Rapid Eye Movement Sleep Deprivation Induces an Increase in Acetylcholinesterase Activity in Discrete Rat Brain Regions

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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 assayed 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.

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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 cholinergic 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 deprived 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 cholinergic 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 enzyme 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. £ of significance set at P 0.05. Specific activity of Achase is expressed as nmol of thiocholine formed min-1 mg protein-1. Data are reported as the mean ± SD (N = 6 for all groups). Final Results acetylthiocholine concentration in the assays was 1 mM.

Brain region Treatment Enzyme activity REM sleep deprivation induced a statistically significant increase in Achase activ- total particulate soluble ity in the brain regions listed in Table 1. Hippocampus Control 127.9 ± 17.2 141.7 ± 15.5 142.0 ± 20.8 Significant increases in soluble Achase ac- REMSD 126.6 ± 14.5 138.0 ± 16.2 123.9 ± 20.1 tivity were obtained in the medulla oblon- Striatum Control 692.0 ± 22.6 815.1 ± 75.4 409.7 ± 10.7 gata (t = 2.12, d.f. = 10, P<0.05), in total (t = REMSD 690.4 ± 20.1 800.1 ± 35.0 403.0 ± 79.9 2.44, d.f. = 10, P<0.025), and particulate Cerebral cortex Control 103.8 ± 8.6 124.3 ± 7.4 43.4 ± 6.5 enzyme activity (t = 1.86, d.f. = 10, P<0.05) REMSD 102.8 ± 8.9 121.5 ± 8.9 46.0 ± 8.6 in the pons and in total Achase activity in the

Braz J Med Biol Res 34(1) 2001 106 M.A.C. Benedito and R. Camarini

sults show that 96 h of REM sleep depriva- The pattern of change in Achase activity tion in rats induced a significant increase in after 96 h of REM sleep deprivation ob- Achase activity in the medulla oblongata, tained in our study showed that the increase pons and thalamus, without affecting the in enzyme activity occurred only in the brain enzymes activity in the striatum, hippocam- region where cholinergic neurons involved pus or cerebral cortex. The present data agree in REM sleep generation are located (the with the reported heterogeneous distribution pons) and in brain regions receiving a cho- of Achase activity in different regions of the linergic input (medulla oblongata, thalamus) rats brain (8,9) and they also indicate the from pontine cholinergic neurons (1,11). The need to carry out assays in discrete brain lack of change in enzyme activity in the areas rather than in large ones. striatum, hippocampus and cerebral cortex There are significant differences in suggests that the other cholinergic nuclei Achase activity among discrete regions (8). which innervate these brain regions (1,11) These differences are also observed in smaller were not affected by REM sleep deprivation. brain areas such as discrete nuclei (9). There- It has been demonstrated that Achase fore, the small significant differences (11 to levels increase in neuromuscular junction 15%) in enzyme activity obtained in our and brain tissue as a result of an increased study may be larger in more restricted brain frequency of Ach-receptor interaction (17, areas, although to explore this possibility 18). Higher output of Ach occurs in the pons will require further experimentation. (2), medulla oblongata (3) and thalamus (5) The increase in Achase activity in the during REM sleep and it is possible that the brainstem regions (pons and medulla oblon- pressure for REM sleep caused by depriva- gata) obtained in our study is in accordance tion leads to an overshoot in Ach release in with previous data which showed an in- an attempt to overcome the REM sleep dep- crease in total enzyme activity in the whole rivation. Therefore, the increase in Achase brainstem of rats after 96 h of REM sleep activity observed after REM sleep depriva- deprivation (7). However, our results differ tion may be the result of an increased release from another report which showed a de- of Ach above the normal levels needed to crease in free Achase activity in the medulla generate REM sleep. A decrease in musca- oblongata and pons and an increase in bound rinic M2-type cholinergic receptor binding enzyme activity in the medulla oblongata in the thalamus of rats deprived of REM after 96 h of REM sleep deprivation in rats sleep for 96 h (19) seems to support this (16). Although at this time there is no expla- hypothesis of increased synaptic Ach re- nation for these discrepancies, it is likely lease. that the different patterns of change obtained The noradrenergic neurons of the locus in the two sets of data are due to different coeruleus, located in the brainstem, decrease methodological approaches in the prepara- their firing rate in anticipation of REM sleep tion of the enzyme fractions used in the and almost cease firing during REM sleep, assays. For instance, we used a 900 g super- whereas a subset of cholinergic neurons in natant (freed of blood vessels and cell de- the pedunculopontine region of the brain- bris) to prepare the soluble and bound en- stem increase their firing rate and seem to be zyme, whereas the other study used the whole involved in the generation of REM sleep homogenate as the source of free and bound (20). Therefore, there is a physiological im- enzyme (16). Furthermore, we did not use balance between cholinergic and noradre- high NaCl concentrations or detergent to nergic neurotransmission in the brainstem release bound Achase, whereas in the other during REM sleep. It has been proposed that study they did (16). an imbalance between brain cholinergic/nor-

