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. 2014 Aug 22;9(8):e105941.
doi: 10.1371/journal.pone.0105941. eCollection 2014.

Optogenetic Recruitment of Dorsal Raphe Serotonergic Neurons Acutely Decreases Mechanosensory Responsivity in Behaving Mice

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Free PMC article

Optogenetic Recruitment of Dorsal Raphe Serotonergic Neurons Acutely Decreases Mechanosensory Responsivity in Behaving Mice

Guillaume P Dugué et al. PLoS One. .
Free PMC article

Abstract

The inhibition of sensory responsivity is considered a core serotonin function, yet this hypothesis lacks direct support due to methodological obstacles. We adapted an optogenetic approach to induce acute, robust and specific firing of dorsal raphe serotonergic neurons. In vitro, the responsiveness of individual dorsal raphe serotonergic neurons to trains of light pulses varied with frequency and intensity as well as between cells, and the photostimulation protocol was therefore adjusted to maximize their overall output rate. In vivo, the photoactivation of dorsal raphe serotonergic neurons gave rise to a prominent light-evoked field response that displayed some sensitivity to a 5-HT1A agonist, consistent with autoreceptor inhibition of raphe neurons. In behaving mice, the photostimulation of dorsal raphe serotonergic neurons produced a rapid and reversible decrease in the animals' responses to plantar stimulation, providing a new level of evidence that serotonin gates sensory-driven responses.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Specificity of ChR2-YFP expression in DRN 5-HT neurons and efficiency of photostimulation in vitro.
A, Schematics of viral injections. B, Fluorescence picture of a parasagittal slice showing restricted ChR2-YFP expression in the DRN. C, Superimposed fluorescence and brightfield images of a ChR2-YFP-expressing coronal slice. Bar: 500 µm. D1–3, Confocal pictures of the area delimited by a black rectangle in C. Aq: aquaduct. Bar: 100 µm. E1–3, Magnified view of the area delimited by a white rectangle in D 3. Bar: 50 µm. F, Schematics of patch-clamp recordings in DRN slices. G, Left: example of a loose cell-attached recording illustrating the protocol used to assess photostimulation thresholds (PTs). Right: blow-up of the trace showing a single photoevoked spike. H, Spike probability versus incident irradiance for 21 cells. I, Kernel density estimate of the distribution of logarithmically-scaled PTs (black) and superimposed normalized cumulative sum (red). J, Spike count per pulse versus pulse duration for 17 cells at twice their PT. Inset: representative firing profiles of 3 different cells identified in the graph by colored arrowheads. K, Response of a ChR2-YFP cell to trains of repeated light pulses (6 ms) at various frequencies (irradiance set at twice its PT). L, Average firing rate (± SEM, shaded area) during a 5 s train versus photostimulation frequency at twice the PT (dashed line; n = 29, 28, 25 and 18 cells for 1–2, 5–10, 20 and 50 Hz respectively) and at a higher irradiance (∼5 mW·mm−2; solid line, n = 14 and 13 cells for 1–20 and 50 Hz respectively).
Figure 2
Figure 2. Photostimulation of DRN 5-HT neurons in vivo.
A, Schematics of the experiment used to assess the spread of blue light in the DRN. B, Example image of DRN illumination. The intensity is color-coded relative to the pixel of maximal intensity (white dot). The 10 and 2% contour lines are shown in white. IC: inferior colliculus. C, Circular profile plot calculated from B, showing pixel intensities along concentric lines centered on the brightest pixel. D, Average intensity profile (± SD, shaded area; n = 3 brains) along the fiber axis. The depth and intensities are calculated relative to the brightest pixel. E, Schematics of the setup used to map YFP fluorescence and photoevoked firing in vivo. F, Example of a combined electrophysiological and fluorescence mapping experiment in a ChR2-YFP-expressing SERT-Cre (ChR2, solid lines) and a wild-type (WT, dotted lines) mouse. Top: normalized fluorescence (green, left axis) and OLFP amplitude (black, right axis) profiles plotted against the optrode position. Bottom: boxplots showing the optrode position at the point of maximal fluorescence (green, n = 8 mice) and largest OLFP amplitude (grey, n = 6 mice). Inset: average OLFP for the ChR2 mouse at the locations marked by the arrowheads. G, Example showing the time course of the effect of the 5-HT1A receptor agonist 8-OH-DPAT on the OLFP amplitude (black), latency (purple) and width (blue). Bin: 158 s. Inset: average OLFP before (black) and after (red) 8-OH-DPAT injection (taken from the periods indicated by arrowheads). H, Top: OLFP amplitude before and after 8-OH-DPAT injection (mean ± SD: 150±121 µV and 121±105 µV respectively, n = 10). Bottom: OLFP peak latency before and after 8-OH-DPAT injection (mean ± SD: 2.4±0.5 ms and 2.8±0.7 ms, n = 10). Changes are significant in both cases (P = 0.037 and P = 0.030 respectively, paired Wilcoxon rank sum test). Error bars represent the SEM. I, Example illustrating the dependence of the OLFP amplitude (grey points) and latency (red points) on the irradiance at the fiber tip. The sum of two exponential functions was fitted to each curve (black lines). Inset: superimposed OLFPs for 0.2, 0.7, 1.6 and 6.0 mW·mm−2. J, Normalized average (± SD, shaded area) OLFP amplitude (black, left axis) and latency (red, right axis) versus photostimulation frequency (n = 7 mice). K, Average OLFP amplitude recovery curve (green; ± SD, shaded area) assessed by delivering test pulses at variable intervals (δt) after 10 Hz, 5 s trains of 6 ms light pulses (n = 4 mice). Blue: average amplitude during the trains.
Figure 3
Figure 3. Decreased responsivity to mechanical stimuli during DRN 5-HT neurons photostimulation.
A, Schematics of the experiment. B, Structure of a session. C, Structure of a trial in the “stim” block. The filament was applied to the hindpaw during a 12 s train of light pulses. D, Average baseline response probability curves for control (infected WTs, solid line, n = 17) and ChR2 (dashed line, n = 17) mice. E, Kernel density estimates of the baseline threshold distribution for control (WT, solid line) and ChR2 (dashed line) mice. F, Average baseline threshold across sessions for control (WT, solid line) and ChR2 (dashed line) mice. G–H, Thresholds for all control and ChR2 mice in the baseline (dark grey), “stim” (blue) and recovery (light grey) blocks. *: P<0.05 (paired Wilcoxon rank sum test); ***: P<0.001 and **: P<0.01 (unpaired Wilcoxon rank sum test). (I) Threshold change for control and ChR2 mice in the “stim” (blue) and recovery (light grey) blocks. ** P<0.01 and *** P<0.001 (paired and unpaired Wilcoxon rank sum test respectively). Shaded areas and error bars represent the SD in all panels.

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Grant support

This work was supported by an European Research Council grant to ZM (N ° 250334), an Intra-European Marie Curie postdoctoral fellowship to GD (N ° 220098), a Human Frontier Science Programme postdoctoral fellowship to MLL (N ° LT001009/2010L), a Human Frontier Science Programme postdoctoral fellowship to EL (N ° LT000881/2011L) and an Agence Nationale pour la Recherche grant to CL (Sensocode 11-BSV4-028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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