Theme : Dreams

Decrease in Muscarinic M2 Receptors from Synaptosomes in the Pons and Hippocampus after REM Sleep Deprivation in Rats

Rafael J. Salín-Pascual, Mauricio Díaz-Muñoz, Lorena Rivera-Valerdi, Leonardo Ortiz-López and Carlos Blanco-Centurión
Departamento de Fisiología, Universidad Nacional Autonoma de Mexico, México, D.F. 04510, México
Abstract
The effects of both REM sleep deprivation and its recovery on pontine and hippocampus muscarinic M2 receptors were investigated in synaptosomes using [3H]-AF-DX 384 as a ligand. Animals were divided into three groups: REM sleep deprivation group (small platforms 6.5 cm of diameter); stress group (large platforms 14 cm of diameter) and cage control group. In a second experiment REM sleep-deprived animals were allowed 48 h of recovery. REM sleep-deprived rats showed a reduction in M2 receptors compared with both intact and stress groups. Changes in M2 receptors were also observed after 48 h of recovery from REM sleep deprivation only in hippocampus. The enhancement of acetylcholine release during both REM sleep deprivation and recovery could explain the present findings.

Current Claim: REM sleep deprivation reduces the density of M2 receptors in the pontine area.

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It is known that selective deprivation of REM sleep increases the propensity for REM sleep, as shown by the shortening of REM sleep latency and lengthening of REM sleep duration (Siegel and Gordon, 1965). The physiological mechanisms responsible for the REM sleep rebound remain obscure, although considerable attention has been given to cholinergic mechanisms since they play such a prominent role in the regulation of REM sleep (Gillin et al., 1993).
In a previous study (Salín-Pascual et al., 1992a) it was shown that low doses of biperiden (selective M1 antagonist), which had no effect on REM sleep during baseline, significantly attenuated or blocked the increase in REM sleep rebound in cats subjected to REM sleep deprivation by the platform method. These data could mean that after REM sleep deprivation a decrease in M1 muscarinic receptors may occur. On the other hand, rats subjected to chronic total sleep deprivation by the disk-over-water method (studied with L-[3H]-nicotine and [3H]-quinuclidinyl benzilate) did not show changes in nicotine receptors while muscarinic receptors were modified mainly in limbic and septal regions (Tsai et al., 1994).

Tufik et al. (1987) reported that REM sleep deprivation for 90 h by the platform method produced a reduction in yawning behavior induced either by physostigmine or pilocarpine. Also, 120 h of REM sleep deprivation produced the same result but revealed no differences with the Stress group (large platforms). When a binding assay was performed, the REM sleep-deprived group showed a reduction in muscarinic receptor density, while in the stress control group the main difference was in the affinity to [3H]-methyl scopolamine (Salín-Pascual et al., 1992b).

Kushida et al. (1995), using in situ hybridization histochemistry to study the effects of REM sleep deprivation on M1, M2 and M3 mRNA expression of muscarinic receptors, found that 72 h of REM sleep deprivation did not affect M1 mRNA expression; however, a significant increase in M3 and decrease in M2 receptors' mRNA expression in pontine nuclei were found. Nuñes Jr. et al. (1994), using an autoradiographic technique with AF-DX-384, an M2 selective antagonist, found a down-regulation of M2 receptors after 96 h of REM sleep deprivation.

The main problem with AF-DX ligands is that they bind with two types of muscarinic sites in homogenates of rat brain; one with high affinity and low capacity, and the second with low affinity and high capacity (Lapchak et al., 1989). This may indicate that two M2 receptor populations can be detected, namely presynaptic and postsynaptic receptors. Synaptosomes can be more selective than membrane preparation and usually possess high density for presynaptic receptors. So we decided to study synaptosomes from two brain regions, the pontine and hippocampus, with the M2 antagonist AF-DX-384.

