Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Dec 4;6(49):eabc6309.
doi: 10.1126/sciadv.abc6309. Print 2020 Dec.

Deep imaging in the brainstem reveals functional heterogeneity in V2a neurons controlling locomotion

Joanna Schwenkgrub  1   2 , Evan R Harrell  1   2 , Brice Bathellier  3   2 , Julien Bouvier  3
Affiliations

Deep imaging in the brainstem reveals functional heterogeneity in V2a neurons controlling locomotion

Joanna Schwenkgrub et al. Sci Adv. .

Abstract

V2a neurons are a genetically defined cell class that forms a major excitatory descending pathway from the brainstem reticular formation to the spinal cord. Their activation has been linked to the termination of locomotor activity based on broad optogenetic manipulations. However, because of the difficulties involved in accessing brainstem structures for in vivo cell type-specific recordings, V2a neuron function has never been directly observed during natural behaviors. Here, we imaged the activity of V2a neurons using micro-endoscopy in freely moving mice. We find that as many as half of the V2a neurons are excited at locomotion arrest and with low reliability. Other V2a neurons are inhibited at locomotor arrests and/or activated during other behaviors such as locomotion initiation or stationary grooming. Our results establish that V2a neurons not only drive stops as suggested by bulk optogenetics but also are stratified into subpopulations that likely contribute to diverse motor patterns.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Activity of V2a neurons can be recorded with micro-endoscopy.
(A) Schematic of viral injections and lens implantation into the Gi of a Chx10-Cre animal for calcium imaging in freely behaving mice. (B) Nissl-stained (magenta) coronal sections of Chx10-Cre mouse showing native GCaMP6s fluorescence (green) and the location of medial (left) or lateral (right) GRIN lens implants. Inset in the yellow box is a magnified view of GCaMP6s expression in V2a neurons. Scale bars in micrometers. (C) Example mouse during treadmill running with miniature microscope attached. Photo credit: Joanna Schwenkgrub, CNRS. (D) Example cell contour map (left) with corresponding raw/deconvolved calcium traces (right). Abbreviations used in (A) and (B): 4V, 4th ventricle; 7N, facial motor nucleus; 7n, facial nerve; py, pyramidal tract; Gi, gigantocellular reticular nucleus; sp5, spinal trigeminal tract; a.u., arbitrary units.
Fig. 2
Fig. 2. Activity of V2a Gi neurons correlates with spontaneous locomotor stops during treadmill running.
Example mouse on treadmill (A) (photo credit: Joanna Schwenkgrub, CNRS). (B) Corresponding smoothed raw calcium traces (right) and cell contour map (left). Red vertical lines, stops; ♦, example stop event. (C) Associated average calcium transients at stop events are shown (left), together with another example animal (right); *, significant difference relative to time-shuffled data (gray shaded areas: SEM, P < 0.05). (D) Top: Fraction of V2a neurons uncorrelated, excited, or inhibited at stops. Distribution at lateral versus medial locations is significantly different (2 × 3 Fisher exact test, P = 0.0139). Bottom: Fraction of excited and inhibited cells per animal; dot size = number of recorded cells. (E) Real and predicted locomotion state (stationary = red bar). The magnification shows stops corresponding to traces in (B). ♦, unpredicted stop. (F) Receiver operating characteristic (ROC) for the same mouse. AUC, area under the curve. Δ = prediction performance in (E). (G) Number of cells per animal against performance of the stop classifier. Trend line in black. (H) Scatter plot of the peak ΔF/F (a.u.) for a stop versus stop-activation probability for all the visible cells (n = 39) recorded in six mice; resampling test, P < 0.05.
Fig. 3
Fig. 3. Activity of V2a neurons correlates with locomotor stops during open-field exploration.
(A) Running (a) and stationary (b) postures of an example mouse (anatomical landmarks used for tracking are indicated with colored squares) in the open-field arena. Photo credit: Joanna Schwenkgrub, CNRS. (B) Example cell contour map (left) with corresponding raw calcium traces smoothed with Gaussian filter (half-width 150 ms) (right). Red vertical lines indicate stops; red shaded areas indicate stationary periods. (a) and (b) correspond to the behaviors shown in (A). (C) Trajectory map showing the position and locomotor state of an example animal during one experimental session; forward locomotion is in blue, backward locomotion is in red, and stationary state is in yellow. (D) Fraction of V2a neurons that are excited, inhibited, or not affected at stops for lateral (orange) or medial (blue) locations. The distribution of excited, inhibited, and uncorrelated cells was significantly different between medial and lateral populations (2 × 3 Fisher exact test, P = 0.017). Inset: Fraction of excited and inhibited cells per animal; the dot sizes correspond to the number of recorded cells. (E) Average calcium transients at stop events for all recorded cells from the example shown in (A) to (C) (raw and deconvolved data); * represents a significant difference relative to time-shuffled data (single shuffle averages in black, gray shaded areas represent SEM), P < 0.05.
