data not shown). These results indicate that the rubrofacial pathway
is a premotoneuronal region crucially involved in the execution of the task.
Discussion
In the present study, we aimed to identify the mechanisms whereby rats keep track of the
position of their vibrissae (Cheung et al., 2019; Kleinfeld and Deschenes, 2011). To address
this question, we conditioned rats to a vibrissa motor sequence task without contact, so that
deafferenting animals was strictly akin to abolishing sensory feedback, one of the two
feedback mechanisms potentially at play along with efference copy. By contrast, tasks
involving touch tangle up reafferent and exafferent signals, making the specific role of
reafferent signals intractable (and deafferentation making such tasks unrealizable).
We draw five main observations from our experimental data. First, intact rats were able to
learn our task, which indicates that they are aware of the position of their vibrissae even
30
without touching objects. This is in line with previous reports in which free whisking
movements were reinforced (Bermejo et al., 1996; Gao et al., 2009; Sehara et al., 2019).
Second, intact rats who reached the expert criterion remained able to execute the task
similarly well after deafferentation. Furthermore, rats could learn the task even when initially
exposed to it after being deafferented. Therefore, an internal model exists and provides
sufficient awareness of vibrissae relative position to allow rats to execute a fine whisker
positioning task. Third, rats whose motor cortex was bilaterally lesioned were also able to
learn the task, indicating that the motor cortex is not a required part of this internal model.
Fourth, deafferentation increased motor variability in decorticated rats but not in corticated
rats. This implies that sensory feedback and motor cortex can interact and compensate for
each other, an illustration of plastic redundancy. Despite these control deficits, the overall
ability to do the task was preserved even in decorticated deafferented rats, implying the
existence of a cortically-independent internal model. As far as we know, this is the first
demonstration of voluntary movements in mammals without motor cortex and without
sensory feedback. Fifth, inactivation of rubrofacial neurons drastically impeded rats’
performance, indicating that the red nucleus is a premotor region critically involved in the
execution of the task. Its inputs make it an ideally situated integrator of reafferent and
efferent (motor cortical and cerebellar) signals.
Motor cortex and sensory feedback
Using lesions, our study reveals the dispensability of sensory feedback and motor cortex in
fine vibrissa movements but leaves open the question of their normal physiological
involvement. The literature remains quite incomplete in this regard. The importance of
sensory feedback has been evidenced in the establishment of vibrissa sensorimotor maps
during development (Carvell and Simons, 1996; Fox and Wong, 2005; Khazipov et al., 2004;
Landers and Philip Zeigler, 2006). In adult rats, sensory feedback was proposed to cancel
self-movement signals in the somatosensory cortex (Gutnisky et al., 2017). As for the
vibrissa motor cortex, it has been shown to modulate whisking’s amplitude and setpoint
(Ebbesen et al., 2017; Gao et al., 2003), even though it is not required for initiating whisking
(Lovick, 1972; Semba and Komisaruk, 1984). Behavioral contexts which would highlight
more fundamental roles remain to be identified. By analogy with the forelimb motor cortex,
one may assume that the vibrissa motor cortex is either required for the execution of “skilled
movements” (Castro, 1972; Miri et al., 2017) or for temporally constrained motor sequences
(Kawai et al., 2015). This first possibility seems unlikely given that rats were able to reach a
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10°-wide reward zone after lesion of the motor cortex (data not shown), which is comparable
to the performance of intact animals. The second possibility has not yet been addressed.
Red nucleus and cerebellum
Contrary to our cortical and sensory manipulations, we chose to transiently inactivate rather
than lesion the red nucleus, thereby testing its involvement rather than its requirement. Yet,
transient manipulations can make it difficult to disambiguate the nature of the involvement,
either instructive or permissive (Otchy et al., 2015), and can even lead to fast compensatory
mechanisms unfolding in the space of single behavioral sessions (Fetsch et al., 2018),
blurring the probed normal physiology. Yet, the deficits observed in our inactivation
experiments are unlikely to be strictly due to a permissive involvement of the red nucleus,
since the targeted cells (rubrofacial neurons) are glutamatergic premotoneurons (personal
communications from Fan Wang and Jun Takato) and the targeted motoneuronal region
(facial nucleus) houses only motoneurons (Courville, 1966; Moore et al., 2015b), whose
single spikes trigger movements (Herfst and Brecht, 2008). The changes observed under
red nucleus inactivation in both sustained movements and whisking suggest a common
regulatory mechanism for setpoint control. This idea is reinforced by the existence of
projections from the red nucleus to the whisking oscillator (personal communications from
Fan Wang and Jun Takato), the inhibitory nature of the whisking oscillator whose inputs
modulate setpoint (Moore et al., 2013) and, in our data, the positive correlation between the
whisking angles in adjacent whisking and non-whisking fragments (data not shown).
The involvement of the red nucleus also implies the motor cortex and/or the cerebellum, its
two major inputs. Both latter regions anticipate vibrissa position, indicating that they are part
of a common network. Elucidating their specific contributions vis-à-vis the red nucleus will
require further investigation. An ideal experiment may consist in recording the red nucleus,
while sequentially or simultaneously manipulating the motor cortex and the cerebellum
optogenetically in a closed-loop fashion.
Since cerebellar activity anticipates vibrissa position even when the motor cortex is
inactivated, it is tempting to speculate that the cerebellum might take control of the red
nucleus in the absence of the motor cortex, possibly resulting in cerebello-rubral synaptic
sprouting (the reciprocal phenomenon, cortico-rubral sprouting, had been observed at the
level of the rubrospinal pathway following cerebellar lesion (Murakami et al., 1976;
Tsukahara, 1974)). Furthermore, since rubrofacial neurons send collaterals to the lateral
reticular nucleus (supplementary 4A), which projects back to the cerebellum, the cerebellum
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receives an efference copy of the vibrissa movements instructed by the red nucleus. This
might grant the cerebello-rubro-lateral reticular nucleus loop with the capability not only to
control but also to correct vibrissa movements, even in the absence of external (sensory)
feedback. A number of studies indicate that the cerebello-rubro-facial pathway is involved in
plastic changes associated with motor learning, especially in the eye blink reflex elicited by
somatic or auditory conditional stimuli. The reward-related signals to which the cerebellum
has access (Giovannucci et al., 2017; Heffley and Hull, 2019; Kostadinov et al., 2019; Larry
et al., 2019) are likely to substantiate these learning abilities.
Beyond vibrissae
Like eye muscles, vibrissa muscles are not load-bearing, hence their lack of proprioceptors.
This property and the dispensability of reafferent signals reported in this study position
vibrissae as a powerful system to study internal models in movement control, without
environmental confounding variables related to the properties of the borne objects.
Methods
This study’s protocol was approved by the Comité de Protection des Animaux de l’Université
Laval (CPAUL). All procedures were caried out in strict accordance with Canadian federally-
prescribed animal care and use guidelines. All surgical procedures were performed under
ketamine-xylazine anesthesia.
Animals
Male Long Evans adult rats, 250 to 350 g in mass (Charles River), were used for combined
behavioral, neurophysiological and anatomical experiments. They were housed in a reverse
dark-light cycle in a facility with controlled temperature and humidity. After a week of daily
handling aimed at getting them habituated to the experimental room and to the manipulator,
they were implanted with a head-plate, following procedures previously described (Moore et
al., 2015a). A week after head-plate implantation, the rats were water-deprived from Sunday
to Friday and watered in the experimental room only. They were progressively head-
restrained over increased periods of time and watered concomitantly. When they tolerated
being watered with a sufficient amount of water (~20 ml per day) while being head-
restrained, we considered that they could be exposed to the vibrissa positioning task
(referred to below as “the task”). From that moment on, all their vibrissae but left C1 were
trimmed weekly under light isoflurane anesthesia, in order to optimize the online detection
of the vibrissa of interest and to prevent tactile contact with any element of the surrounding
33
environment. The choice of C1 was motivated by the relative stable dorso-ventral
(azimuthal) level of this vibrissa along its whole retracted-to-protracted range. Rats were
then trained to the task twice a day, 20 minutes per session, from Monday to Friday.