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Animals
Male Wistar rats (180-200 g) were housed in a temperature-controlled room with 12:12 h light-dark cycle (08:00 - 20:00 hours). They were divided into three groups: (a) REM sleep deprivation group (-REM) (small platforms of 6.5 cm diameter); (b) stress group (Stress group, large platforms of 14 cm diameter); and (c) cage control group (Intact group). Animals remained on the platforms for 96 h. After that, all animals were sacrificed, the brain removed and pontine and hippocampus regions were obtained. In a second experiment REM sleep-deprived animals were allowed 48 h of recovery after the 96 h of platforms (48 h-rebound (REB)). They were also sacrificed and the same brain regions were obtained.
Materials
[2,3-Dipropylamino-3H]-AF-DX 384 (133 Ci/mmol) was obtained from DuPont-NEN Research Products (Boston, MA). Atropine sulphate was obtained from Sigma Chemical Co. (St Louis, MO). Other chemicals used were reagent grade of the best quality available.

Synaptosomal fraction
Synaptosomes were isolated from the region mentioned above, as described by Löscher et al. (1985). This procedure allows a purified synaptosomal fraction from several regions to be obtained using only one rat brain. Previous studies showed that synaptosomes were in a good state of preservation and essentially free from myelin and mitochondria (Díaz-Muñoz and Tapia, 1988).

[2,3-Dipropylamino-3H]-AF-DX 384 binding to muscarinic M2 sites in synaptosomal fractions
[3H]-AF-DX 384 binding was determined essentially as described by Lapchak et al. (1989) with some minor modifications (Hamilton et al., 1990). [ 3H]-AF-DX 384 was incubated for 60 minutes at 4°C with 80 µg of synaptosomal fraction protein in 0.25 ml binding buffer containing: NaCl 120 mM; MgSO4 1.2 mM; KH2PO4 1.2 mM; glucose 5.6 mM; NaHCO3 25 mM; CaCl2 2.5 mM; KCl 4.7 mM (pH 7.4). [3H]-AF-DX 384 was used at concentrations ranging from 1 to 150 nM. Non-specific binding was defined in the presence of 10 µM of unlabeled atropine sulfate. At the end of the incubation time, the samples were filtered through Whartman GF/F glass fiber filters using a multifilter apparatus (Brandel, Gaithesburg, MD). The filters were washed with 5 ml aliquots of cold 0.3 M KCl and counted in liquid scintillation, after the addition of 10 ml of Trisol (Fricke, 1975). Scatchard plots were analyzed by linear regression. Previous studies had shown that equilibrium conditions were reached with this experimental protocol (data not shown).

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Figure 1

Figure 2

Figure 3

Figure 4

As shown in Figure 1 a significant reduction in [3H]-AF-DX 384 binding was observed in pons synaptosomes after 96 h of REM sleep deprivation, while no changes were observed in the Stress group or after 48 h of REM sleep recovery (48 h-REB group). Figure 2 shows the hippocampus synaptosome data. Changes in [3H]-AF-DX 384 binding were observed only after 48 h of recovery. Scatchard plots are depicted in Figures 3 and 4 for pons and hippocampus, respectively. As illustrated, the reduction in the binding of M2 receptors in pons (Figure 3) was due to a reduction in their Bmax (density of neurotransmitter receptors) (Intact: 124.7; -REM: 85.9 fmol/mg of protein, Students "t" test p < 0.001), without significant changes in the Kd (dissociation constant). The Scatchard analysis for hippocampus (Figure 4) also showed that the binding differences were due to the reduction in the number of M2 muscarinic receptors after 48 h of recovery (Intact: 139.9; 48 h REB: 117.1 fmol/mg of protein).

