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. 2023 May 26;26(6):106985. doi: 10.1016/j.isci.2023.106985

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© 2023 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Opposite effects of the optogenetic activation of L5 sublayer neuronal populations on appetitive licking

(A) Illustrations depicting AAV injections for retrograde ChR2 expression in the L5a or L5b populations (left) and the placement of a chip LED (right).

(B) An illustration depicting a freely moving mouse with a single water spout (left) and the protocol for water delivery and illumination (right). Water (∼2 μL) is delivered when the mouse licks the spout. The illumination (10 ms, 20 Hz) is presented simultaneously at the beginning of water delivery only at random bouts.

(C and H) Three typical examples of licking across a series of bouts with (blue) or without (white) illumination for mice expressing ChR2 in the L5a (C) and L5b (H) populations. Only a part of the session (25 bouts) is shown.

(D and I) Comparisons of mean licks per bout between the bouts with (blue) and without (gray) LED illumination in ChR2-expressing (ChR) and control (tdTom) mice (L5a: 15.7 ± 3.0 for [LED-] versus 7.4 ± 1.1 for [LED+], Wilcoxon signed rank test, p < 0.01 for ChR, D-left; 14.6 ± 2.5 for [LED-] versus 13.9 ± 2.2 for [LED+], Wilcoxon signed rank test, p = 0.84 for tdTom, D-right; L5b: 9.7 ± 2.0 for [LED-] versus 19.2 ± 2.8 for [LED+], Paired t-test, p < 0.01 for ChR, I-left; 13.8 ± 1.6 for [LED-] versus 15.5 ± 1.0 for [LED+], Paired t-test, p = 0.30 for tdTom, I-right).

(E and J) Ratio of the mean licks per bout with illumination relative to those without illumination (L5a: 0.53 ± 0.08 for ChR versus 0.99 ± 0.09 for tdTom, Student’s t test, p < 0.01, E; L5b: 2.41 ± 0.40 for ChR versus 1.22 ± 0.14 for tdTom, Welch’s t test, p = 0.022, J).

(F and K) Proportion of long drinking bouts with >10 licks per bout in the session (L5a: 44.6 ± 5.9% for [LED-] versus 25.2 ± 5.2% for [LED+], Paired t test, p = 0.028 for ChR, F-left; 44.0 ± 7.4% for [LED-] versus 43.5 ± 6.6% for [LED+], Paired t test, p = 0.93 for tdTom, F-right; L5b: 23.6 ± 5.0% for [LED-] versus 49.5 ± 5.0% for [LED+], Paired t test, p < 0.01 for ChR, K-left; 39.3 ± 4.0% for [LED-] versus 46.9 ± 4.5% for [LED+], Paired t test, p = 0.12 for tdTom, K-right).

(G and L) Percentage of bouts with illumination in the top 10 longest bouts in the session (L5a: 34.5 ± 4.9% for ChR versus 50.0 ± 4.3% for tdTom, Student’s t test, p = 0.034, G; L5b: 66.4 ± 4.6% for ChR versus 53.1 ± 2.5% for tdTom, Student’s t test, p = 0.022, L). Lines in D, F, I, and K and dots in E, G, J, and L represent individual mice (L5a: n = 10 for ChR, n = 8 for tdTom; L5b: n = 8 for ChR, n = 8 for tdTom). Dotted lines in E, G, J, and L indicate the chance level. ∗p < 0.05, ∗∗p < 0.01. See also Figure S5.