Fig. 4
Fig. 4. The correlation of V2a neuron activity with locomotor stops can be context independent.
(A) Aligned cell contour maps of an example animal recorded in two behavioral contexts: treadmill running and open-field exploration. (B) Average calcium transients at stop events for all recorded cells from the example [raw data, cell numbering is the same as (A)] in both behavioral contexts; n corresponds to the number of detected stop events, and blank spaces indicate cells not detected in one of the conditions; * and # represent significant differences relative to time-shuffled data for excitation and inhibition, respectively (single shuffle averages in black, gray shaded areas represent SEM), P < 0.05. (C) Scatter plot of the peak ΔF/F for treadmill (ordinate) versus open-field (abscissa) stop-related calcium transients. Cells included in the plot are those visible in both and significantly correlated to stops in at least one of the behavioral contexts (n = 25 cells in six animals). Open circles denote cells with significant correlation to stops only in one behavioral context. Filled circles indicate neurons with significant correlation in both treadmill and open-field conditions. Inset: Definition of peak ΔF/F and time differences between locomotion arrest and the peak ΔF/F (ΔT stop).
Fig. 5
Fig. 5. The activity of V2a neurons correlates with locomotor onsets during open-field exploration.
(A) Average calcium transients at locomotor start and stop events from six example cells recorded in three different animals (raw data). Red lettering indicates stop-correlated cells; * and # represent significant differences relative to time-shuffled data for excitation and inhibition, respectively (single shuffle averages in black, gray shaded areas represent SEM), P < 0.05. (B) Scatter plot of the peak ΔF/F for start-related (ordinate) versus stop-related (abscissa) calcium transients in the open field. Cells included in the plot (n = 31 cells in six animals) are significantly correlated to at least one of the two behaviors, i.e., starts or stops of locomotion. Filled circles indicate neurons with significant correlation to both behaviors. Open circles denote cells significantly correlated only to one of them (start cells in green, stop cells in blue). Inset: Definition of peak ΔF/F. (C) Fraction of V2a neurons that are excited, inhibited, or uncorrelated at start events for lateral (orange) or medial (blue) locations. The absolute number of recorded cells is given in parentheses. Inset: Fraction of excited and inhibited cells per animal; the dot sizes correspond to the number of recorded cells. The distribution of excited, inhibited, and uncorrelated cells was not significantly different between medial and lateral populations (2 × 3 Fisher exact test, P = 0.24).
Fig. 6
Fig. 6. A fraction of V2a neurons are active during grooming.
(A) Grooming postures (frame nos. 1 to 5 and 7) of example animal no. 1. (B) Top right panel: Calcium traces from three cells excited during grooming from the animal shown in (A). Frame numbers correspond to posture numbering from (A). Top left panel: Average of calcium transients at stop events of the same cells; n.s., nonsignificant correlation compared to time-shuffled data (P > 0.05). Bottom right panels: Calcium traces of the two example cells (grooming-correlated in turquoise, stop-correlated in red) recorded in animal no. 2 in treadmill (upper) and open-field (lower) conditions showing increased activity during grooming periods (shaded areas). Example grooming posture (lower right panel) from this animal. Bottom left panels: Stop event averages for example cells (raw data). * and # represent significant differences relative to time-shuffled data for excitation and inhibition, respectively (single shuffle averages in black, gray shaded areas represent SEM), P < 0.05. Photographs from (A) and (B) by Joanna Schwenkgrub, CNRS. (C) Scatter plot of the peak ΔF/F after grooming onset versus locomotor stops. The 46 cells included in the plot are all cells recorded in seven mice during free exploration (treadmill not engaged or open field). Inset: Scatter plot of the peak ΔF/F for a grooming period versus locomotor stops.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kiehn O., Development and functional organization of spinal locomotor circuits. Curr. Opin. Neurobiol. 21, 100–109 (2011). - PubMed
    1. Grillner S., Jessell T. M., Measured motion: Searching for simplicity in spinal locomotor networks. Curr. Opin. Neurobiol. 19, 572–586 (2009). - PMC - PubMed
    1. Goulding M., Circuits controlling vertebrate locomotion: Moving in a new direction. Nat. Rev. Neurosci. 10, 507–518 (2009). - PMC - PubMed
    1. Grillner S., El Manira A., Current principles of motor control, with special reference to vertebrate locomotion. Physiol. Rev. 100, 271–320 (2020). - PubMed
    1. Jordan L. M., Liu J., Hedlund P. B., Akay T., Pearson K. G., Descending command systems for the initiation of locomotion in mammals. Brain Res. Rev. 57, 183–191 (2008). - PubMed

Publication types

LinkOut - more resources