Figure 6.

Peptidergic EW neuron activation precedes nesting and controls behavioral transitions to nesting

(A) Fiber photometry recording of EWcp neurons calcium activity with GCaMP7s.

(B) Motion-corrected (isosbestic point) calcium-dependent fluorescence over a full sequence of behavior, with zooms at the level of the waking, nesting, and sleep transitions in virgin females. Two different modes of activity are visible, during wakefulness and during sleep.

(C) Peri-event plots of awakening, nesting, and sleep transitions in virgin females. Activity of the EWcp^CART+ neurons peaks after the animal wakes up and rises before nesting onset and falls at sleep onset.

(D) Acute inhibition of EWcp^CART+ neurons with hM4Di in Cart-Cre mice.

(E) Cumulative nestlets interaction time in E14–E15 pregnant females injected with an AAV2 expressing hM4Di or mCherry, following saline or CNO IP injections over 5 h. CNO-injected females spend less time nesting and more time sleeping than saline injected mice for the hM4Di injected group only.

(F) Pairwise comparisons of total nesting and sleep times in mice injected with the control or hM4Di virus, following saline and CNO injections.

(G) Behavioral transitions following saline or CNO injections in hM4Di-injected pregnant females, with a significant reduction in switches toward nesting.

(H) Regimen of acute stimulations of the EWcp^CART+ neurons with ChrimsonR in virgin Cart-Cre females: five sessions of 1 h are distributed around the switch from the dark to the light phase. During each 1-h session, trains of 20-Hz stimulations for 30 s are initiated every 5 min.

(I) Proportion of stimulations in virgin females that elicit a switch in behavior. Stimulations randomly occur during either sleep or active behaviors, and the ratio of stimulation leading to a switch over ineffective stimulations is evaluated.

(J) Switches of behavior evoked by the stimulation in control and opsin-injected virgin female mice. Random stimulations evoked a switch toward nesting in 23% of the time in awake animals.

(K) Behavioral state of the animals around stimulations, showing an increase in the proportion of time spent nesting in ChrimsonR-injected animals during the stimulation.

(L) Cumulative time spent nesting in control and ChrimsonR-injected animals over the 1 h of stimulations, as in (H). The stimulated animals spend more time nesting but only during the first half of the stimulation periods. Scale bars are 50 (A) and 150 μm (D). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. All data are presented as mean ± SEM. Wilcoxon’s signed rank test (C, F, and K) and unpaired Mann-Whitney ranked U test (G, I, J, and L). See also Figure S6.