Neuroscience Letters, 130 (1991) 221-224 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100514W NSL 08032 221 Short-term REM sleep deprivation increases acetylcholinesterase activity in the medulla of rats Birendra Nath Mallick and Mahesh Thakkar School of Life Sciences, Jawaharlal Nehru University, New Delhi (India) (Received 15 April 1991; Revised version received 10 June 1991; Accepted 10 June 1991) Key words: Acetylcholinesterase; Rapid eye movement sleep; Deprivation; Platform; Brainstem; Medulla Involvement of cholinergic ponto-medullary brainstem mechanism regulating rapid eye movement (REM) sleep is known. Recently it was found that though short term REM deprivation influenced brainstem neuronal excitability, the activity of the brainstem acetylcholinesterase was not affected until after 96 h deprivation. Therefore, it was hypothesized that short-term REM deprivation might influence acetylcholinesterase in a restricted brainstem region. Results of this study show that the enzyme activity increased only in the medulla after 24 and 48 h REM deprivation. The flower pot technique was used for depriving the experimental rats of REM sleep. Suitable control experiments were conducted to rule out the possibility of non-specific effects. Thus, the medullary cholinergic mechanism probably is more important for REM. The brainstem mechanism for rapid eye movement (REM) sleep is well documented [9, 13, 17, 24--26, 29]. It is difficult to comment on which of the REM related neurons, REM-on or REM-off, are more important for REM generation. The importance of the ponto-medul- lary region [16, 24--26, 29] and/or, an interaction between the brainstem cholinergic and aminergic neurons [13, 14] has been put forward. The role of acetylcholine (ACh) in relation to REM sleep has been extensively studied [1- 3, 5, 7-10, 12, 15, 17, 28, 32, 35]. Recently, we have shown that the acetylcholinesterase (ACHE) level in- creases first in the brainstem of REM sleep-deprived rat [30]. Though 48 h REM deprivation did not affect the brainstem AChE activity [30], only 20 h deprivation was sufficient to affect the brainstem single neuronal activity [19, 20]. It was hypothesized that if the brainstem choli- nergic mechanism does play a significant role in REM sleep, the level of AChE might increase first at the site of the REM generator. In an attempt to shed light on this matter, the effect of 24 and 48 h REM sleep depriva- tion was investigated on midbrain, pons and medulla AChE activity. Experiments were conducted on male albino rats weig- hing between 220 and 280 g. The animals were main- tained in the animal house under 12:12 h light-dark cycle and food and water ad libitum. REM deprivation was continued on experimental (E) group of rats for 24 and Correspondence: B.N. MaUick, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India. 48 h by the flower pot technique [33]. Free moving con- trol (FMC), large platform control (LPC) and recovery (R) experiments were performed. Details of experimen- tal procedures are mentioned in a previous report [30]. In brief, FMC rats were maintained in rat cages in the same room along with E, LPC and R rats. LPC and E rats were subjected to similar environmental conditions. Each rat of the former group was maintained on a 6.5 cm diameter island while that of the latter group on an island of 13.5 cm diameter projecting above a pool of water. For the R group, rats were subjected to a treat- ment similar to that of the E group (48 h) followed by maintaining them in normal rat cages for 48 h. Brains were removed after decapitation. Medulla, pons and midbrain were separately dissected out and homogenized in 1 M saline buffer containing 1% Triton X-100 (v/v). The AChE activity was estimated by the method of Ellman et al. [6] and the protein concentration was estimated by the method of Lowry et al. [18] as reported earlier [30]. In short, for AChE estimation the reaction mixture contained 0.168 M phosphate buffer (pH 8.0), 0.01 mM DTNB (Sigma) and 0.01 mM acetyl- thio-choline (Sigma) and increase in the absorbance was observed spectrophotometrically (Shimadzu UV 260) at 412 nm for 5 min. For protein estimation the reaction mixture contained 5 ml of Lowry's reagent (48 ml of 2% Na2CO3 in 0.1 N NaOH + 1 ml 0.5% CuSO4 + 1 ml of 1% Na/K tartarate) and 0.5 ml of Folin's reagent (SRL, India). The mixture was incubated for 30 min at room temperature and the colour developed was read at