title: Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.
abstract: Precise control of speed during locomotion is essential for adaptation of behavior in different environmental contexts [1-4]. A central question in locomotion lies in understanding which neural populations set locomotor frequency during slow and fast regimes. Tackling this question in vivo requires additional non-invasive tools to silence large populations of neurons during active locomotion. Here we generated a stable transgenic line encoding a zebrafish-optimized botulinum neurotoxin light chain fused to GFP (BoTxBLC-GFP) to silence synaptic output over large populations of motor neurons or interneurons while monitoring active locomotion. By combining calcium imaging, electrophysiology, optogenetics, and behavior, we show that expression of BoTxBLC-GFP abolished synaptic release while maintaining characterized activity patterns and without triggering off-target effects. As chx10(+) V2a interneurons (V2as) are well characterized as the main population driving the frequency-dependent recruitment of motor neurons during fictive locomotion [5-14], we validated our silencing method by testing the effect of silencing chx10(+) V2as during active and fictive locomotion. Silencing of V2as selectively abolished fast locomotor frequencies during escape responses. In addition, spontaneous slow locomotion occurred less often and at frequencies lower than in controls. Overall, this silencing approach confirms that V2a excitation is critical for the production of fast stimulus-evoked swimming and also reveals a role for V2a excitation in the production of slower spontaneous locomotor behavior. Altogether, these results establish BoTxBLC-GFP as an ideal tool for in vivo silencing for probing the development and function of neural circuits from the synaptic to the behavioral level.
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France.
authors.forename: Jenna R
authors.initials: JR
authors.lastname: Sternberg
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France.
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Severi
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France.
authors.forename: Kevin
authors.initials: K
authors.lastname: Fidelin
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France.
authors.forename: Johanna
authors.initials: J
authors.lastname: Gomez
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France.
authors.forename: Yara
authors.initials: Y
authors.lastname: Alcheikh
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Hubbard
authors.affiliation: Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Shizuoka 411-8540, Japan. Electronic address: kokawaka@nig.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
authors.affiliation: Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Shizuoka 411-8540, Japan; Neural Circuits and Behaviour Group, Uni Research AS, Thormølensgate 55, Bergen 5008, Norway.
authors.forename: Maximiliano
authors.initials: M
authors.lastname: Suster
authors.affiliation: Institut du Cerveau et de la Moelle Épinière (ICM), 47 Boulevard de l'Hôpital, 75013 Paris, France; INSERM UMRS 1127, 75013 Paris, France; CNRS UMR 7225, 75013 Paris, France; UPMC Université Paris 06, 75005 Paris, France. Electronic address: claire.wyart@icm-institute.org.
authors.forename: Claire
authors.initials: C
authors.lastname: Wyart
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000254:growth & development / Q000502:physiology; D001905:Botulinum Toxins / Q000494:pharmacology*; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000502:physiology; D007395:Interneurons / Q000502:physiology; D008124:Locomotion / Q000187:drug effects* / Q000502:physiology; D009498:Neurotoxins / Q000494:pharmacology*; D013550:Swimming / Q000502:physiology*; D015027:Zebrafish / Q000196:embryology / Q000254:growth & development / Q000502:physiology*
chemical_list: D009498:Neurotoxins; D001905:Botulinum Toxins
keywords: V2a interneurons; botulinum toxin; chx10; locomotion; zebrafish