Brazilian Journal of Medical and Biological Research (2001) 34: 103-109 Acetylcholinesterase activity after REM sleep deprivation 103 ISSN 0100-879X Rapid eye movement sleep deprivation induces an increase in acetylcholinesterase activity in discrete rat brain regions M.A.C. Benedito Departamento de Psicobiologia, Universidade Federal de São Paulo, and R. Camarini São Paulo, SP, Brasil Abstract Correspondence Some upper brainstem cholinergic neurons (pedunculopontine and Key words M.A.C. Benedito laterodorsal tegmental nuclei) are involved in the generation of rapid · REM sleep deprivation · Departamento de Psicobiologia eye movement (REM) sleep and project rostrally to the thalamus and Acetylcholinesterase Universidade Federal de São Paulo · caudally to the medulla oblongata. A previous report showed that 96 Brain regions Rua Botucatu, 862 · Thalamus h of REM sleep deprivation in rats induced an increase in the activity 04023-062 São Paulo, SP · Medulla oblongata Brasil of brainstem acetylcholinesterase (Achase), the enzyme which inacti- · Pons vates acetylcholine (Ach) in the synaptic cleft. There was no change in Research supported by FAPESP and the enzymes activity in the whole brain and cerebrum. The compo- Associação Fundo de Incentivo à nents of the cholinergic synaptic endings (for example, Achase) are Psicofarmacologia (AFIP). not uniformly distributed throughout the discrete regions of the brain. R. Camarini was the recipient of In order to detect possible regional changes we measured Achase a FAPESP fellowship. activity in several discrete rat brain regions (medulla oblongata, pons, thalamus, striatum, hippocampus and cerebral cortex) after 96 h of Received December 6, 1999 REM sleep deprivation. Naive adult male Wistar rats were deprived of Accepted September 25, 2000 REM sleep using the flower-pot technique, while control rats were left in their home cages. Total, membrane-bound and soluble Achase activities (nmol of thiocholine formed min-1 mg protein-1) were as- sayed photometrically. The results (mean ± SD) obtained showed a statistically significant (Student t-test) increase in total Achase activity in the pons (control: 147.8 ± 12.8, REM sleep-deprived: 169.3 ± 17.4, N = 6 for both groups, P<0.025) and thalamus (control: 167.4 ± 29.0, REM sleep-deprived: 191.9 ± 15.4, N = 6 for both groups, P<0.05). Increases in membrane-bound Achase activity in the pons (control: 171.0 ± 14.7, REM sleep-deprived: 189.5 ± 19.5, N = 6 for both groups, P<0.05) and soluble enzyme activity in the medulla oblongata (control: 147.6 ± 16.3, REM sleep-deprived: 163.8 ± 8.3, N = 6 for both groups, P<0.05) were also observed. There were no statistically significant differences in the enzymes activity in the other brain regions assayed. The present findings show that the increase in Achase activity induced by REM sleep deprivation was specific to the pons, a brain region where cholinergic neurons involved in REM generation are located, and also to brain regions which receive cholinergic input from the pons (the thalamus and medulla oblongata). During REM sleep extracellular levels of Ach are higher in the pons, medulla oblongata and thalamus. The increase in Achase activity in these brain areas after REM sleep deprivation suggests a higher rate of Ach turnover. Braz J Med Biol Res 34(1) 2001 104 M.A.C. Benedito and R. Camarini Introduction brain. Striatum cholinergic innervation, on the other hand, is mostly due to local inter- Brainstem cholinergic neurotransmission neurons (for reviews, see 1,11). is involved in the generation and mainte- nance of rapid eye movement (REM) sleep. Material and Methods There is strong evidence that pontine cholin- ergic and cholinoceptive neurons, interact- Animals ing in coordination, trigger and maintain REM sleep (for a review, see 1). Higher Three-month-old, naive adult male Wistar cholinergic activity during REM sleep has rats, bred in our colony, and weighing 250- been shown by means of microdialysis. 300 g were used in this study. After weaning, Higher acetylcholine (Ach) output occurs in the rats were housed in wire-mesh cages in the pons (2), medulla oblongata (3), hippo- groups of 3 and kept in a room with a con- campus and cerebral cortex (4) in cats, and trolled light-dark cycle (lights on from 7 a.m. in the thalamus in rats (5), during REM sleep to 7 p.m.) and temperature (22 ± 2oC). The as compared to synchronized sleep. animals had free access to tap water and Previous data have shown an increase in Purina® lab chow until the time of sacrifice. the activity of acetylcholinesterase (Achase), The handling of animals was limited to room the enzyme that inactivates Ach in the syn- and cage cleaning. aptic cleft (6), in the brainstem of rats de- prived of REM sleep for 96 h (7). There