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. Author manuscript; available in PMC: 2021 Nov 9.
Published in final edited form as: Cell Rep. 2020 Jun 16;31(11):107779. doi: 10.1016/j.celrep.2020.107779

Figure 1. Tube confinement induces sighing through Nmb neurons in RTN.

Figure 1.

(A) Sigh rate (bin 4 min, slide 1 min) determined by plethysmography of wild-type C57BL/6 mice (n=6) before, during, and after placement in a 50 ml conical tube. Grey, confinement period. (B) Quantification of sigh rate before, during, and after tube confinement (mean +/− SEM). *, p<0.05 (ANOVA followed by post hoc t-test). (C and D) Quantification of respiratory rate (C) and sigh-to-eupneic-breath ratio (D) before and during tube confinement (mean +/− SEM). *, p<0.05 (paired t-test). (E) Map of Nmb-CreERT2 mouse BAC transgene used for marking and manipulating Nmb-expressing sigh control neurons in medulla retrotrapezoid nucleus (RTN). The three exons of the Nmb locus are numbered (black fill, coding region). WPRE, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Response Element; pA, polyadenylation signal. (F) Scheme for chemogenetic silencing of Nmb-positive RTN neurons. RTN of Nmb-CreERT2 adult mouse is infected with recombinant AAV-DIO-hM4Di-mCherry virus. Expression of CNO-dependent neuronal silencer hM4Di and mCherry is induced by tamoxifen (Tam), and then hM4Di is activated by CNO. CAG, cytomegalovirus early enhancer/chicken beta actin promoter; triangles, Cre recombination sites. (G) Expression of mCherry (red) in Nmb-CreERT2-expressing neurons in RTN (dashed outline) at day 20 of scheme in F. DAPI (nuclear marker), blue. Inset, close up of boxed region showing two labeled Nmb-expressing neurons (arrowheads) and schematic diagram of one of them. Scale bar, 100 um (50 um, inset). (H) Sigh rate during tube confinement in mice with (+, n=6) and without (−, n=5) hM4Di expression in Nmb neurons (Nmb-hM4Di) treated with (+) or without (−, vehicle only) CNO injection as shown in F. Note the residual sighing during confinement could due to incomplete silencing of Nmb neurons or to Grp neurons in RTN, which have similar function. *, p<0.05; n.s., not significant.