Our unique approach allows us to break the cycle of the motor command to sensory stimulation into individual components and observe the individual impact of each component on the cerebellum. This will allow us to test the hypothesis that the cerebellar cortex of mammals adapts to cancel the effect of any sensory stimulus that consistently accompanies a motor command and provide an understanding of those mechanisms.
The initial objectives of this project were to recruit a postdoc to the project, build a new two-photon microscope, implement the fictive locomotion preparation underneath the microscope, and begin to image populations of identified cells in the cerebellar cortex. This initial phase would involve strong collaboration between the two partners because they each have expertise in one aspect of the two components of the experiment. Marin Manuel would be responsible for implementing and training the postdoc to perform the decerebration and fictive locomotion and Brandon Stell would be responsible for building the microscope, the imaging and daily oversight of the cerebellum experimentation with the postdoc. This unique approach of combining the fictive-locomotion preparation with experiments probing cerebellar physiology allows us to break the cycle of the motor command to sensory stimulation into individual components and observe the individual impact of each component on the cerebellum.
We have constructed and now routinely use a new 2-photon microscope in the lab (Fig 1) that is dedicated to the SpinoCereLoco project. This microscope has been designed for the fictive locomotion preparation and employs resonant scanning galvanometers that allows us to image large populations of neurons in the cerebellum (Fig 1) with 0.03 s between image frames.
Fictive locomotion preparation
One hurdle that we had to overcome was to transfer the knowledge and expertise of the fictive locomotion preparation from the team of Marin Manuel to the team of Brandon Stell. This became of even more extreme importance when we learned that Marin would be leaving France for the United States and would not be able to give daily hands-on help with the preparation. However, despite the difficulty presented by the health crisis Murat successfully learned the preparation and is now independently performing experiments on the preparation for the last 6 months.
2-photon imaging during fictive locomotion and stimulation of identified limb nerves
To observe the effect of individual sensory and motor components involved in locomotion on the neurons of the cerebellar cortex, we began by imaging Purkinje cells expressing the calcium indicator GCaMP7f. In figure 1 we show an example of how these experiments quickly bore fruit. A train of stimuli delivered to the sural nerve of the right hind-limb produces a delayed and maintained activation of a large population of Purkinje cell somata imaged in lobule IV/V of the vermis (Fig 1A). In the dendrites of the same cells (Fig 1B) the response of the stimulus is localized to a small subset of dendrites and produces an immediate and brief increase in dendritic [Ca2+].
Electrophysiological recordings during fictive locomotion
To determine the origin of the calcium increases observed in Purkinje cells resulting from the nerve stimuli we are routinely performing cell-attached recordings from the various cell-types found in the cerebellar cortex.
Shortly after the recruitment of the postdoc to work on this project the COVID19 pandemic started and we were forced to close the lab for several months. Furthermore, at the beginning of the project, Marin Manuel accepted a position at the University of Rhode Island in the United States, and moved his lab at the beginning of 2021.
Fortunately, the major contribution of Marin Manuel to this project was planned for the very beginning of the project when he would train Brandon and the postdoc how to perform the surgery necessary for the decerebrate preparation. The combination of the lockdown with the departure of Marin could have been disastrous for the project but we were able to complete the training before Marin’s departure in early 2021. The project is now proceeding as planned with the recruited postdoc performing experiments with Brandon Stell in regular consultation with Marin Manuel.
Theories of cerebellar function have proposed that the mammalian cerebellum uses internal models, similar to those described in control theory, to distinguish external from self-generated stimuli. In control theory, a forward model uses copies of a motor command together with sensory information to predict and cancel the sensory consequences of the self-generated motor command. This model has been shown to fit with observations in cerebellar-like structures of lower vertebrates (Bodznick et al., 1999) and mice (Singla et al., 2017). However, the cellular correlate in the mammalian cerebellum remains unknown because the reafferent sensory input is not readily isolated from the corollary discharge. Consequently it is not known how the cerebellar cortex processes information to coordinate movements in even a routine behavior such as walking (Medina, 2011). We propose to combine the expertise of two teams in our laboratory to separate sensory and motor components in a simple locomotor behaviour. Crucially the experiments will be in decerebrate preparations without the implications and complications associated with anesthesia and permitting separation of sensory input.We will use electrical recording and 2-photon imaging to understand how the separate components of the behavior are processed by populations of cells in the cerebellar microcircuits and then use this knowledge of the individual components to see how they are combined in the cerebellum of awake mice running on a treadmill.
Our two teams have independently made recent advances that put us in a unique position to distinguish the effect of the copy of the motor command sent to the cerebellum from the effect of the sensory response elicited by the movement itself. The spinal cord and brainstem contain neuronal networks, called central pattern generators (CPGs), capable of generating stereotyped rhythmic locomotor activity (e.g. walking, breathing, whisking, licking). Since CPGs generate rhythmic motor activity in the absence of cortical command, the activity persists when the forebrain is surgically removed (unanesthetized decerebrate preparations) from paralyzed animals (generating “fictive” locomotion), and thereby removing sensory feedback. We will use the fictive locomotion to separate the sensory from the motor inputs into the cerebellum to better understand how they are processed by the neuronal networks of the cerebellar cortex.
We will particularly test the hypothesis that the microcircuits of the cerebellar cortex adapt by adjusting synaptic weights to cancel sensory input that is consistently paired with the motor command in order to distinguish input arriving from the environment.
Monsieur Brandon STELL (Institut des Neurosciences Paris Saint-Pères)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
SPPIN Institut des Neurosciences Paris Saint-Pères
SPPIN Team3 Institut des Neurosciences Paris Saint-Pères
Help of the ANR 592,990 euros
Beginning and duration of the scientific project: December 2019 - 48 Months