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mise à jour du
25 octobre 2009
Brain Res.
Decreased muscarinic receptor binding in rat brain after paradoxical sleep deprivation: an autoradiographic study
Nunes Júnior GP, Tufik S, Nobrega JN.
Departamento de Psicobiologia, Escola Paulista de Medicina, São Paulo, Brazil.


Previous work demonstrated that paradoxical sleep deprivation (PSD) leads to a decrease in yawning behavior elicited by cholinergic agonists, suggesting that a downregulation of cholinergic muscarinic receptors may occur after PSD. More recent work using intracerebral injections of muscarinic agonists has suggested a critical role for M2 receptors in paradoxical sleep. In this study [3H]AF-DX 384 was used to investigate the effects of PSD on M2-type cholinergic receptors throughout the brain using quantitative autoradiography.
After 96 h of paradoxical sleep deprivation, [3H]AF-DX 384 binding was generally reduced throughout the brain, and significantly so in the olfactory tubercle (-20%), n. accumbens (-23%), frontal caudate-putamen (-16%), islands of Callejas (-20%), piriform cortex (-24%), lateral (-26%) and medial (-24%) septum, anteromedial (-19%), ventrolateral (-22%), and lateral geniculate (-15%) nuclei of thalamus, deep layers of the superior colliculus (-15%), entorhinal cortex (-12%) and subiculum (-23%). [3H]AF-DX 384 binding was reduced in pontine structures, but not to a higher degree than in other brain areas. The observed downregulation of M2-type muscarinic receptors after PSD may be causally related to the previously reported decrease in cholinergically induced behaviors after PSD.
I. Introduction
Considerable evidence has established an important role for brain cholinergic neurons in paradoxical sleep mechanisms and in the functional consequences of paradoxical sleep deprivation. Paradoxical sleep deprivation induces a number of behavioral alterations in rats, including a decrease in yawning behaviour induced by cholinergic agonists such as pilocarpine or physostigmine. This has led to the hypothesis that PSD induces a down-regulation of muscarinic receptors in brain. The discovery of different muscarinic receptors subtypes and the availability of drugs with relative selectivity for subtypes of muscarinic receptors, has prompted efforts to characterize the involvement of the various muscarinic receptor subtypes in the regulation of paradoxical sleep.
Intrapontine injections of muscarinic agonists such as oxotremorine-M and cis-dioxolane, which have higher selectivity for M2 than for M1 receptors, have been shown to elicit paradoxical sleep. This was not observed with agonists with higher selectivity for M1 than M2, such as McN-A-343, suggesting that M2-type muscarinic receptors play an important role in paradoxical sleep induction and maintenance. The aim of the present study was to examine the effects of paradoxical sleep deprivation on regional brain M2-type receptor binding, using quantitative receptor autoradiography. A variety of procedures have been used to label M2 muscarinic sites in the rat brain, most of which have relied on addition of nicotinic and M1 blockers to radiolabeled non-selective muscarinic ligands such as [3H]acetylcholine, [3H]QNB and [3H]N-methylscopolamine [7,20,24,27,28]. The first antagonist used to label M2 sites directly was [3H]AF-DX 116 . However, this compound lacks stability and shows a poor signal-to-noise ratio in binding assays.
More recently, the related tricyclic compound [3H]AFDX 384 (5,11-dihydro-ll-[[(2-(2-[(dipropylamino)methyl]- 1-piperidinyl)ethyl)amino]cabonyl-6H-pyrido-[2,3-b]- [1,4]-benzodiazepin-6-one) has been shown to possess good stability, excellent signal-to-noise ratio, and good selectivity for M2 sites. It was therefore chosen to examine the effects of paradoxical sleep deprivation on M2-1ike sites in the present study.
