Fig. 2: Kcnk9 knockdown in the locus coeruleus induces elevated nocturnal activity.

a Bilateral virus injection with pAAV-Syn-shRNAmir-Kcnk9-EF1a-eGFP (KD) or Syn-shRNAmir-scrambled-EF1a-eGFP (SC) in the locus coeruleus (LC): Top left: schematic coronal section of the mouse brain at the position −5.4 relative to bregma⁴³; red triangles represent the LC; top right: Immunofluorescence staining of a coronal section. Scale represents 1000 µm (4×). Bottom left: Images of the LC. Scale represents 200 µm (20×); bottom right: Magnification of LC cells. Scale represents 50 µm (60×), majority of tyrosine hydroxylase (TH; red) expressing cells co express GFP (green) (yellow arrows), while also GFP negative TH expressing cells could be observed (white arrow). Blue, DAPI; green, GFP; red, TH (TH serves as a norepinephrine marker). b Cell-based quantification of Kcnk9 gene knockdown (Kcnk9 KD) using Kcnk9-specific shRNAs by RT-qPCR compared with negative control (NT). Validated shRNAs with KD > 80%: 2, 3, 4, 5, 6, 7, 8, 9, and 10. ShRNA 6 was utilized for AAV generation in 293T cells. Arithmetic means of Kcnk9 expression of presented IDs were provided by Sirion Biotech. c Left: Kcnk9 knockdown efficiency validation in vivo. Kcnk9 expression analysis in the prefrontal cortex (PFC, blue circle, SC n = 8, KD n = 4), hippocampus (pink circle, SC n = 7, KD n = 4) and LC (red circle, SC n = 7, KD n = 4) by RT-qPCR revealed a significant down-regulation of Kcnk9 gene expression in the LC of Kcnk9 KD mice compared with mice injected with scrambled controls. Mann–Whitney U: P = 0.004. Right: Tyrosine hydroxylase (TH) RT-qPCR expression analysis in the LC to validate accuracy of tissue collection. LC samples of Kcnk9 KD (n = 4) and scrambled controls (n = 7) exhibit similar TH levels demonstrating sample collection accuracy. n = biologically independent mice. d Total locomotor activity in dark (12 h) phase, 21 days after AAV-injections. Mice injected with pAAV-Syn-shRNAmir-Kcnk9-EF1a-eGFP (n = 10) display increased nocturnal activity compared with pAAV-Syn-shRNAmir-scambled-EF1a-eGFP (n = 9) injected controls. Mann–Whitney U: P = 0.0101. e Y-maze percentage alternation analysis reveals tendency toward impaired working memory in Kcnk9 KD mice (n = 10) compared with age-matched controls (n = 10); n = biologically independent mice. Mann–Whitney U: P = 0.0797. Values are means ± SEM. Statistical analyses and approaches are provided in Supplementary Table 1. Source data are provided as a Source Data file.