Detailed description of the vibrissa positioning task
All behavioral experiments were carried out with head-restrained rats, in a silent and dark
room. The task was implemented with customized codes written under Matlab (Mathworks),
operating two cameras: one to detect tongue movement used as a lickometer, the other one
to detect vibrissa position with respect to head’s antero-posterior axis (i.e., monitoring of the
absolute vibrissa angle). An infrared light source illuminated the behavioral setup.
Task’s trials were self-initiated by the rat when its vibrissa was detected in the “go zone” (70-
90°). Trial initiation was accompanied by an auditory cue (4 kHz, 300 ms), after which the
rat had 10 seconds to succeed in the trial by moving its vibrissa to the “reward zone” (100-
130°) and maintaining it within it for a given required hold time. Over training, the required
hold time increased from 10 ms up to 1 s in an adaptative way, depending on the success
rate of previously “attempted trials”. An attempted trial is defined as a trial during which the
vibrissa went beyond 5° above the upper limit of the go zone (i.e., beyond 95°). If the mean
success rate of the 50 past attempted trials reached 50%, the hold time was increased by
5% of its previous value, up to 1 s (the expert level). At the end of every trial, one of two
possible sounds was played depending on whether the trial was successful or failed (1 kHz
and 8 kHz respectively, 300 ms). In case of success, a pump was concomitantly activated,
delivering 75 µl of water. After a successful trial, a pause of random duration (between 5
and 8 seconds), during which it was impossible to initiate another trial, allowed animals to
lick the delivered water without interfering with the task. After an unsuccessful trial, there
was a 1-s pause to prevent the juxtaposition of trials in case the vibrissa was in the go zone
as a trial ended, which allowed to play distinctly the sounds announcing the end of the failed
trial and the one announcing the onset of the following one.
With some of the rats which reached the expert criterion (hold time: 1 seconds), we further
investigated their ability to finely control their vibrissa by keeping the hold time constant but
adaptatively diminishing the upper limit of the reward zone (by 1° in case of at least 70% of
successes in the 50 previous trials). This allowed us to quantify rats’ maximum angular
precision. Finally, some of the rats which went through this precision stage were also tested
in terms of their ability to adjust when shifting the whole reward zone, while keeping its width
34
constant (typical shift: from 100-110° to 105-115°). This allowed to test rats’ dynamic
adaptative capabilities.
Deafferentation
Our deafferentation procedure took advantage of the fact that the vibrissae’ sensory and
motor nerves are distinct: on the side of the tracked vibrissa (left), we transected the
infraorbital (sensory) branch of the infraorbital nerve at its entrance to the orbit (Gao et al.,
2001; Hill et al., 2011); on the opposite side (right), we transected the branches of the facial
(motor) nerve (Henstrom et al., 2012) innervating the musculature of the mystacial pad
(namely, the buccal and marginal branches (Henstrom et al., 2012)). Thus, the mysticial pad
does not convey any information related to self-motion. A comparable result could have
been achieved by bilaterally transecting the infraorbital nerves, but this latter possibility
would have led to bilateral anesthesia of the face, diminishing rats’ well-being by making the
use of all their mystacial vibrissae as sensors impossible. Four days after deafferentation,
the rats were exposed back to the task. The effectiveness of the facial nerve lesion was
assessed by the absence of vibrissa movement on the related side. Once the behavioral
training ended, the effectiveness of the deafferentation was verified by the absence of a
long-term potential (LFP) in the vibrissa lemniscal sensory thalamus (ventral posteromedial
nucleus, VPM) (Castro-Alamancos, 2002), upon electrical stimulation of the pad
(supplementary figure 2A). To confirm the emplacement of the LFP pipette in the VPM,
Chicago Sky Blue dye was iontophoretically injected through the recording pipette, the rat
was perfused, and its brain tissue processed for histology.
Some concern may be raised about the completeness of our deafferentation model, since
there exist three non-mystacial moving vibrissae (two supra-orbital and one genal) on each
side of rats’ face, whose sensory innervation is not provided by the infraorbital nerve, and
whose afferents encode amounts of information about whisking setpoint comparable to
mystacial vibrissae’ afferents (Severson et al., 2019). Yet, the much higher number of
mysticial vibrissae (30 in total) (Brecht et al., 1997) and the requirement of infraorbital-
dependent information for the proper development of whisking movements (Landers and
Philip Zeigler, 2006) suggest a primacy involvement of mysticial vibrissae’ sensory
information in reafference.
35
Cortical lesion
The vibrissa motor cortex was unilaterally or bilaterally lesioned by the application, over the
pia mater, of a small crystal of silver nitrate, a strong cauterizing agent (Lavallee et al., 2005)
(with respect to the bregma, centered to 2 mm on the antero-posterior axis and 2 mm on the
medio-lateral axis). The crystal was left in place for 5 min to allow diffusion of the chemical
to the deep layers. Then, the cortical surface was abundantly rinsed with saline and sucked
out. A head-plate was then implanted.
Transient inactivation of the red nucleus
Inhibitory DREADDs were expressed in rubrofacial neurons via dual viral injections (100
nanoliters each): AAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene) in the right parvocellular
red nucleus and retrogradeAAV-hSyn.Cre.WPRE.hGH (Addgene) in the vibrissa subpart of
the left facial nucleus. Both regions were identified based on their standard stereotaxic
coordinates (Paxinos and Watson, 2007) (red nucleus: 1 mm lateral, 5.5 mm behind the
bregma, 6.5 mm below the dura; facial: 2 mm lateral, 2 mm behind the bregma, 7.5 mm
below the dura) and functional criteria (the red nucleus is vibrissa-responsive and the micro-
stimulation of vibrissa motoneurons elicits vibrissa micro-twitches even in anesthetized
animals). A head-plate was implanted immediately after the viral injections were completed.
Given the recovery period following this surgery (1 week) and the time needed for rats to
reach the expert level (at least 3 weeks), the inactivation experiments were caried out at
least 4 weeks after the viral injections, a sufficient time for an optimal AAV-mediated
expression. To inactivate the rubrofacial neurons expressing DREADDs, Clozapine N-oxide
dihydrochloride (Tocris) was intraperitoneally injected (2 mg/kg) one hour prior to the
behavioral experiment. The exact same procedure was employed for rats who did not
express DREADDs (SHAM group).
When all the behavioral sessions were completed, the animals were perfused and their brain
processed for histology. Images of the red nucleus and brainstem containing labelled
neurons were acquired using a confocal microscope (Zeiss) and a slide scanner (Huron
Digital Pathology).
36
Data analysis
All data analyses were carried out under Matlab (Mathworks).
Licking learning
In order to isolate the vibrissa positioning task learning, the vibrissa data was analyzed only
from the moment when rats had learnt to lick reliably upon water delivery. This moment was
defined as the trial before which the likelihood of licking occurrence in the 250 previous post-
reward pauses was higher than 80%. Licking had to start between 0 and 2 s following reward
delivery to be considered.
Vibrissa task learning
As per our behavioral protocol, required hold times do not interfere with the way the vibrissa
position is evaluated during intertrials; thus, intertrials’ mean angles can be compared over
learning. This is not the case for trials, given that, during trials, rats are not given the
possibility to protact their whisker in the reward zone for more than the required hold time,
a parameter which changes over learning. Intertrial pauses following successful trials
dedicated to licking were excluded from the analyses (vibrissa retraction typically
accompanies licking), as well as the 1-s pause following unsuccessful trials.
“False alarm” licks during the performance stage
For each “false alarm” lick, we computed the maximum hold time in the reward zone in the
1-s fragment preceding the lick’s occurrence. To compare these hold times between trials
and intertrials, we excluded from the analysis the licks occurring during trials, whose the 1-
s preceding window included intrusion in the go zone (as this would not have been possible
during intertrials, leading to their end).