were REM sleep deprivation no changes in the enzymes activity in the whole brain or cerebrum. There are signifi- REM sleep deprivation started at 9 a.m. cant regional differences in the levels of the Animals were placed individually in water components of cholinergic nerve endings in tanks on a small flower-pot for 96 h. The the brain. Levels of choline and Ach (8), flower-pot was 6.5 cm in diameter surrounded choline acetyltransferase and Achase activ- by water 2 cm below its surface. The control ity (8,9) and cholinergic receptors (10) are rats were kept individually in their home not uniformly distributed throughout the re- cages. A large platform control group, to gions of the brain. These regional differ- control the stress of the procedure, was not ences indicate differences in the density and included as it has been shown that there is no activity of cholinergic nerve endings. This change in Achase activity in such groups suggests that regional studies are most ap- (7,12). propriate to detect changes in brain cholin- ergic neurotransmission after REM sleep Enzyme assay deprivation. In order to address this issue we studied the effect of 96 h of REM sleep After decapitation, the brains were ex- deprivation on Achase activity in several cised and kept on a Petri dish cooled with discrete brain areas (medulla oblongata, pons, crushed ice. The brains were washed with thalamus, striatum, hippocampus and cere- cold isotonic saline to remove blood and the bral cortex) in rats. These brain regions were brain regions were then immediately dis- chosen because of their different cholinergic sected and weighed. The tissue was kept in inputs. Cholinergic neurons located in the cold 0.32 M sucrose (pH 7.4 with Tris base). pons project to the thalamus and medulla Homogenates (5% w/v) were prepared oblongata, whereas the cholinergic inputs to using glass homogeneizer tubes and a the hippocampus and cerebral cortex arise in Teflon motor-driven pestle. The homoge- cholinergic nuclei located in the basal fore- nates were centrifuged at 900 g for 10 min at Braz J Med Biol Res 34(1) 2001 Acetylcholinesterase activity after REM sleep deprivation 105 4oC and the supernatants were collected and thalamus (t = 1.82, d.f. = 10, P<0.05). centrifuged at 100,000 g for 60 min at 4oC There were no statistically significant dif- (13). The supernatants from this step were ferences in Achase activity (total, particulate the source of soluble Achase and the result- or soluble) in the hippocampus, striatum or ing pellets were resuspended in the original cerebral cortex (P>0.05 for all of the com- volume with cold 0.32 M sucrose. The latter parisons) (Table 2). preparation was the source of membrane- bound Achase. Enzyme activity was also Discussion assayed in the 900 g supernatant (total en- zyme activity). Homogenates were kept at It has previously been shown that 96 h of -20oC until they were assayed. REM sleep deprivation does not change total Acetylcholinesterase activity was assayed Achase activity in the whole brain or cere- according to Ellman et al. (14) as described brum of rats (7). However, the present re- in detail elsewhere (12). Acetylthiocholine was used as the substrate in a 1-mM final Table 1 - Specific activity of total, particulate and soluble acetylcholinesterase (Achase) assay concentration. The thiocholine formed in discrete brain regions involved in the generation of, or affected by, REM sleep, after was measured spectrophotometrically at 412 REM sleep deprivation (REMSD). nm. Proteins were assayed using bovine al- Specific activity of Achase is expressed as nmol of thiocholine formed min-1 mg bumin as the standard (15). protein-1. Data are reported as the mean ± SD (N = 6 for all groups). Final The enzyme activity is expressed as nmol acetylthiocholine concentration in the assays was 1 mM. *P<0.05 and **P<0.025 vs control (Student t-test). thiocholine formed min-1 mg protein-1. Brain region Treatment Enzyme activity Reagents total particulate soluble All reagents were analytical grade from Medulla oblongata Control 122.1 ± 7.1 139.6 ± 7.5 147.6 ± 16.3 Sigma Chemical Co. (St. Louis, MO, USA) REMSD 122.5 ± 5.5 138.7 ± 5.4 163.3 ± 8.3* or Merck S.A. (Rio de Janeiro, RJ, Brazil). Pons Control 147.8 ± 12.8 171.0 ± 14.7 141.5 ± 31.0 Twice-distilled water prepared in an all-glass REMSD 169.3 ± 17.4** 189.5 ± 19.5* 158.3 ± 27.2 apparatus was used throughout the assays. Thalamus Control 167.4 ± 29.0 158.8 ± 32.5 106.7 ± 23.0 REMSD 191.9 ± 15.4* 173.1 ± 11.2 128.1 ± 39.5 Statistical analysis The unpaired Student t-test was used for Table 2 - Specific activity of total, particulate and soluble acetylcholinesterase (Achase) comparisons between groups, with the level in discrete brain regions of REM sleep-deprived (REMSD) rats.