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. 2022 Feb 28;11:e71965. doi: 10.7554/eLife.71965

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© 2022, De Saint Jan

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A) Schematic of the virus injection in Chat^Cre mice (top) and coronal section (bottom), at about bregma –0.1 mm, 20 days after injection. (B) ChR2-eYFP expression (green) in choline acetyltransferase (ChAT)-expressing cholinergic neurons (magenta) in the BF. Scale bar 50 µm. Right panels: zoom on the boxed region. (C) ChR2-eYFP-expressing axons in the olfactory bulb (OB). Scale bar 100 µm. DAPI staining (blue) delimits layers (ONL: olfactory nerve layer; GL: glomerular layer; EPL: external plexiform layer; GCL: granule cell layer). Higher-resolution image in Figure 2—figure supplement 1. (D) Experimental design for recording BF-evoked responses in OB slices. (E) Top: two representative 2-s-long loose cell-attached (LCA) recording episodes (scale bar 200 pA) and raster plot of spiking activity in control condition and when BF axons were photostimulated every 2 s (blue arrow, 1 ms flash, 490 nm). Photostimulation started at episode 31 (one flash/episode, blue line). Bottom: raster plot for the same cell, same experiment in the presence of blockers. (F) Average firing rate per episode (2 s each) in artificial cerebrospinal fluid (ACSF) (green) or in the presence of blockers (violet). Low-frequency photostimulation (0.5 Hz) started at episode 31. (G) Representative recording (scale bar 50 pA), raster plot, and peri stimulus time histograms (PSTH) (20 consecutive trials, bin 200 ms) of an excitatory response evoked in ACSF by a single photostimulation of the BF cholinergic axons at blue arrow and dotted line. Pale areas within the PSTH indicate the two periods that were compared in (I). The nonselective muscarinic ACh receptor (mAChR) antagonist scopolamine (10 µM) blocked the evoked excitation (bottom, scale bar 50 pA). (H) Average firing rate per bin and per 15-s-long episode for 21 cells recorded in ACSF. Each gray line corresponds to a cell; the black line indicates the ensemble average. Photostimulation at blue arrow. (I) Firing rate before (pre) and after (post) photostimulation of BF axons in ACSF (green) in the presence of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), D-2-amino-5-phosphonopentanoic acid (D-AP5), and mecamylamine (violet) or in the presence of the mAChR antagonist atropine (n = 4) or scopolamine (n = 4) (red). Each line indicates a cell; blue circles are the means. Paired t-test or Wilcoxon signed-rank-sum test (for atro/scopo).

Figure 2—figure supplement 1. — (A) Schematic of the virus injection in Chat^Cre mice (top) and coronal section (bottom), at about bregma –0.1 mm, 20 days after injection. (B) ChR2-eYFP expression (green) in choline acetyltransferase (ChAT)-expressing cholinergic neurons (magenta) in the BF. Scale bar 50 µm. Right panels: zoom on the boxed region. (C) ChR2-eYFP-expressing axons in the olfactory bulb (OB). Scale bar 100 µm. DAPI staining (blue) delimits layers (ONL: olfactory nerve layer; GL: glomerular layer; EPL: external plexiform layer; GCL: granule cell layer). Higher-resolution image in Figure 2—figure supplement 1. (D) Experimental design for recording BF-evoked responses in OB slices. (E) Top: two representative 2-s-long loose cell-attached (LCA) recording episodes (scale bar 200 pA) and raster plot of spiking activity in control condition and when BF axons were photostimulated every 2 s (blue arrow, 1 ms flash, 490 nm). Photostimulation started at episode 31 (one flash/episode, blue line). Bottom: raster plot for the same cell, same experiment in the presence of blockers. (F) Average firing rate per episode (2 s each) in artificial cerebrospinal fluid (ACSF) (green) or in the presence of blockers (violet). Low-frequency photostimulation (0.5 Hz) started at episode 31. (G) Representative recording (scale bar 50 pA), raster plot, and peri stimulus time histograms (PSTH) (20 consecutive trials, bin 200 ms) of an excitatory response evoked in ACSF by a single photostimulation of the BF cholinergic axons at blue arrow and dotted line. Pale areas within the PSTH indicate the two periods that were compared in (I). The nonselective muscarinic ACh receptor (mAChR) antagonist scopolamine (10 µM) blocked the evoked excitation (bottom, scale bar 50 pA). (H) Average firing rate per bin and per 15-s-long episode for 21 cells recorded in ACSF. Each gray line corresponds to a cell; the black line indicates the ensemble average. Photostimulation at blue arrow. (I) Firing rate before (pre) and after (post) photostimulation of BF axons in ACSF (green) in the presence of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), D-2-amino-5-phosphonopentanoic acid (D-AP5), and mecamylamine (violet) or in the presence of the mAChR antagonist atropine (n = 4) or scopolamine (n = 4) (red). Each line indicates a cell; blue circles are the means. Paired t-test or Wilcoxon signed-rank-sum test (for atro/scopo).