Recovery from deafferentation
The session from which rats were considered to have recovered from deafferentation is the
first of the three consecutive sessions whose pooled success rate was not statistically
different from the pooled success rate of the last three sessions preceding deafferentation
(chi-squared test, p>.05). All subsequent analyses aimed at comparing steady-state
execution before and after deafferentation were carried out with the post-recovery data.
37
Spectral analyses
All spectral analyses were done using multitaper estimates (Kleinfeld and Mitra, 2014), as
implemented by the Chronux Matlab toolbox (Bokil et al., 2010).
At least 500-ms long whisking fragments were detected using spectrograms. The extraction
of whisking parameters (setpoint, amplitude and phase) was done, as previously described,
using the Hilbert transform (Hill et al., 2011; Moore et al., 2013).
All analyses on animal groups were weighted, so that every animal influences the final
measure equivalently. All error bars and shaded regions correspond to 95% confidence
intervals.
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Figure legends
Figure 1: Intact animals can learn the vibrissa positioning task
A: Scheme of the vibrissa positioning task. Rats are trained to move their vibrissa from a
retracted zone to a protracted zone, and maintain it in the reward zone for a given duration
(the “required hold time”) which adaptatively increases over training sessions.
B: Average vibrissa traces of successful hold times in the reward zone, throughout learning
(data from an illustrative rat).
C: Latencies between trials’ onset and the first at least 50 ms long holds in the reward zone
over learning.
D: Mean vibrissa angle over the 500 ms preceding trials’ onset over learning.
Figure 2: Sensory feedback is neither required before, nor after learning
A: Scheme of the deafferentation procedure. The infraorbital nerve is transected on the side
of the tracked vibrissa, the buccal and marginal branches of the facial nerve are transected
on the opposite side.
B: Mean angles over trials before and after deafferentation.
C: Empirical cumulative distribution of the maximum hold times in the reward zone across
trials, before and after deafferentation (each color represents a specific rat).
D: 10°-wide holds before and after deafferentation.
E: Traces of the vibrissa position during 1-sec successful hold times, before and after
deafferentation.
F: Angular standard deviation across 1-sec successful hold times, before and after
deafferentation.
Figure 3: Motor cortex is not required to learn the task
A: Empirical cumulative distribution of the maximum hold times in the reward zone across
trials in bilaterally decorticated rats, before and after deafferentation (each color represents
a specific rat).
B: Mean angles over trials in bilaterally decorticated rats, before and after deafferentation.
C: Traces of the vibrissa position during 1-sec successful hold times in bilaterally
decorticated rats, before and after deafferentation
D: Angular standard deviation across 1-sec successful hold times in bilaterally decorticated
rats, before and after deafferentation
Figure 4: Inactivation of the rubro-facial pathway disrupts performance
A: Fluorescent microscopy image of the parvocellular red nucleus and its surroundings
(PAG: periaqueductal gray; RPC: red parvocellular nucleus; RMC: red magnocellular
nucleus; ML: medial lemniscus; SNR: substantia nigra pars reticulata).
B: Empirical cumulative distribution of the maximum hold times in the reward zone across
trials in DREADDs and SHAM groups, before and after CNO administration (each color
represents a specific rat).
C: Mean angles over trials in DREADDs and SHAM groups, before and after CNO
administration.
39
D: Circuit diagram of rubrofacial neurons’ inputs and outputs.
Supplementary Figure 1 A: Likelihood of licking occurrence up to 2 seconds after reward delivery over successive trials (data from an illustrative rat). B: Average vibrissa traces of trial initiation, throughout learning (data from an illustrative rat).
C: At the performance level (required hold time: 1 sec), hold times in reward zone preceding “false alarm” licks. Supplementary Figure 2 A: Post-hoc assessment of the effectiveness of the infraorbital nerve lesion, through bilateral LFP recording in the vibrissa ventral posteromedial nucleus of the thalamus, during electrical stimulation of pad muscles. B: Power spectrum during 1-s successful hold times, before and after deafferentation. C: Average vibrissa traces of successful hold times in the reward zone throughout learning,
for a deafferented rat.
D: Latencies between trials’ onset and the first at least 50 ms long holds in the reward zone
over learning, for deafferented rats.
E: Mean vibrissa angle over the 500 ms preceding trials’ onset over learning, for deafferented rats. F: Empirical cumulative distribution of the maximum hold times in the reward zone across
trials, for rats deafferented before learning and for rats deafferented after learning (each
color represents a specific rat).
Supplementary Figure 3 A: Fluorescent microscopy images and pictures of a bilateral motor cortical lesion. B: Average vibrissa traces of successful hold times in the reward zone throughout learning,
for a bilaterally decorticated rat.
C: Latencies between trials’ onset and the first at least 50 ms long holds in the reward zone
over learning, for bilaterally decorticated rats.
D: Mean vibrissa angle over the 500 ms preceding trials’ onset over learning, for bilaterally decorticated rats. Supplementary Figure 4: A: Fluorescent microscopy images of collaterals of rubrofacial neurons. B: Success rates across trials at the performance level in DREADDs and SHAM groups,
before and after CNO administration, and the associated mixed-effect logistic regression
estimates.
C: At the performance level (required hold time: 1 sec), mixed-effect linear regression
estimates for hold times in reward zone preceding “false alarm” licks, in DREADDs and
SHAM groups, before and after CNO administration.
40
Figure 1: Intact animals can learn the vibrissa positioning task
eward zone
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Figure 2: Sensory feedback is neither required before, nor after learning
42
Figure 3: Motor cortex is not required to learn the task
43
Figure 4: Inactivation of the rubro-facial pathway disrupts performance
44
Supplementary Figure 1
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Supplementary Figure 2
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Supplementary Figure 3
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Supplementary Figure 4
48
Chapitre 2 A Vibrissal Pathway that Activates the
Limbic System
2.1 Résumé
Nous décrivons un circuit du tronc cérébral convertissant des entrées sensorielles
orofaciales en réactions émotionnelles. Des expériences de traçage viral chez les rongeurs
révèlent que les cellules sensibles aux vibrisses dans le sous-noyau interpolaris du
complexe trigéminal, qui donnent naissance à la voie paralemniscale, projettent
massivement vers le noyau réticulaire médullaire dorsal et vers la corne dorsale de la moelle
cervicale. Les neurones interpolaires projettent également vers le complexe Kölliker-
Fuse/parabrachial, traditionnellement associé aux réactions aversives et au contrôle des
fonctions respiratoires et cardiovasculaires. Les cellules parabrachiales qui reçoivent une
entrée de l’interpolaris, projettent vers les régions limbiques du cerveau antérieur, tandis
que les cellules Kölliker-Fuse donnent lieu à des projections descendantes vers les régions
autonomes, respiratoires et motrices de la moelle. Nous avons également découvert une
population distincte de cellules de Kölliker-Fuse qui ne reçoivent pas d'entrée de
l’interpolaris mais qui projettent vers les noyaux sensoriels du trijumeau. Les données
électrophysiologiques et de microscopie électronique suggèrent que ce dernier groupe de
cellules du Kölliker-Fuse contrôle le relais de l'entrée sensorielle dans la voie paralemniscale
par un mécanisme d'inhibition présynaptique.
2.2 Abstract
We describe a brainstem circuit for the conversion of orofacial sensory input into emotional
reactions. Viral tracing experiments in rodents reveal that vibrissa-responsive cells in the
interpolaris subnucleus of the trigeminal complex, which give rise to the paralemniscal
pathway, send a massive input to the dorsal medullary reticular nucleus and the dorsal horn
of the cervical cord. Interpolaris neurons also project to the Kölliker-Fuse/parabrachial
complex, traditionally associated with aversive reactions and the control of respiratory and
cardiovascular functions. Parabrachial cells that receive interpolaris input project to limbic
regions of the forebrain while Kölliker-Fuse cells give rise to descending projections to the
autonomic, respiratory, and motor regions of the medulla. We also found a separate
population of Kölliker-Fuse cells that do not receive interpolaris input but project to the
trigeminal sensory nuclei. Electrophysiological, and electron microscopic data suggest that
49
the latter pool of Kölliker-Fuse cells gate the relay of sensory input in the paralemniscal
pathway by a mechanism of presynaptic inhibition.
