Experimental protocol used in the study
| Groups | Stereotaxic surgery | Experimental procedure | ||
|---|---|---|---|---|
| Group I (n = 5) | Microinjection of Ctrl-shRNA and TH-shRNA into the left and right LC, respectively, during surgery | Postsurgical recovery for 8 d | 8 d after injection, LC punched out for TH mRNA estimation by qRT-PCR | |
| Group II (n = 4) | 8 d after injection LC punched out for TH protein estimation by Western blot | |||
| Group III (n = 5) | 8 d after injection 40 μm brain sections through LC were immunostained with TH and positive neurons counted | |||
| Group IV (n = 6) | Microinjection of TH-shRNA bilaterally into LC during surgery and implantation of cannula bilaterally into PPT | 8 d after TH-shRNA injection into LC, on day 1 saline was injected bilaterally into PPT and sleep wakefulness recorded for 12 h.On day 2, NA was injected into PPT and sleep–wakefulness recorded in the same rat for 12 h.3 weeks after TH-shRNA injection, sleep wakefulness was recorded for 12 h | ||
| Group V (n = 5) | Microinjection of Ctrl-shRNA bilaterally into LC during surgery | 8 d postsurgery injection sleep–wakefulness recording was done for 12 h | ||
| Group VI (n = 6) | Implantation of cannula bilaterally into LC | After postsurgical recovery, first day either Ctrl-siRNA or TH-siRNA was microinjected bilaterally into LC and sleep–wakefulness recorded next day for 12 h.After 7 d Ctrl-siRNA (which was not injected on the first day) was injected and recording continued. | ||
| Group VII (n = 10) | FMC | FMC home cage without any microinjection | Whole-brain homogenate was prepared and(1) Na-K ATPase activity was estimated(2) TH protein estimation by Western blot | |
| Group VIII (n = 5) | Implantation of cannula into LC bilaterally | REMSD for 96 h with microinjection of Ctrl-siRNA at 24 and 48 h | Whole-brain homogenate was prepared, and Na-K ATPase activity was estimated | |
| Group IX (n = 5) | Implantation of cannula into LC bilaterally | REMSD for 96 h with microinjection of TH-siRNA at 24 and 48 h | Whole-brain homogenate was prepared and Na-K ATPase activity was estimated | |
| Group X (n = 5) | Microinjection of Ctrl-shRNA during surgery into LC bilaterally | After recovery REMSD for 96 h | Whole-brain homogenate was prepared and(1) Na-K ATPase activity was estimated(2) Western blot analysis of TH protein was performed | |
| Group XI (n = 5) | Microinjection of TH-shRNA during surgery bilaterally into LC | After recovery REMSD for 96 h | Whole-brain homogenate was prepared and(1) Na-K ATPase activity was estimated and(2) Western blot analysis of TH protein was performed | |
| Total no. of Rats | 61 | |||