Fig. 4: The projections from pPVT targeted nNOS-expressing neurons in the vmPFC.

a–c Dot plots showing the ratio of c-Fos and nNOS double-positive neurons to total nNOS-positive neurons (a), the number of c-Fos⁺ cells (b), and the ratio of c-Fos and nNOS double-positive neurons to total c-Fos-positive neurons (c) in the vmPFC when pPVT-vmPFC projections were excited by chemogenetics. n = 6. d–f Dot plots showing the ratio of c-Fos and nNOS double-positive neurons to total nNOS-positive neurons (d), the number of c-Fos⁺ cells (e), and the ratio of c-Fos and nNOS double-positive neurons to total c-Fos-positive neurons (f) in the vmPFC of CFA-injected mice when pPVT-vmPFC projections were inhibited by chemogenetics. n = 4. g Action potentials were evoked in a ChR2-eYFP-expressing neuron in pPVT in current-clamp mode by photostimuli pulses (blue light: 465 nm, 43.7 mW, 5 ms duration) at various frequencies (blue vertical lines). h Scheme indicating the pPVT area where AAV-CaMKIIα-ChR2-eYFP (0.2 μl) was microinjected and the vmPFC area where AAV-hSy-DIO-mCherry (0.4 μl) was microinjected. i Experimental setup for recording photostimulation-induced synaptic responses. ChR2-eYFP⁺ projections from the pPVT were excited by blue light (465 nm, 43.7 mW, 5 ms duration), and evoked EPSCs were recorded on mCherry⁺ nNOS-expressing neurons in the vmPFC. j, k Traces of light-evoked EPSCs recorded on nNOS-expressing neurons in the vmPFC, blocked by CNQX (10 μM) and AP-5 (50 μM) (j), or blocked by TTX (0.5 μM) and rescued by 4-AP (100 μM) (k). l Scheme indicating the pPVT area where AAV-CaMKIIα-hM3Dq-mCherry (0.2 μl) was microinjected, and the vmPFC area where AAV-hSyn-DIO-hM4Di-eGFP or AAV-hSyn-DIO-eGFP (0.4 μl) was microinjected into nNOS-Cre mice. m–o Distance in center (m, left) and total distance (m, right) in an OF test, time in open arms (n, left) and total entries in arms (n, right) in an EPM test, and withdrawal threshold of hind paw in a von Frey test (o) 30 min after CNO intraperitoneal injection (2 mg per kg) at day 21 after AAV microinjections. n = 15 and 16, respectively. Data are the mean ± SEM; **p < 0.01, and ***p < 0.001 (unpaired two-tailed Student’s t-test for (a–c) and (m–o); one-way ANOVA followed by Tukey’s post hoc test for (d–f)). Scale bar, 50 μm in (i). Source data are provided as a Source data file. Exact p values and additional statistical information can be found in Source data.