INTRODUCTION
Most sensory systems comprise parallel pathways of sensory information that encode
different features of a stimulus and take part in the control of sensor motion (Igarashi et al.,
2012; Lomber and Malhotra, 2008; Merigan and Maunsell, 1993; Nassi and Callaway, 2009;
Niu et al., 2013). The vibrissa system of rodents is no exception, as it comprises two main
trigeminothalamic pathways (reviewed in (Urbain and Deschênes, 2009)): (1) a lemniscal
pathway that arises from the trigeminal nucleus principalis (PrV), transits through the ventral
posterior medial nucleus (VPM) of the thalamus, and projects to the primary somatosensory
cortex; (2) a paralemniscal pathway that arises from the rostral part of trigeminal nucleus
interpolaris (SpVIr), transits through the posterior group (Po) of the thalamus, and projects
to the somatosensory cortical areas and to the vibrissa motor cortex.
In contrast with PrV cells, which innervate principally VPM thalamus, interpolaris cells that
project to Po thalamus also innervate a number of additional regions by means of branching
axons (Bellavance et al., 2017; Pierret et al., 2000; Veinante et al., 2000). These include the
superior colliculus, the anterior pretectal nucleus, the ventral division of zona incerta, and
the dorsal lateral sector of the facial nucleus. Tract tracing studies by means of classic
tracers also reported interpolaris projections to the brainstem and spinal cord (Jacquin et
al., 1986; Phelan and Falls, 1991). Whether the latter projections also arise from Po-
projecting cells remains an open question.
While the lemniscal pathway conveys tactile information and information about the relative
position (phase) of the vibrissae during whisking (Moore et al., 2015; Yu et al., 2006), the
role of the paralemniscal pathway remains puzzling. It was proposed that this pathway
conveys information about whisking kinematics (Yu et al., 2006), but later studies found that
encoding of whisking along the paralemniscal pathway is relatively poor (Moore et al., 2015;
Urbain et al., 2015). It was also proposed that the paralemniscal pathway is specifically
activated upon noxious stimulation (Frangeul et al., 2014; Masri et al., 2009), but it has never
been shown that interpolaris cells that respond to vibrissa deflection are also activated by
noxious stimuli. Thus the general function of the paralemnical pathway remains unresolved.
50
Here we used virus-based tract tracing methods and electrophysiology to document the full
extent of the collateral projections of interpolaris cells that give rise to the paralemniscal
pathway. We searched for new pathways of ascending information to forebrain regions as
well as for feedback projections to brainstem nuclei as a means to uncover the role of the
paralemniscal pathway in rat’s behavior.
RESULTS
Collateral Projections of Interpolaris Cells that Project to Po Thalamus
To map the collateral projections of interpolaris cells that innervate Po thalamus, we injected
the retrograde virus G-pseudotyped Lenti-Cre in Po thalamus, and AAV2/8-hSyn-DIO-GFP
in the vibrissa-responsive sector of SpVIr (5 juvenile rats). This approach revealed an
unexpected diversity of axonal projections (Figure 1). In accordance with prior studies,
terminal fields are found in Po thalamus, the ventral division of zona incerta, the anterior
pretectal nucleus, the superior colliculus, the perirubral region, the dorsal lateral part of the
facial nucleus, and in the other subdivisions of the sensory trigeminal complex (Bellavance
et al., 2017; Jacquin et al., 1989; Jacquin et al., 1986; Veinante et al., 2000). Profuse
projections are also found in the ipsilateral dorsal medullary reticular nucleus (MdD) and
further caudally in the dorsal horn of the cervical cord. An ascending projection terminates
ipsilaterally in the intertrigeminal region (ITr) and in the Kölliker-Fuse/parabrachial complex
(KF/PBc). Whether individual interpolaris cells project to each of the above-mentioned target
regions remains an open issue. What is clear, however, is that interpolaris cells that project
to Po broadcast sensory messages to multiple midbrain and hindbrain regions.
Injection of the anterograde virus AAV1-hSyn-eGFP-WPRE in the vibrissa-responsive
sector of the SpVIr (3 rats) yields similar results (Figure S1); yet in these cases anterograde
labeling is also found in the cerebellum. This is consistent with prior studies, which reported
that different populations of SpVI cells project to the thalamus and cerebellum (Jacquin et
al., 1986; Steindler, 1985). Of note, in sagittal sections of the brainstem, the terminal field of
interpolaris axons in KF/PBc covers an extensive crescent-shaped territory at the rostral
border of PrV and the trigeminal motor nucleus (Figure S1B).
51
KF/PBc and MdD Cells Respond to Vibrissa Deflection
Interpolaris cells that give rise to the paralemniscal pathway respond to vibrissa deflection
(Veinante et al., 2000). Thus, one expects KF/PBc and MdD neurons to also respond to
vibrissa deflection. We indeed found vibrissa-responsive neurons in the KF/PBc (latency:
8.0 ± 1.2 ms; mean ± SD; 22 cells) and in the MdD (latency: 6.0 ± 1.4 ms; mean ± SD; 33
cells) (Figure 2). The location of recorded units was assessed by single cell juxtacellular
labeling in the KF/PBc (8 cells), and by iontophoretic deposit of Chicago Sky Blue in the
MdD (3 rats). Like interpolaris cells, the receptive field of KF/PBc and MdD cells includes
multiple vibrissae. In KF/PBc, vibrissa-responsive cells are intermingled with cells that
discharge in phase with the respiratory rhythm. Yet the spontaneous activity of vibrissa-
responsive cells displays no statistically significant coherence with respiration (mean
coherence <C> in the spectral band of breathing is |<C>| = 0.16 for vibrissa-responsive cells
versus |<C>| = 0.72 for respiratory units (Figure 2D).
Axonal Projections of KF/PBc Cells that Receive Interpolaris Inputs
Among the targets of the paralemniscal pathway, we focused on the KF/PBc because of the
well-documented hodology of its axonal projections (see review by Saper, 2015). As a
means to visualize the axonal projections of KF/PBc cells that receive interpolaris input, we
injected AAV2/1 that expresses Cre in the vibrissa-responsive sector of the SpVI to achieve
anterograde transsynaptic expression of Cre (Zingg et al., 2017), and AAV-DIO-hChR2-
eYFP was injected in the KF/PBc (3 rats). We observed anterograde labeling in the lateral
part of the facial nucleus, throughout the ventral lateral medulla, the MdD, the nucleus of the
solitary tract, and the cervical cord (Figure 3). Ascending projections were found in the dorsal
raphe, the Edinger-Wesphal nucleus, the periaqueductal gray, the posterior lateral
hypothalamus, the paracentral and central medial thalamic nuclei, the parvicellular part of
the ventral posterior thalamic nucleus, the ventral division of zona incerta, the central
amygdala (CeA), and the lateral part of the bed nucleus of stria terminalis (Figure 3 and S2).
In summary, KF/PBc cells that receive interpolaris input in turn project to most of the
brainstem and forebrain regions previously identified by means of anterograde tracer
injections in the KF/PBc (Saper, 2015). Yet, it is worth noting that no anterograde labeling
is observed in the trigeminal sensory nuclei. This negative result is significant, as it indicates
that KF/PB cells that receive interpolaris input do not feed back to the relay cells that give
rise to the paralemniscal pathway (see below).
