Fig. 5: Optogenetic activation of the Gal+ neurons leads to BSM activity in an anesthetized preparation. | Nature Communications

Fig. 5: Optogenetic activation of the Gal+ neurons leads to BSM activity in an anesthetized preparation.

From: A galanin-positive population of lumbar spinal cord neurons modulates sexual arousal and copulatory behavior in male mice

Fig. 5

A Optogenetic stimulations (200 Hz, 100 pulses/10 ms, 20 mW) were performed on top and along the rostrocaudal lumbar spinal cord in adult anesthetized and spinalized Gal-Chr2 males (animals expressing Channerrhodopsin 2 in Galanin-positive cells, left panel) while performing electromyogram (EMG) recordings in the bulbospongiosus muscle (BSM) and a leg muscle (TA - Tibialis anterior). B Electrolytic lesions were placed at the location where optogenetic stimulation led to the most prominent BSM potentials (upper panel, see inset and white arrow; Green: Galanin-ChR2, Purple: Nissl stain). Scale bar of spinal cord section 200 μm, inset, 20 μm. Lower panel: representative traces of the EMG activity (BSM and TA) during optogenetic application on top of the lesion site. Note that it led to a similar activity pattern compared to the electrical stimulations: while the first laser application led to high amplitude and high frequency discharges in the BSM, but not in the leg muscle (TA), the second round of light delivery led to a reduced response, and BSM activity was not detected during the third round of optogenetic stimulation. This was consistent for the 10 animals tested in this experiment. C Diagram showing the light-triggered BSM activity along the rostrocaudal spinal cord axis. Largest BSM responses were encountered at the L2/L3 segments (mean amplitude 5.61 ± 2.31 mV, N = 10 mice). Note that responses are more restricted to the L2/L3 spinal segments where the cluster of Gal+ cells was found, around the central canal (mean amplitudes: 500 μm rostral to L2/L3 0.99 ± 0.58 mV, 500 μm caudal to L2/L3 1.35 ± 1.06 mV; represented as mean amplitude values +/− SEM). P-values result from a two-tailed Mann-Whitney-U Test. Different colored dots represent different animals (C, D). D Violin plots with boxplots illustrating the latencies with which BSM responses were triggered as a function of distance (N = 10 mice). Shorter latencies were achieved at 0 μm which corresponds to the L2/L3 spinal segments (0.02 ± 0.006 s). P-values result from a two-tailed Mann-Whitney-U Test. In 7 out of 10 animals BSM responses were triggered at 500 μm rostral to L2/L3 whereas only in 4 animals BSM responses were triggered at 500 μm caudal to L2/L3 (see individual dots). Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. E Violin plots illustrating the amplitudes of optogenetically triggered BSM signal during 1st (mean amplitude 5.5 ± 2.6 mV), 2nd (mean amplitude 1.48 ± 0.86 mV) and 3rd rounds (mean amplitude 0.41 ± 0.23 mV) of laser application, with boxplots (N = 8 mice). P-values result from a two-tailed Mann-Whitney-U Test. Every dot represents the BSM responses obtained from an animal. Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. F Same as (E), but the duration of BSM activity is plotted. Mean durations: after 1st 29.33 ± 7.28 s; 2nd 15.59 ± 6.43 s; and 3rd 2.27 ± 1.24 s, laser application. P-values result from a two-tailed Mann-Whitney-U Test. Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. G Same as (F), but the onsets with which BSM activity was initiated are plotted. Mean onset of BSM EMG: after 1st 1.18 ± 0.02 s; 2nd 25.25 ± 0.1; and 3rd 4.28 ± 0.1 s, laser application. P-values result from a two-tailed Mann-Whitney-U Test. Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. H In addition to tonic BSM responses as observed during the electrical stimulations, optogenetic stimulations also led to timely locked BSM responses, capable of following stimulation frequencies up to 20 Hz (N = 12 mice). In a subset of animals (4 out of 12) we recorded photo-identified single Gal-ChR2 neurons (N = 7 cells). Upper left panel: Juxtacellular recording of a Gal-ChR2 cell in parallel with EMG recordings of the BSM (2nd trace) and the TA muscle of the leg (3rd trace) during a 5 Hz laser stimulation. Upper right panel: zoom in of the indicated box illustrating the spike and EMG onset. Middle panels: same as upper panel but for a 10 Hz and 20 Hz (pulses of 10 ms length) laser application of the same cell shown in the upper panel. I Violin plot with boxplots illustrating the latency for optogenetically triggered spikes in Gal-ChR2 single cells (Gal spike), the latency between spike onset of Gal-ChR2 single cells and BSM EMG onset (Gal EMG), and the latency to spike of single photo-identified BSM motor neurons (BSM-MNs) (BSM-MN spike). Data refers to 7 single Galanin cells obtained from 4 Gal-ChR2 animals and to 7 single BSM-MNs obtained from 6 animals that received retro AAV injections with ChR2 as pups (see Fig. 1M). Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. J Same as (L), but the Gal-ChR2 spike and Gal-EMG fidelity (with which a laser pulse triggered a single spike or EMG response) is plotted. Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. K Violin plots illustrating the amplitudes of optogenetically time-locked (5 Hz) BSM signals (N = 12 mice) during 1st (mean amplitude 7.45 ± 1.64 mV), 2nd (mean amplitude 3.59 ± 1.17), 3rd (mean amplitude 2.93 ± 1.26 mV) and last rounds (mean amplitude 3.98 ± 1.98) of laser application, with boxplots (elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles). P-values result from a two-tailed Mann-Whitney-U Test. Every dot is an individual animal. 11 out of 12 animals showed BSM responses upon 2nd laser application, in 9 out of 12 animals BSM responses were triggered upon 3rd laser application and in 8 out of 12 animals, laser application led to BSM responses during last rounds of stimulation (5th laser applications). L Same as (I), but the duration of BSM signals is plotted. Mean durations: after 1st 0.06 ± 0.01 s; 2nd 0.05 ± 0.01; 3rd 0.03 ± 0.01; and last 0.03 ± 0.06 laser application. P-values result from a two-tailed Mann-Whitney-U Test. Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. M Same as (I), but the onset with which locked laser light triggered a BSM response. Data refers to 5 averaged onsets. Mean onsets: after 1st 0.03 ± 0.005 s; 2nd 0.03 ± 0.006; 3rd 0.03 ± 0.006; and last 0.03 ± 0.005 s, laser application. Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. N Optogenetic activation of photo-identified BSM-MNs (N = 7 cells from 6 mice; same experimental approach as in Fig. 1G). Upper left panel: Juxtacellular recording of a BSM-MNs in parallel with EMG recordings of the BSM (2nd trace) during a 20 Hz laser stimulation. Upper right panel: zoom in of the indicated box illustrating the BSM-MNs spike and the EMG onset. O Repeated optogenetic activation of BSM-MNs (N = 10 mice) led to BSM activity of stable amplitude (mean amplitude: after 1st 7.65 ± 0.91 mV; 2nd 8.66 ± 0.93 mV; and 3rd 6.99 ± 0.59 mV laser applications). Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles. P Same as (O), but the duration of BSM activity in response to optogenetic activation of BSM-MNs (N = 10 mice) is shown (mean durations: after 1st 15.28 ± 0.47 ms; 2nd 15.85 ± 0.39 ms; and 3rd 15.61 ± 0.47 ms laser application). Elements of violin plot: center line, median; box limits, upper (75) and lower (25) quartiles.

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