Periodic Reporting for period 1 - FunCoSpeedSpine (Mapping the functional connectome for speed control in spinal motor circuits)
Berichtszeitraum: 2018-02-01 bis 2020-01-31
Locomotion, be it by land, air or sea enables all creatures to navigate their environment. Animals move at a diverse speed to search for mates, catch prey and escape predation. A key aspect of this fundamental behavioural repertoire is the selection of an appropriate gait and speed that would guarantee the successful completion of the task at hand. While the decision to perform an action is initiated in the brain, the precise timing of motor neuron recruitment relies on neuronal networks in the spinal cord. Within the spinal cord, descending inputs are first parsed by assemblies of premotor interneurons, which convey correct patterns of excitation to pools of motor neurons innervating axial and limb muscles. An evolutionarily conserved source of ipsilateral excitatory premotor drive arises from cells called V2a neurons. These cells express the ChX10 transcription factor and have been described in terrestrial and aquatic vertebrates. V2a neurons are found both in the hindbrain and along the entire span of the spinal cord where they make direct connections with motor neurons and modulate the speed of locomotor actions.
In this project, we aimed to use a combination of in vivo electrophysiology recordings, functional calcium imaging of network activity together with state of the art 3D sculped optogenetic stimulation restricted to single cells in vivo. Specifically, we wanted to tackle the following questions:
1. What are the synaptic connections among V2as and between V2a and MNs within the slow and fast range?
2. What is the degree of convergence and divergence between V2a neurons and MNs?
3. Finally, do supraspinal inputs to the spinal cord segregate based on speed?
Performing this work would give us access to an unprecedented level of detail of how locomotor speed modules function in vivo, and would lead to a better understanding of the basic biological networks that allow adaptive locomotor flexibility.
In this project, we aimed to use a combination of in vivo electrophysiology recordings, functional calcium imaging of network activity together with state of the art 3D sculped optogenetic stimulation restricted to single cells in vivo. Specifically, we wanted to tackle the following questions:
1. What are the synaptic connections among V2as and between V2a and MNs within the slow and fast range?
2. What is the degree of convergence and divergence between V2a neurons and MNs?
3. Finally, do supraspinal inputs to the spinal cord segregate based on speed?
Performing this work would give us access to an unprecedented level of detail of how locomotor speed modules function in vivo, and would lead to a better understanding of the basic biological networks that allow adaptive locomotor flexibility.
The crux of the proposed project was based on having access to efficient optogenetic actuators. For this reason, I have performed an in-depth characterisation and calibration of most opsin excitatory and inhibitory transgenic lines. This work represents the first in vivo calibration of optogenetic tools with precise electrophysiological methods and has been published earlier in 2020 in Elife. The unexpected complexity of the calibration of the optogenetic methods, delayed the start of data collection to directly address our original aims. However, these questions were in fact addressed by other colleagues in the field via three recent and standout publications from the McLean, El Manira and Koyama lab [1-3].
[1] Pujala A, Koyama M. Chronology-based architecture of descending circuits that underlie the development of locomotor repertoire after birth. Elife 2019;8.
[2] Menelaou E, McLean DL. Hierarchical control of locomotion by distinct types of spinal V2a interneurons in zebrafish. Nat Commun 2019;10:4197.
[3] Song J, Pallucchi I, Ausborn J, Ampatzis K, Bertuzzi M, Fontanel P, et al. Multiple Rhythm-Generating Circuits Act in Tandem with Pacemaker Properties to Control the Start and Speed of Locomotion. Neuron 2020;105:1048–61.e4.
[1] Pujala A, Koyama M. Chronology-based architecture of descending circuits that underlie the development of locomotor repertoire after birth. Elife 2019;8.
[2] Menelaou E, McLean DL. Hierarchical control of locomotion by distinct types of spinal V2a interneurons in zebrafish. Nat Commun 2019;10:4197.
[3] Song J, Pallucchi I, Ausborn J, Ampatzis K, Bertuzzi M, Fontanel P, et al. Multiple Rhythm-Generating Circuits Act in Tandem with Pacemaker Properties to Control the Start and Speed of Locomotion. Neuron 2020;105:1048–61.e4.
The opsin calibration part of the project was originally planned to happen over a short period of several months. Instead, we realised this part of the project is in fact important, not only for the experiments planned here, but could in fact be extremely useful to the general systems neuroscience zebrafish community. For this reason, together with two newly established collaborations with the Bianco and Baier Lab, we decided to perform the critical work necessary for calibrating opsin efficiency for the first time in vivo, in the zebrafish in an exhaustive and comprehensive manner. The publication that stemmed from this work will be highly influential and will become a key resource for any future zebrafish based optogenetic manipulation experiment.