52
As PB cells that project to the CeA receive input from vibrissa-responsive SpVI neurons,
one expects to find vibrissa-evoked responses in the amygdala. We addressed this issue by
recording the local field potential evoked by air jet deflection of the vibrissae (Figure 4).
These experiments were carried out under urethane instead of ketamine/xylazine
anesthesia as 2 agonists (e.g. xylazine) strongly depress synaptic transmission in the
parabrachial-amygdaloid pathway (Delaney et al., 2007). Vibrissa deflection evoked a clear
negative field potential in the CeA (peak latency, 12 ms; Figure 4). This response was
abolished following electrolytic lesion of the ipsilateral PB (3 rats). Together, our anatomical
(Figure 3) and electrophysiological (Figure 4) data delineate a pathway of vibrissa
information that reaches the CeA and several limbic regions of the forebrain via a relay in
the KF/PBc.
Differential Projections of KF and PB Cells
Prior studies in mice have shown that separate pools of KF/PB neurons project to the
forebrain and to lower brainstem (Barik et al., 2018; Geerling et al., 2017). We thus examined
whether descending and ascending projections of KF/PBc in rats arise from different or
overlapping neurons. We made large injections (100 nl) of the retrograde labels retroAAV-
CAG-eGFP and retroAAV-CAG-mCherry in the CeA and the facial nucleus, respectively (2
rats; Figure 5 A-D). Although a number of retrogradely labeled cells were found in the
KF/PBc, none was doubly labeled. Amygdala-projecting cells (326 in rat 1, and 312 in rat 2)
were mainly concentrated in lateral and medial PB, while the majority of facial projecting
cells (299 in rat 1, and 140 in rat 2) were located in KF. In complementary experiments, we
injected retroAAV expressing Cre in the ventral respiratory group and AAV2/8-hSyn-DIO-
GFP in KF/PBc (2 rats). Anterograde labeling was observed in the facial nucleus, the ventral
respiratory group and the MdD (Figure S3), but not in midbrain and forebrain regions.
Together these results confirm that neighboring but separate populations of KF/PB neurons
project to the forebrain and to the lower brainstem.
GABAergic KF cells gate sensory transmission in the SpVI
In rodents, KF projection to the brainstem arises from two neuronal populations:
glutamatergic cells that project to the autonomic, respiratory, and motor regions of the
medulla, and GABAergic neurons, which project principally to the SpVI (Geerling et al.,
2017). To determine whether GABAergic KF cells inhibit interpolaris cells, we made large
injections (200 nl) of anteroAAV-expressing Cre in KF/PB and AAV-Ef1a-Dio-eYFP in the
53
SpVI (3 rats). Unexpectedly, few interpolaris neurons were labeled (n = 44, 48, 53
respectively in each of the rats). Anterograde labeling was observed in the KF/PB complex
and in the parvicellular part of the ventral posterior thalamic nucleus, a region known to
process input from the tongue and oral cavity (Ogawa and Nomura, 1988; Verhagen et al.,
2003). No labeling was observed in the cerebellum, or in brainstem and midbrain regions
that receive input from the paralemniscal pathway (cf. Fig. 1). Yet, labeled cells were present
in the parvicellular reticular formation at the medial border of the trigeminal nuclei, which
indicates that the paucity of cell labeling in SpVI is hardly ascribable to a technical pitfall.
Together these results indicate that projections from the KFn do not innervate the SpVI
interneurons or the projection cells that give rise to the paralemniscal pathway.
To further assess these anatomical results, we injected AAV-hSyn-ChR2-eYFP in KF and
used an optrode to concurrently stimulate KF axons and record the activity of SpVI neurons.
Interpolaris cells either discharged spontaneously or were driven by jiggling a single vibrissa
with a piezoelectric stimulator. Optogenetic stimulation suppressed sensory-evoked
discharges in monovibrissa-responsive cells (n=26) but did not affect the firing of
multivibrissa-responsive units (n=32). Together with the lack of transsynaptic labeling of
local circuit cells from the KFn, these results raise the possibility that KF-induced
suppression of sensory responses in local circuit interpolaris cells is mediated by
presynaptic or extrasynaptic inhibition.
DISCUSSION
The present study reveals a hitherto unsuspected diversity of axonal projections of
interpolaris cells that give rise to the paralemniscal pathway. We identified two new collateral
projections that target the MdD and the KF/PBc. Furthermore, we show that the KF/PBc
contains two populations of vibrissa-responsive neurons; one gives rise to an ascending
pathway, which projects to limbic regions of the forebrain, and the other one projects to the
autonomic, respiratory, and motor regions of the medulla. Lastly, KF contains a pool of
GABAergic neurons, which do not receive interpolaris input, but project locally and to the
trigeminal sensory nuclei. The diagram in Figure 8 summarizes the first-order axonal
projections of interpolaris cells that give rise to the paralemniscal pathway, and the second-
order projections that derive from the KF/PBc.
54
Descending Projection to the MdD
By far the most abundant collateral projection of vibrissa-responsive interpolaris cells is to
the MdD and the dorsal horn of the cervical cord. The finding of vibrissa-responsive cells in
the MdD is unexpected as most studies reported that MdD neurons best respond to noxious
stimuli (reviewed in Martins and Tavares, 2017). Although there is no reason to believe that
vibrissa deflection per se is painful, an unexpected air puff directed toward the head of a rat
elicits fear-related behavior such as a startle response (Engelmann et al., 1996), 22 kHz
ultrasonic vocalization (Knapp and Pohorecky, 1995) and avoidance behavior (Cimadevilla
et al., 2001). As the MdD sends profuse projections to the cervical cord and the facial
nucleus (Bernard et al., 1990; Takatoh et al., 2013), these results suggest that vibrissa
messages to the MdD may elicit avoidance or flight behaviors. This conclusion is in line with
that of the recent study, which reported that the MdD is part of a brainstem-spinal circuit to
control escape responses to noxious stimuli (Barik et al., 2018).
The Trigemino-Parabrachial Amygdaloid Pathway
A number of studies have reported trigeminal projections to the KF and PB subnuclei
(Cechetto et al., 1985; Chamberlin, 2004; Dallel et al., 2004; Feil and Herbert, 1995;
Rodriguez et al., 2017; Slugg and Light, 1994). Most of these studies focused on ascending
projections from subnucleus caudalis, which convey pruriceptive and nociceptive inputs to
the KF/PBc (Jansen and Giesler, 2015). Thus, it came as a surprise to find vibrissa-
responsive neurons in KF/PBc, and further find that vibrissa signals are conveyed to the
limbic regions of the forebrain via the medial and lateral PB. One study previously reported
short-latency (about 11 ms) activation of amygdala neurons upon electrical stimulation of
the mystacial pad (Bernard et al., 1992). Such a short latency is consistent with the activation
of a trigemino-parabrachial-amygdaloid pathway by A primary vibrissa afferents.
The Paralemniscal Pathway and Threat
Although vibrissa-responsive interpolaris cells do not respond to noxious stimuli, many of
the regions they innervate contain cells that process nociceptive or aversive inputs. This is
the case for the MdD (reviewed in (Martins and Tavares, 2017)), the KF/PBc (Jansen and
Giesler, 2015), the superior colliculus (Dean et al., 1989; Redgrave et al., 1996), the zona
incerta (Masri et al., 2009), and Po thalamus (Frangeul et al., 2014; Masri et al., 2009;
Sobolewski et al., 2015). These anatomical data suggest that the paralemniscal pathway
55
conveys signals from threatening or alarming sensory inputs, perhaps through association
with aversive contexts.
