Extended Data Figure 7 : Distribution of AAV-DREADD infection sites in the zona incerta of Lhx6-cre mice and CNO activates Fos expression in AAV-DIO-hM3Dq-injected Lhx6-Cre mice, whereas CNO does not affect sleep in AAV-DIO–mCherry-injected Lhx6-Cre mice.

From: Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep

Extended Data Figure 7

Continued from Fig. 4. a, Schematics showing distribution of DREADD-infected (hM3Dq or hM4Di mice) neurons (red) across the rostrocaudal extent of the zona incerta after AAV-DREADD was injected into the zona incerta of Lhx6-cre mice. Infection is specific to the zona incerta in 12 Lhx6-cre mice (red colour indicates infection site from all 12 mice). Yellow colour indicates additional infected areas seen in 4 out of 12 mice. bd, Representative images showing co-expression of hM3Dq DREADD in the zona incerta of Lhx6-cre mice (b, red) with Lhx6 immunostaining (c, green). The merged image is shown in d, inset shows a magnified image of d. eg, Representative images showing co-expression of AAV-hM4Di infection in the zona incerta of Lhx6-cre mice (e, red) with Lhx6 immunostaining (f, green). A merged image is shown in g; inset shows a magnified image of g. Scale bar, 100 μm. hj, Representative images showing hM3Dq–mCherry expression (red) and Fos immunostaining in the zona incerta of Lhx6-cre mice after saline (h) or CNO (i) injection. A magnified image of i is shown in j. k, The percentage Fos+ neurons in hM3Dq–mCherry+ Lhx6-expressing neurons in the zona incerta in mice injected with saline or CNO. ***P < 0.001, two-tailed paired t-test; n = 4 mice, 8 sections for saline; n = 5 mice, 8 sections for CNO. ln, Representative images showing hM4Di–mCherry expression (red) and Fos immunostaining (green) in the zona incerta of Lhx6-Cre mice after saline (l) or CNO (m) injection. A magnified image of l is shown in n. Scale bars, 100 μm. o, The percentage Fos+ neurons in hM4Di–mCherry+ Lhx6-expressing neurons in the zona incerta in mice injected with saline or CNO. *P < 0.05, two-tailed paired t-test; n = 3 mice, 8 sections for saline; n = 3 mice, 8 sections for CNO. Data are mean ± s.e.m. oq, Saline- or CNO-injection at ZT5 in the AAV8-DIO–mCherry group shows that CNO injection does not affect sleep. EEG analysis showing amount of time spent across ZT in wake (o), REM sleep (p) and NREM sleep (q). P > 0.1 for all behavioural readouts, two-way ANOVA followed by Sidak’s post hoc test, n = 4 mice for saline, n = 4 mice for CNO.