Transient inhibition and long-term facilitation of locomotion by phasic optogenetic activation of serotonin neurons
- Champalimaud Centre for the Unknown, Portugal
- University of California, United States
Figures
Figure 1
Optogenetic DRN 5-HT activation reduces spontaneous locomotion in the open field.
(A) Schematic of the optogenetic approach. DRN neurons are infected with AAV2/9-Dio-ChR2-EYFP. In SERT-Cre mice, 5-HT neurons will express ChR2-YFP (green cells) and can be photoactivated with blue light delivered through an implanted optical fiber. (B) Fluorescence image of a parasagittal section showing ChR2-YFP expression (green) localized to the DRN. Scale bar, 500 μm. (C) Schematic drawing of the open field paradigm. (D) Schematic diagram of photostimulation protocol. Each 30 min session consisted of stimulated (stim, blue) and non-stimulated (nostim, white) blocks of 5 min. A session always starting with a non-stimulated block and blocks always alternated, for a total of 30 min. During stim blocks, 3 s pulse trains of light were delivered every 10 s. Pre and post intervals shown were used to calculate stimulation effects. (E) Probability of being in a specific behavioral state for non-stimulated (pre) and stimulated (post) intervals for the population of SERT-Cre mice (N = 15). Note that probabilities do not sum to 100% because scoring does not include all time points. Individual mice shown in grey lines and averages across mice in filled circles. Error bars indicate SEM. In some cases, the error bars are too small to be visible. n.s: not significant. *p<0.05, **p<0.01, ***p<0.001, with paired t-test. (F) Probability of being in a mobile state (walking, rearing, jumping), as a function of time relative to stimulation onset. The shaded area indicates SEM across mice. (G) Same as (F) but for the immobile states (resting, digging, grooming and scratching).
Figure 2
Optogenetic DRN 5-HT activation slows down animals in the open field, independently of previous locomotion speed.
(A) Position tracking of example WT (top) and SERT-Cre (bottom) mice. All positions visited in the session are shown in gray; the positions visited during each 3 s stimulation periods are shown for one WT (green) and one SERT-Cre (blue) mouse. (B) Time course or speed relative to stimulation onset. Here, and below, WT (N = 9) are shown in green and SERT-Cre mice (N = 15) in blue and data mean ± SEM across animals is shown. Pre and post intervals are indicated by horizontal lines. (C) Average speed in pre- and post-stimulation intervals for individual mice (gray lines) and for the population of mice (mean ± SEM). In some cases, the error bars are too small to be visible. n.s: not significant. ***p<0.001 with paired t test. (D) Probability distribution of speed in pre- and post-stimulation intervals for the population of WT and SERT-Cre mice. (E) Difference between speed in post- and pre-stimulation intervals (delta speed) ***, p<0.001 with two-sample t test. (F) Dependence of delta speed on frequency of stimulation for the individual mice (gray lines) and for the subset of SERT-Cre mice tested (mean ± SEM, N = 3). (G) Speed probability distribution in the pre interval for all stimulation periods within stimulated blocks (as well as equivalent measures for non-stimulated blocks) for an example SERT-Cre mouse. (H) Average post speed in stimulated blocks, conditioned by pre speed for SERT-Cre mice. The four colors indicate the speed ranges used for pre conditioning, as indicated in the distribution in (G). (I) The same as (H) but for equivalent period in non-stimulated blocks. (J) The difference between the stimulated and non-stimulated blocks shows the effect of stimulation conditioned on pre speed. (K) Average difference between stimulated and non-stimulated blocks for delta speed (difference between post- and pre-intervals) for each quartile for individual mice (gray lines) and for the population of SERT-Cre mice (mean ± SEM, N = 15). In some cases, the error bars are too small to be visible.
Figure 3
Optogenetic DRN 5-HT activation does not affect motor coordination in the rotarod assay.
(A) Schematic of the accelerating rotarod assay. (B) Latency to fall for one example SERT-Cre mouse, with randomly interspersed stimulated trials. (C) Average latency to fall, showing learning and testing period. Note, stimulation (cyan bar) only occurred after training, WT (green, n = 5) and SERT-Cre (blue, N = 7) mice (mean ± SEM) are shown. (D) Average latency to fall in the testing session for individual mice (gray lines) and for the population of WT mice (green, N = 5) and SERT-Cre mice (blue, N = 7, mean ± SEM). In some cases, the error bars are too small to be visible. n.s: not significant.
Figure 4 with 1 supplement
Optogenetic DRN 5-HT activation does not induce motor impairment in the LocoMouse assay.
(A) Schematic drawing of the LocoMouse apparatus. Water deprived animals walk freely across a glass corridor connected to two boxes with water ports. A mirror below at 45° angle allows a single high-speed camera to capture side and bottom views at 400 frames per second. DRN 5-HT photostimulation occured randomly in 50% of crossings. (B) Paws, nose and tail segments were automatically segmented and tracked in 3D. Individual strides were divided into swing and stance phases for further analysis. (C) Average time to cross the linear track for individual mice (gray lines) and for the population of SERT-Cre mice (N = 7, mean ± SEM) in non-stimulated (black) and stimulated (blue) trials. In some cases, the error bars are too small to be visible. n.s: not significant. (D) Average whole-body speed (center of mass) for individual mice (gray lines) and for the population of SERT-Cre mice (N = 7, mean ± SEM) in non-stimulated (black) and stimulated (blue) trials. In some cases, the error bars are too small to be visible. n.s: not significant. (E) Instantaneous forward speed of front-right paw during swing phase at stride speed of 15–20 cm·s-1 for stimulated (blue) and non-stimulated (black) crossings. (F) x-y position of four paws relative to the body center during swing. (G) Vertical (z) position of front-right paw relative to ground during swing.