In light of the evidence that interpolaris cells innervate brain regions that process nociceptive
or aversive stimuli, we postulate that the KF is primarily involved in orchestrating defensive
reactions to aversive and threatening stimuli. Four additional lines of evidence support this
hypothesis. First, the KF projects to the cardio-respiratory medullary centers, the facial,
hypoglossal and ambiguous motor nuclei, the nucleus of the solitary tract, and to the
preganglionic neurons of the sympathetic nervous systems (reviewed in Saper and
Stornetta, 2015). Second, activation of KF cells elicits changes in respiration, heart rate,
blood pressure (Chamberlin, 2004; Chamberlin and Saper, 1992; Guo et al., 2002; Lara et
al., 1994), and contraction of facial muscle. Third, individual KF neurons project to many of
the above-mentioned targets by means of branching axons, which might form the anatomical
substrate for coordinating autonomic and somatic reactions (Song et al., 2012).
Conclusion
Our results clearly indicate that the paralemniscal pathway broadcasts vibrissa-based
sensory signals to a number of brainstem and forebrain regions that are involved in the
expression of emotional reactions. They provide a different viewpoint on the role of the
Kollliker-Fuse nucleus, which is currently considered to control the mechanics of respiratory
and cardiac rhythms, but no more. We propose that the Kollliker-Fuse nucleus is involved in
the larger role of orchestrating the expression of emotional reactions, e.g., facial
expressions, autonomic and somatic reactions, of which respiration is one of many players.
ACKNOWLEDGMENTS
We thank Fan Wang for critically reading the manuscript and sharing retrograde-Lenti-Cre.
This work was supported by grants from the Canadian Institutes of Health Research (grant
MT-5877) and the National Institutes of Health (U19 NS107466 and R35 NS097265).
AUTHOR CONTRIBUTIONS
D.K. and M. Deschênes supervised the project. M.E. and A.C-P. performed the experiments.
M. E. and M. Demers analyzed data. M.E., D.K. and M. Deschênes wrote the manuscript.
56
DECLARATION OF INTEREST
The authors declare no competing interests
FIGURE LEGENDS
Figure 1. Axonal Projections of Interpolaris Cells that Give Rise to the Paralemniscal
Pathway
(A) Viral method used for labeling paralemniscal projections.
(B) Labeling of interpolaris cells after injection of G-pseudotyped Lenti-Cre virus in Po
thalamus, and a Cre-dependent AAV that expresses GFP in the vibrissa-responsive sector
of SpVIr.
(C) Anterograde labeling of terminal fields in Po thalamus and zona incerta.
(D) Anterograde labeling in KF/PB.
(E) Anterograde labeling in the ITr.
(F) Anterograde labeling in the dorsal sector of the facial nucleus.
(G) Anterograde labeling in the MdD and cervical cord.
See Figure S1 for additional data. Abbreviations: 5N, trigeminal motor nucleus; 7n, facial
nerve tract; ITr, intertrigeminal region; KF/PB, Kolliker-Fuse/parabrachial complex; MdD,
dorsal part of the medullary reticular formation; Po, posterior nuclear group of the thalamus;
PrV, principal trigeminal nucleus; VPM, ventral posterior medial nucleus; SpVC, caudalis
division of the spinal trigeminal complex; SpVIr, rostral division of the interpolaris nucleus;
ZIv, ventral division of zona incerta.
Figure 2. Vibrissa-Evoked Responses in KF and MdD
(A) Population peristimulus time histogram of spike discharges evoked in KF (22 cells) by
air puff deflection of the vibrissae. A representative response is shown in the insert.
57
(B) Example of a vibrissa responsive KF cell labeled by juxtacellular delivery of Neurobiotin.
(C) Location of 8 juxtacellularly labeled KF cells. Horizontal brainstem sections in (B and C)
were counterstained for cytochrome oxidase.
(D) Coherence of spontaneous discharges of KF cells with the respiratory cycle. Note that,
in contrast with the respiratory units (blue dots), spontaneous discharges of vibrissa-
responsive cells (red triangles) display low coherence with respiration.
(E) Population peristimulus time histogram of spike discharges evoked in MdD (33 cells) by
air puff deflection of the vibrissae.
(F) Recording site in the MdD labeled by an iontophoretic injection of Chicago Sky Blue.
This coronal section was counterstained for cytochrome oxidase and a negative image was
generated.
Abbreviations: 5N, motor trigeminal nucleus; KF, Kolliker-Fuse nucleus; mcp, middle
cerebellar peduncle; MdD, dorsal part of the medullary reticular formation; MdV, ventral part
of the medullary reticular formation; PrV, principal trigeminal nucleus; s5, sensory root of the
trigeminal nerve; Sol, nucleus of the solitary tract; SpVC, caudalis division of the spinal
trigeminal complex; VLL, ventral nucleus of the lateral lemniscus.
Figure 3. Axonal Projections of KF/PB Cells that Receive Interpolaris Input
(A) AAV2/1-hSyn-Cre-WPRE was injected in the vibrissa-responsive sector of the SpVIr,
and AAV2/1-EF1a-DIO-hChR2-eYFP was injected in KF/PB. This parasagittal section
shows labeling in KF/PB. The section was immunostained for choline acetyltransferase. The
green and red framed areas are enlarged in (B) and (C).
(D) Terminal labeling in the ventral respiratory group underneath the ambiguus nucleus.
(E) Anterograde labeling in the central amygdala.
(F) Anterograde labeling in the paracentral and central medial thalamic nuclei.
58
(G) Anterograde labeling in the parvocellular division of the ventral posterior medial nucleus
of the thalamus (see Figure S2 for additional projection sites).
Frontal sections in (E-G) were counterstained for cytochrome oxidase. Abbreviations: 5n,
root of the trigeminal motor nucleus; 7N, facial nucleus; 7n, facial nerve tract; Amb,
ambiguus nucleus; CeA, central amygdala; CM, central medial thalamic nucleus; mt,
mammillothalamic tract; PC, paracentral thalamic nucleus; PrV, principal trigeminal sensory
nucleus; SpVI, interpolaris division of the spinal trigeminal complex; VPPc, parvocellular part
of the ventral posterior thalamic nucleus; VRG, ventral respiratory group.
Figure 4. Vibrissa-Evoked Responses in Central Amygdala
(A) Average response (n = 50) evoked in the CeA by air puff stimulation of the vibrissae
before (red trace) and after (black trace) an electrolytic lesion of the PB complex.
(B) Recording site in the amygdala labeled by an iontophoretic injection of Chicago Sky Blue
(cytochrome oxidase counterstaining).
(C) Electrolytic lesion of the PB complex.
Abbreviations: 5N, trigeminal motor nucleus; CeA, central amygdala; PB, parabrachial
nuclei; scp, superior cerebellar peduncle.
Figure 5. Separate Cellular Populations in KF/PB Project to the CeA and the Facial
Nucleus
(A) Injection site of retroAAV-CAG-eGFP in the CeA.
(B) Injection site of retroAAV-CAG-mCherry in the facial nucleus.
(C) Retrogradely labeled cells in KF/PB.
(D) 3D reconstruction showing the distribution of amygdala-projecting cells (green dots) in
the medial and lateral PB, and facial-projecting neurons in the KF (magenta dots). Note that
59
a few cells in lateral PB project to the facial nucleus; yet, none of the KF/PB cells is doubly
labeled.
(E) Wiring diagram of the projections of KF and PB cells that receive vibrissa input from the
interpolaris nucleus (see also Figure S3 for additional evidence). Abbreviations: 5N,
trigeminal motor nucleus; 5n, motor root of the trigeminal nucleus; 7N, facial motor nucleus;
7n, facial nerve tract; CeA, central amygdala; CPu, caudate putamen; opt, optic tract; scp,
superior cerebellar peduncle.
Figure 6. Transsynaptic Labeling of Brainstem Cells that Receive Input from the KFn
The viral method used to label cells that receive input from the KFn is shown in (A). (B)
Transsynaptic labeling in SpVI is restricted to a small population of cells (C) which project
to VPCC (B). Yet, numerous cells are transsynaptically labeled in the parvicellular reticular
formation (D). Abbreviations: 5t, trigeminal tract; Hab, habenula; PCRT, parvicellular
reticular formation; Po, posterior group of the thalamus; PrV, principal trigeminal sensory
nucleus; Rt, reticular thalamic nucleus; SpVC, caudalis division of the trigeminal sensory
nuclei; SpVIc, caudal sector of the interpolaris trigeminal nucleus; VPL, ventral
posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; VPCC,
parvicellular sector of the ventral posteromedial thalamic nucleus.
