Additional file 1 of Investigations of brain-wide functional and structural networks of dopaminergic and CamKIIα-positive neurons in VTA with DREADD-fMRI and neurotropic virus tracing technologies
Creators: name: Zheng, Ning
name: Gui, Zhu
name: Liu, Xiaodong
name: Wu, Yang
name: Wang, Huadong
name: Cai, Aoling
name: Wu, Jinfeng
name: Li, Xihai
name: Kaewborisuth, Challika
name: Zhang, Zhijian
name: Wang, Qitian
name: Manyande, Anne
name: Xu, Fuqiang
name: Wang, Jie
Publisher: name: figshare
Year: 2024
Subjects: Medicine
Neuroscience
Science Policy
Mental Health

description: Additional file 1: Methods. Animal behavioral testing. Figure S1. Expression of hM3D(Gq) in CamKIIα-positive neurons in the VTA. (A) the cre-dependent neurons expressing hM3D (Gq) and mCherry proteins (red); (B) CamKIIα neurons were stained by the antibody of Anti-CamKII (green); (C) co-locations of the CamKIIα neurons (yellow) by merging the results of the virus labeling (A) and immunohistochemistry (B). Panels (D), (E), (F) are higher-magnification images of boxed regions in (A), (B), (C), respectively. The nuclei were stained blue by DAPI, and the scale bar: 200 μm. Figure S2. Immunofluorescent c-Fos staining in the VTA of CamKIIα-cre rats infected with hM3D(Gq) encoding rAAV after the activation with CNO (chemogenetic activation) or saline. (A–C) Representative coronal sections show the virus-labeled CamKIIα neurons (red, A), and the immunofluorescent CamKIIα neurons with c-Fos staining (green, B) and the co-localization neurons (yellow, C) of in the VTA after the chemogenetics activation of VTA CamKIIα neurons; Panels A1, B1 and C1 are higher-magnification images of the boxed regions in panels of A, B and C, respectively; (D–F) the saline injection did not cause obvious c-Fos signal in the region of VTA of CamKIIα-cre transgenic rats; (D) virus-labeled CamKIIα neurons (red); (E) results of immunofluorescent c-Fos staining; (F) the merged results of the virus labeling (D) and c-Fos immunohistochemistry (E); Panels D1, E1 and F1 are higher-magnification images of boxed regions in the panels of D, E and F, respectively. Note: The nuclei were stained blue by DAPI. The scale bar: 200 μm. Figure S3. Chemogenetics activation of dopaminergic neurons in VTA resulted in hyperactivity. (A, D) The heatmap illustrates the position of the animals in the open field after the injection of CNO (A) or Saline (D). The color gradient from blue to red represents the duration of time the animal spends in a particular position, with red indicating a longer duration. (B–C) The distance traveled after CNO injection was significantly higher compared to the distance traveled after saline injection (B), there was no significant difference in the duration traveled (C). (E–F) There was no significant difference in locomotion distance (E) or duration (F) before the injection of CNO and Saline. N = 5, Two-tailed t-test, Ave. ± STD; *p &lt; 0.05; **p &lt; 0.01. Figure S4. Chemogenetics activation of CamKIIα-positive neurons in the VTA resulted in hyperactivity. (A, D) The heatmap depicts the animal’s position in the open field after the injection of CNO (A) or Saline (D). (B–C) The distance (B) and duration (C) traveled after CNO injection were significantly higher compared to those after saline injection. (E–F) There were no significant differences in locomotion distance (E) or duration (F) before the injection of CNO and Saline. Note: N = 5, Two-tailed t-test, Ave. ± STD; *p &lt; 0.05; **p &lt; 0.01.