Figure 4—figure supplement 1
DRN 5-HT activation does not affect motor coordination and locomotion.
(A) Polar plots indicating the phase of the step cycle in which each limb enters stance, aligned to stance onset of front-right paw (FR, red), for the population of SERT-Cre mice (mean, N = 7). Distance from the origin represents walking speed. Left, non-stimulated trials; right, stimulated trials. (B) Nose trajectory in the z dimension, corresponding to height above the floor, for SERT-Cre mice (N = 7), conditional on walking speeds of 15–20 cm·s-1. Dark lines and shading show mean ± SEM. (C) Same as (B), but for tail segment eight trajectory along the y dimension.
Figure 5
Effect of DRN 5-HT optogenetic activation does not induce anxiety-like behavior in the open field.
(A) Position tracks of an example SERT-Cre mouse, depicting the main areas of the open field: corners (dark pink), periphery (light blue), center (light pink) and edges (gray). Filled circles represent the position of the mouse at the beginning of each stimulus train. For this and subsequent panels, blue indicates stimulated blocks and black indicates equivalent times in non-stimulated blocks. (B) Speed probability distributions during pre and post stimulation intervals that began when the mouse was in the center area of the open field mice. For this and subsequent panels, data is pooled or averaged across all SERT-Cre mice (N = 15). (C, D) The same as (B) but for the periphery and corners areas. (E) Average speed across blocks within a session for individual mice (gray lines) and for the population of SERT-Cre mice (N = 15, mean ± SEM) in non-stimulated (black) and stimulated (blue) blocks. In some cases, the error bars are too small to be visible. Each pair of stimulated and non-stimulated block was compared (***p<10−3, paired t test with Bonferroni correction for multiple comparisons). (F) Fractional occupancy in each area of the open field, for the population of SERT-Cre mice (N = 15), color code as in (A). (G) Fraction center area occupancy as a function of duration within the session. (H) Same as G but showing individual mice averaged over the entire session duration. (I) The cumulative distribution of the average distance from the geometric center point of the open field normalized to the distance of the walls to the center. (J) Fraction of the total distance travelled that was in the center area as function of time within the session. (K) Same as J but showing individual mice averaged over the entire session duration.
Figure 6
Optogenetic DRN 5-HT activation in a specific region of interest does not produce aversive or appetitive responses.
(A) Position tracks for an example SERT-Cre mouse for sessions before (pre), during (T1, T2, T3) and after (post) photostimulation. Red square indicates the ROI in which photostimulation occurred (indicted by blue lines). (B, C) Heat maps depicting the normalized occupancy and average speed for the same sessions depicted in A. (D–G) Population data (mean ± SEM) for SERT-Cre mice (N = 4; above, blue are stimulated sessions) or WT mice (N = 5; below, green are stimulated sessions). Different measures as indicated.
Figure 7 with 1 supplement
Long-term optogenetic DRN 5-HT activation induces an increase in speed in the open field.
(A) Schematic diagram of the photostimulation protocol, as in Figure 1D; a total of 270 s of 20 Hz stimulation is delivered over a 30 min session. (B) Experimental protocol. Group 1 (G1, 3 SERT-Cre and 2 WT mice) received photostimulation from session 1 to 24. Group 2 (G2, 3 SERT-Cre and 2 WT mice) was exposed to the arena for 23 days, receiving photostimulation beginning only on the 24th day for six consecutive days. (C) Average speed across sessions for Group 1 (N = 3 SERT-Cre mice). Non-stimulated (pre, black) and stimulated (post, blue) intervals. Lines and shading indicate mean ± SEM here and E, G, I, J. (D) Speed in early and late sessions (as indicated in (B)) for mice in (C). Dark lines and error bars indicate (mean ± SEM). Error bars are too small to see in some cases. Gray lines indicate individual mice. *p<0.05; n.s: not significant, with paired t test. Same applies to F, H, K, L. (E, F) Same as (C, D) but for Group 2 (N = 3 SERT-Cre mice). (G) Group one stimulation effect (delta speed, difference between post- and pre-stimulation intervals) across all sessions. (H) Stimulation effect in early vs. late sessions. (I) Same as (G) but for Group 2. (J) Group one speed across sessions assessed during the pre interval only in the first block, i.e. prior to receiving any stimulation during that session (K) Speed in the first block as in (I) for early vs. late sessions (as indicated in (B)). (L) Center occupancy in early vs. late sessions for Group one and Group 2.
Figure 7—figure supplement 1
Lack of correlation between short and long-term effects of DRN 5-HT activation.
(A) Difference in speed during the pre-stimulation interval between sessions n+1 and n-1 is plotted against the stimulation effect on day n (delta speed, difference between post- and pre-stimulation intervals) for all mouse-session pairs (excluding the first and last sessions; n = 66). Red line indicates linear regression curve. R2=3.89 × 10−3, p=0.270 against a constant model. (B) Residuals of a linear regression between pre-stimulation speeds and sessions are plotted against the stimulation effect (delta speed, difference between post- and pre-stimulation intervals) for all mouse-session pairs (excluding the last session; n = 69). Red line indicates linear regression curve. R2=0.030, p=0.080 against a constant model.
Videos
Video 1
Example SERT-Cre mouse behavior in the open field experiment during a photostimulation block.
Also shown are the behavioral state (red), photostimulation condition (on/off, filled/empty blue square respectively), and speed (green rectangle, normalized to the maximum value in that session).
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Transient inhibition and long-term facilitation of locomotion by phasic optogenetic activation of serotonin neurons
eLife 6:e20975.
https://doi.org/10.7554/eLife.20975