Figure 7. Optogenetic Stimulation of KF Axons Inhibits Vibrissa-Evoked Responses
in Monovibrissa-Responsive Interpolaris Cells
Injection of AAV2/1-hSyn-hChR2-eYFP in KF results in anterograde labeling in SpVI (A).
The framed region in A is shown in B; arrowheads point to labeled cell bodies. Note the
dense network of axon collaterals within the KF/PB complex. (C) Representative responses
of a monovibrissa-responsive cell (biphasic unit) and a multivibrissa-responsive cell (positive
unit) upon optogenetic stimulation of KF axons. (D) Population peristimulus time histogram
60
of responses of 26 monovibrissa-responsive units upon optogenetic stimulation of KF axons.
Abbreviations: KF, Kölliker-Fuse nucleus; PrV, principal trigeminal sensory nucleus; SpVI,
interpolaris division of the trigeminal sensory nuclei; vsc, ventral spinocerebellar tract.
Figure 8. Summary of the First-Order Axonal Projections of Interpolaris Cells, and the
Second-Order Projections that Derive from the KF/PB Complex
(A) Projection sites in the framed area are from prior studies (see review by Saper and
Stornetta, 2014). Abbreviations: APT, anterior pretectal nucleus; BSTL, bed nucleus of the
stria terminalis; CeA, central amygdala; Cerv Cord, cervical cord; CM/PC, central
medial/paracentral thalamic nuclei; DR, dorsal raphe; EW, Edinger-Westphal; IML,
intermedio-lateral column of the spinal cord; ITr, intertrigeminal nucleus; KF, Kölliker-Fuse;
MdD, medullary reticular nucleus dorsal part; NA, nucleus ammbiguus; NTS, nucleus of the
solitary tract; PB, parabrachial nuclei; PLH, posterior lateral hypothalamus; PAG,
periaqueductal gray; Po. posterior group of the thalamus; RN, red nucleus; SC, superior
colliculus; SpVIr, rostral division of the interpolaris nucleus; TG, trigeminal ganglion; VPCC,
ventral posterior thalamic nucleus (parvicellular part); VRG, ventral respiratory group; ZIv,
ventral division of zona incerta.
EXPERIMENTAL PROCEDURES
Animals
Experiments were carried out in Long Evans rats of both sexes (250-350 g in mass),
according to the National Institutes of Health Guidelines. All experiments were approved by
the Institutional Animal Care and Use Committee at Laval University and the University of
California at San Diego.
Viruses
61
G-pseudotyped Lenti-Cre virus was designed and produced as described (Stanek et
al., 2016). AAV2/8-hSyn-DIO-GFP and AAV-Flex-ChR2-mCherry were obtained from the
Neurophotonics Centre, Laval University. AAV2/1-EF1a-DIO-hChR2-EYFP-WPRE-
HGHpA, AAV1-hSyn-Cre WPRE, AAV1-hSyn-eGFP-WPRE, retroAAV-CAG-tdTomato,
retroAAV-CAG-eGFP, retroAAV-pmSyn1-EBFP-Cre, and AAV5-hSyn-Flex-ChrimsonR-
tdTomato were obtained from Addgene.
Surgery and virus injections
All, but three rats were anesthetized with ketamine (75 mg/kg) and xylazine (5
mg/kg), and body temperature was maintained at 37 C with a thermostatically controlled
heating pad. Three rats were anesthetized with urethane (1.4 g/kg) to record the local field
potential evoked in the amygdala by vibrissa deflection. All virus injections were carried out
after electrophysiolological identification of the target regions. When AAV injections were
made in the interpolaris nucleus, we targeted the lateralmost sector of the nucleus based on
the well-known somatotopic representation of the vibrissae in this nucleus. This precaution
proved useful to avoid infecting cells in neighboring regions. Viruses were pressure injected
(40-100 nl) using micropipettes of 15-20 m tip diameter. After 3 to 4 weeks of survival
animals were either perfused with saline and paraformaldehyde (4% (v/v) in PBS), or
anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg) for optogenetic stimulation.
In three rats the vibrissa-responsive sector of the KF/PB was first located by
electrophysiological recording, and then lesioned by passing 0.5 mA through a stainless
steel electrode with a tip (10 m) de-insulated over a length of 500 m.
Electrophysiology
Single unit recordings were carried out in KF/PB and MdD with micropipettes (tip
diameter, 1 m) filled with either 0.5 M potassium acetate and 2% (w/v) Neurobiotin, or 0.5
M potassium acetate and 4% (w/v) Chicago Sky Blue. To maximize the chance of cell
recovery after juxtacellular labeling in the KF/PB, we injected one cell per animal and
perfused the rat thereafter. Vibrissae were deflected in the dorsocaudal direction with air-
puffs. The time delay between the command voltage and the onset of vibrissa deflection
was measured with a piezoelectric film positioned at the same distance from the puffer tip.
Respiration was monitored with a cantilevered piezoelectric film (LDT1 028K; Measurement
Specialties) resting on the rat’s abdomen just caudal to the torso. To monitor vibrissa motion
62
we used a Basler A602f camera operated in line-scan mode with a 1-kHz scan rate. All
signals were sampled at 10 kHz and logged on a computer using the Labchart acquisition
system (AD Instruments).
Glutamate stimulation of KF/PB neurons
Glass micropipettes with an outer tip diameter of 10 - 15 m were used to pressure-
inject glutamate (10 mM in PBS) in KF/PB. Injections were carried out after locating the
vibrissa-responsive sector of KF/PB. The injected volume (20 - 40 nl) was estimated by
measuring the movement of the meniscus with an operating microscope. At the end of each
experiment another pipette filled with 4% (w/v) Chicago Sky Blue in 0.5 M potassium acetate
was lowered at the very same location to label the glutamate injection site by iontophoresis
(negative current pulses of 4 A, 2 s duration, half duty cycle for 5 min).
Optogenetic stimulation in alert head-restrained rats
On the day of virus injections, a head-restraining post was secured to the skull with
screws and acrylic cement, and a NTS thermistor (MEAS-G22K7MCD419, Measurement
Specialties) was positioned over the nasal epithelium to record respiration (McAfee et al.,
2016). A fiber-optic cannula (OD, 125 m; core diameter, 100 m; NA, 0.22; Doric Lens)
was positioned about 200 m above the site of virus injection in KF/PB, and fixed in place
with acrylic cement. Three weeks after virus injections rats were head restrained and
recordings were performed under near-infrared illumination (760 nm). Optogenetic
stimulation was carried out with laser diode fiber light sources (473 nm for ChR2, Doric
Lenses; 637 nm for Chrimson, Coherent). Stimulus parameters were as follow: pulse
duration, 5 - 10 ms; frequency, 50 - 100 Hz; train duration 300 - 800 ms; intensity 2 - 5 mW.
A high speed video camera (HiSpec 2G; Fastec Imaging) was used to record nose and
vibrissa motion at a rate of 500 fps. A 45 degree mirror installed below the rat enabled
tracking nose motion in all planes.
Histology
Following perfusion brains were postfixed for 1 h, and cryoprotected overnight in 30%
(w/w) sucrose in PBS. Brains were cut at thickness of 50 µm on a freezing microtome.
Labeled material was processed for either fluorescence or brightfield microscopy. For
fluorescence microscopy, sections were immunoreacted with a chicken anti-GFP antibody
63
(1:1000; Abcam), and a donkey anti-chicken Alexa 488 IgG (Abcam). NeuN immunostaining
was carried out with a rabbit anti-NeuN antibody (1:1000; EM Millipore) and an anti-rabbit
IgG conjugated to Alexa 594. For brightfield microscopy, sections were first counterstained
for cytochrome oxidase, and then immunoreacted with a rabbit anti-GFP antibody (1:1000;
Novus Biological), a biotinylated anti-rabbit IgG (1:200: Vector Labs), the avidin/biotin
complex (Vectastain ABC kit, Vector Labs), and the SG peroxidase substrate (Vector Labs).