Braz J Med Biol Res 34(1) 2001 Acetylcholinesterase activity after REM sleep deprivation 107 adrenergic neurotransmission is involved in rons in the control of sleep and wakefulness. mood disorders. According to this hypo- Magnocellular regions of the basal forebrain thesis, depression is the result of overactive are involved in the regulation of the sleep- brain cholinergic neurotransmission and wake cycle and arousal seems to be medi- mania is the result of overactive noradrener- ated by basal forebrain cholinergic neurons gic neurotransmission (21). REM sleep dep- (36). Brainstem cholinergic neurons (pedun- rivation has antidepressant effects in hu- culopontine and laterodorsal tegmental nu- mans (22) and in rats it decreases immobility clei) project to the basal forebrain (37) and in the behavioral despair test, which has cholinergic basalis neurons project mono- been used for the screening of antidepres- synaptically to the neocortex and regulate sive drugs (23). REM sleep deprivation in- the EEG patterns characteristic of wakeful- duces a state of higher brain excitability (24) ness and REM sleep (36,38). Microinjection and a decreased sensitivity to cholinergic of carbachol, a cholinergic agonist, into the agonists, as demonstrated by a decrease in basal forebrain increases wakefulness, yawns (25). Depressed patients have dis- whereas microinjection into the pons induces turbed REM sleep, as shown by a shorter a state resembling REM sleep (39). We have REM sleep latency, more REM sleep epi- previously shown a significant decrease in sodes in the first part of the night and more membrane-bound Achase activity in the fron- eye movements during REM sleep (26). This tal cortex of REM sleep-deprived rats (12). disturbance in REM sleep is suggestive of a In the present study there was no difference dysregulation of cholinergic neurotransmis- in Achase activity in the cerebral cortex. sion in depression. Antidepressant treatments Cholinergic cerebral cortex innervation arises increase brain noradrenergic neurotransmis- in the basal forebrain neurons (11). The sion (27) and REM sleep deprivation also frontal cortex receives its cholinergic input has this effect; for instance, it induces an from cholinergic neurons located in the ros- increased availability of noradrenaline as tral nucleus basalis and substantia innomi- evidenced by an increase in whole brain nata, whereas the other cortical areas are noradrenaline turnover (28), an increase in innervated by cholinergic neurons located tyrosine hydroxylase mRNA and enzyme more caudally in the nucleus basalis and activity (29-31), a decrease in the activity of nucleus of the ansa lenticularis (11). The monoaminoxidase A (32,33), a downregula- decrease in Achase activity in the frontal tion of beta-adrenergic receptors (34), and a cortex (12) and the lack of change in enzyme decrease in the synthesis of cAMP stimulat- activity in the cerebral cortex suggest an ed by noradrenaline (35). These data may effect of REM sleep deprivation on a sub- suggest a counteracting effect of REM sleep population of forebrain basal cholinergic deprivation on the normal imbalance be- neurons. The pattern of the effect of REM tween cholinergic/noradrenergic neurotrans- sleep deprivation on Achase activity (de- mission observed during REM sleep. The creased activity in frontal cortex with no increase in Achase activity obtained in the change in cerebral cortex and increased ac- brainstem and thalamus and the reported tivity in pons, medulla oblongata and thala- decrease in cholinergic receptor binding (19) mus) seems to represent the functional com- may be the expression of the changed inter- plexity of cholinergic nuclei in the basal action between cholinergic/noradrenergic forebrain and brainstem and their role in neurotransmission in rats deprived of REM different brain functions (1,11,36,37,40). sleep. In conclusion, we observed an increase There is a relationship between brain- in Achase activity after 96 h of REM sleep stem and basal forebrain cholinergic neu- deprivation which was restricted to brain

Braz J Med Biol Res 34(1) 2001 108 M.A.C. Benedito and R. Camarini

regions either involved in REM sleep gen- needed to better understand the effects of eration or receiving cholinergic inputs di- REM sleep deprivation on cholinergic neu- rectly from cholinergic nuclei involved in rotransmission. REM sleep generation. Further studies are

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