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The main findings of the present study were that M2 muscarinic receptors in pontine synaptosomes were reduced after 96 h of REM sleep deprivation and also in hippocampus synaptosomes after 48 h of recovery from REM sleep deprivation.
M2 reduction was related to their density without significant changes in receptor affinity in both pons and hippocampus. One possible explanation of the pontine findings is that during REM sleep deprivation an increase in acetylcholine availability may occur, thus causing the down-regulation of M2 receptors. Kodama et al. (1990) have observed an increase in acetylcholine production when cats are in REM sleep. If this sleep stage is suppressed, acetylcholine production may increase and be responsible for the REM sleep pressure that results from its deprivation. Several groups have shown that some cholinergic-related behavior such as yawning is reduced after REM sleep deprivation (Tufik et al., 1987; Salín-Pascual et al., 1992b). On the other hand, studies in which M2 receptors were measured after REM sleep deprivation found a reduction in the binding sites using in situ hybridization and autoradiography (Kushida et al., 1995; Nuñes Jr. et al., 1994). To the best of our knowledge the present is the first study to show a reduction of M2 receptors in synaptosomes taken from pontine regions after REM sleep deprivation. These data support the assumption that acetylcholine is important in REM sleep regulation and also that REM sleep suppression may have some effect on these receptors, but down-regulation of muscarinic receptors could not be related to REM sleep rebound. Sutin et al. (1986) reported that chronic administration of scopolamine to rats produce an up-regulation of muscarinic receptors, assessed by binding assay, and also an important increase in REM sleep time when scopolamine injections were stopped. Also, different cholinergic agonists such as carbachol (Datta et al., 1991), arecoline (Gillin et al., 1991) or even nicotine (Salín-Pascual et al., 1995), produced a REM sleep enhancement effect.

The down-regulation of M2 in hippocampus could be the result of an increase in acetylcholine release in this region when animals are in REM sleep rebound. In fact, theta rhythms that are very robust during REM sleep have been related to this brain region (Bland, 1986). Marrosu et al. (1995), measured acetylcholine release in the cerebral cortex and dorsal hippocampus by microdialysis during the sleep-waking cycle in freely moving cats, and found about a four-fold increase in acetylcholine release during REM sleep in the hippocampus over the level obtained during slow wave sleep. In our data, animals with REM sleep rebound could have enough acetylcholine release to down-regulate M2 receptors in this area. One of the physiological relationships between the hippocampus and REM sleep is with memory processing (Smith, 1995); REM sleep deprivation results in memory deficits that could be related to some degree to changes in the cholinergic transmission in hippocampus.

Several lines of evidence suggest that the M2 receptor subtypes may have a presynaptic location on cholinergic terminals. The distribution of M2 receptors corresponded to the histochemical visualization of acetylcholinesterase (Mash and Potter, 1986). In rats there is also a co-localization of M2 with the high-affinity choline uptake site density, which is found at presynaptic terminals (Aubert et al., 1996). Muscarinic regulation of acetylcholine release has been shown to be mediated by M2 receptor subtype (Reiteri et al., 1984). The effect of oxotremorine and pilocarpine on striatal acetylcholine release were investigated using brain microdialysis techniques, oxotremerine, a preferential M2 agonist, dose-dependently decreased acetylcholine release in the striatum, and this effect was blocked by scopolamine but not by pirenzepine. These results suggest that oxotremorine-induced decrease in striatal acetylcholine release is due to stimulation of presynaptic M2 autoreceptors (Murakami et al., 1996). Our results may indicate that the cholinergic system is adapted to REM sleep deprivation and the possible increase in acetylcholine release through a reduction in the number of M2 presynaptic receptors in order to reduce the release of this transmitter.

The down-regulation of M2 receptors could be the result of the way in which animals are REM sleep deprived, which is by waking them each time they enter REM sleep. Another possible explanation is that M2 receptors are not involved in REM sleep rebound but are affected by its suppression. This seems to be the case with dopamine, in which REM sleep deprivation up-regulates its receptors but these changes are not related to the REM sleep rebound (Salín-Pascual et al., 1997).

Further investigations on the behavior of acetylcholine during REM sleep deprivation are necessary in order to clarify why muscarinic M2 receptors are reduced after REM sleep deprivation and the possible role of this system in the REM sleep recovery phenomenon.

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This work has been supported by DGPA-UNAM grant IN-200895 to RJS-P.
This work has been presented as an abstract at the 11th Annual Meeting of the Associated Professional Sleep Societies (San Francisco, CA, June, 1997).

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