In three rats brainstem sections were first immunoreacted with a goat anti-choline
acetyltransferase antibody (1:1000; Millipore Sigma) and a rabbit anti-goat IgG conjugated
to horseradish peroxidase (Vector Labs), which was revealed with diaminobenzidine (brown
reaction product). Sections were then immunoreacted with a mouse anti-GFP antibody
(1:2000; Abcam), a biotinylated anti-mouse IgG, which was revealed with streptavidin
horseradish peroxidase conjugate and the Ni-DAB substrate (black reaction product).
Finally, the extent of electrolytic lesions was assessed on material stained with Neutral Red.
Sections were scanned at a resolution of 0.5 µm/pixel (TissueScope LE; Huron Digital
Pathology), and imported in Fiji or Photoshop for color and contrast adjustments.
Data analysis
Three-dimensional maps of retrogradely labeled KF/PB cells were constructed with
the Neurolucida software (Microbrightfield).
Peristimulus time histograms were built using the LabChart 8.0 spike histogram
module (AD Instruments), and the Matlab Chronux toolbox (http://www.chronux.org) was
used to compute the spectral coherence between respiration and the firing rate of KF/PB
cells.
Vibrissa and nose movements were extracted from video recordings using home
scripts in Matlab.
SUPPLEMENTAL INFORMATION
Figure S1. Interpolaris cells project to KF/PB and MdD (Supplementary information related
to Figure 1).
(A) Injection site of AAV1-hSyn-eGFP-WPRE-hgh in SpVIr.
64
(B) Anterograde labeling in KF/PB.
(C) Anterograde labeling in the MdD. The framed region is enlarged in (D).
(E) Terminal field in the dorsal lateral sector of the facial nucleus.
(F) Anterograde labeling in the cerebellum.
Sections were immunostained for NeuN (A-E), or counterstained with DAPI (F).
Abbreviations: icp, inferior cerebellar peduncle; LRT, lateral reticular nucleus; SpVC,
caudalis division of the spinal trigeminal complex.
Figure S2. Additional projection sites of KF/PB cells that receive interpolaris input
(Supplementary information related to Figure 3).
(A-D) AAV2/1-hSyn-Cre-WPRE was injected in the vibrissa-responsive sector of the SpVIr,
and AAV2/1-EF1a-DIO-hChR2-eYFP was injected in KF/PB. Anterograde labeling is
present in the posterior lateral hypothalamus (A), the Edinger-Westphal nucleus (B), the
parvocellular division of the red nucleus (C), and in the dorsal raphé and ventral lateral
periaqueductal gray (D). Sections were counterstained for cytochrome oxidase.
Abbreviations: 3N, oculomotor nucleus; cp, cerebral peduncle; DR, dorsal raphe; EW,
Edinger-Westphal nucleus; PAG, periaqueductal gray; PLH, posterior lateral hypothalamus;
RMC, red nucleus magnocellular part; RPC, red nucleus parvocellular part; STh,
subthalamic nucleus.
Figure S3. Labeling of KF cells that project to lower brainstem (Supplementary information
related to Figure 4).
(A) Labeling of KF cells after injection of retroAAV-pmSyn1-EBFP-Cre in the ventral
respiratory group, and AAV2/8-hSyn-DIO-GFP in the KF/PB. Note the absence of
anterograde labeling in the sensory trigeminal nuclei.
(B) Anterograde labeling in the lateral part of the facial nucleus and in the ventral respiratory
group.
65
No anterograde labeling is observed in midbrain or forebrain regions. Horizontal sections
were immunostained for NeuN. Abbreviations: 5N, trigeminal motor nucleus; 7N, facial
nucleus; 7n, facial nerve tract; Amb, ambiguous nucleus; ITr, intertrigeminal region; SpVI,
interpolaris division of the spinal trigeminal complex; VRG, ventral respiratory group.
66
Figure 1:
Axonal Projections of Interpolaris Cells that Give Rise to the Paralemniscal Pathway
67
Figure 2: Vibrissa-Evoked Responses in KF and MdD
68
Figure 3: Axonal Projections of KF/PB Cells that Receive Interpolaris Input
69
Figure 4: Vibrissa-Evoked Responses in Central Amygdala
70
Figure 5:
Separate Cellular Populations in KF/PB Project to the CeA and the Facial Nucleus
71
Figure 6: Transsynaptic Labeling of Brainstem Cells that Receive Input from the KF
72
Figure 7:
Optogenetic Stimulation of KF Axons Inhibits Vibrissa-Evoked Responses in
Monovibrissa-Responsive Interpolaris Cells
73
Figure 8:
Summary of the First-Order Axonal Projections of Interpolaris Cells, and the
Second-Order Projections that Derive from the KF/PB Complex
74
Supplementary Figure 1:
Interpolaris cells project to KF/PB and MdD
(Supplementary information related to Figure 1)
75
Supplementary Figure 2:
Additional projection sites of KF/PB cells that receive interpolaris input
(Supplementary information related to Figure 3)
76
Supplementary figure 3:
Labeling of KF cells that project to lower brainstem
(Supplementary information related to Figure 4)
77
Conclusion
Que des causes et mécanismes distincts mènent à des mouvements semblables, et que
des stimuli semblables soient interprétés diversement selon le contexte comportemental :
ce sont là les potentialités d’un réseau dont les multiples voies parallèles sensorielles,
motrices et sensori-motrices accèdent à la même information sensorielle, ou bien
communiquent avec les mêmes motoneurones, mais en vertu de principes distincts.
L’interaction de cette cohorte de régions en termes computationnels reste à définir. In fine,
nous déduirons de nos études que :
1) Les rats peuvent finement apprendre à contrôler la position de leurs vibrisses à la
faveur d’un modèle interne ne requérant pas même le cortex moteur. Cela
approfondit encore le mystère qui entoure cette région corticale de la lésion de
laquelle les rongeurs se remettent invariablement, sans déficits manifestes.
Puisqu’ils permettent de distinguer activités ré-afférentes d’efférentes, le modèle des
vibrisses et notre modèle expérimental ouvrent la voie à une meilleure
compréhension des déterminants du modèle interne, notamment des interactions
fondamentales prévalant entre ses composantes.
2) Les vibrisses ne servent pas qu’à l’exploration : les projections de régions répondant
aux vibrisses vers le système limbique en sont un témoignage anatomique. Il
demeure, par des manipulations spécifiques de ces projections en divers contextes
comportementaux, à en cerner l’implication physiologique.
Par ces études visant le même système sensorimoteur mais envisagé dans son étonnante
diversité fonctionnelle, nous voulions rendre hommage à la polysémie vibrissale. Nous
reconnaîtrons toutefois que nos études relèvent strictement de ce que David Chalmers avait
qualifié de « problèmes faciles », par opposition à celui, à notre connaissance inentamé, du
« problème difficile », celui du contenu phénoménal des expériences (qualias). Que plus
d’un demi-siècle d’études scientifiques, « à la troisième personne », du système des
vibrisses, ne nous permette un tant soit peu d’apprécier ce que cela fait que d’explorer le
monde par l’intermédiaire de moustaches en mouvement, pourrait avoir de quoi interpeller
même les plus optimistes partisans d’un programme fort des sciences cognitives. Pour
comprendre ce qui sous-tend la possibilité que les mots que nous agençons désormais à
peine mieux que les traducteurs automatiques contrairement à ces derniers nous parlent, le
78
principal reste encore à penser et à faire. Pour peu que ce problème, qui nous apparaît
principal, ne soit occulté par la communauté neuroscientifique.
79
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