4. Discussion
Tufik et al. first hypothesized that the decrease of cholinergically-induced yawning behaviour after paradoxical sleep deprivation could be due to a downregulation of central muscarinic receptors. The present results provide direct support for that hypothesis by demonstrating that PSD induces a generalized decrease in [3H]AF-DX 384 binding throughout the brain. This is in line with recent evidence implicating brain muscarinic M2 receptors in paradoxical sleep mechanisms. However, several points should be noted in connection with possible functional implications of the present findings.
4.1. M2 specificity of [3H]AF-DX 384 binding Although [3H]AF-DX 384 appears at present to be the ligand of choice for the characterization of M2 receptors in various tissues, it should be noted that this ligand also shows activity at cloned m4 receptors. In addition, recent in situ hybridization work suggests that most of the muscarinic receptors in areas such as the caudate-putamen and olfactory tubercle belong to the m4 class. Although the identity and function of a putative central M4 receptor corresponding to the m4 gene is still not fully established, the preceding considerations suggest that at least in some brain areas the observed decreases in binding may not be exclusively attributable to a decrease in M2 sites. Thus at present the most conservative interpretation is that the observed binding changes reflect alterations in M2/M4 receptors.
4.2. Anatomical circuitry of functional effects Assuming that M2-type receptors are importantly involved in altered cholinergic responses seen after PSD, it had been expected that autoradiographic mapping would provide important information regarding the neuroanatomical substrates of such responses. However, statistically significant changes in [3H]AF-DX 384 were seen in limbic (accumbens, piriform and entorhinal cortices, septum, anteromedial thalamus and subiculum), motor (frontal caudate-putamen and ventrolateral thalamus) visual (lateral geniculate nucleus), ocular (superior colliculus) and olfactory (olfactory tubercle) areas. Thus, the widespread nature of the observed effects does not provide clear clues as to the specific neuroanatomical circuitry involved in altered cholinergic responses associated with PSD. Among other brain systems, mediodorsal pontine structures have since long been known to be important in paradoxical sleep [18], and recent work suggests an important role for M2-mediated processes in the medial pontine reticular formation in the generation of paradoxical sleep [33,34]. In the present study reductions of approximately 22% in [3H]AF-DX 384 binding were observed in pontine structures such as the reticulotegmental pontine n. and the gigantocellular reticular n. These effects were among the highest observed in this study, although they did not reach statistical significance. As noted above, it is conceivable that [3H]AFDX 384 binding to non-M2 sites could have partially obscured alterations specific to M2 receptors. Nevertheless, the present results indicate that, in comparison to other brain areas, M2-type receptors in pontine areas are not exclusively or especially affected by PSD. Thus, while cholinergic processes in the pons seem to have a unique role in the generation and maintenance of paradoxical sleep [18], the effects of paradoxical sleep deprivation clearly extend well beyond pontine structures.
4.3. [3H]AF-DX 384 binding and reduced cholinergic responses Paradoxical sleep deprivation has been found to result in an overall attenuation of yawning behaviour induced by the muscarinic receptor agonist pilocarpine, and to cause a shift to the right in yawning induced by the acetylcholinesterase blocker physostigmine. This pattern of results suggests a decrease in postsynaptic cholinergic receptor sensitivity as a consequence of PSD. However, evidence from ACh release studies, as well as recent in situ hybridization work, suggest that a significant proportion of M2 receptors are located presynaptically in cholinergic nerve terminals and act as autoreceptors. Therefore, caution should be taken in ascribing PSD-induced changes in cholinergically induced responses to decreased M2 binding, as suggested by the present results with [3H]AF-DX 384. M1 and M3 subtypes of muscarinic receptors have been recently implicated in paradoxical sleep processes [17,38], and the possibility must remain open that changes in those receptors may also contribute to the behavioural effects after PSD.
4.4. Stress effects Since PSD necessarily involves a stressful component, this raises the question of the extent to which the observed changes in [3H]AF-DX 384 binding might be simply due to stress effects. Both up- and down-regulation of cholinergic receptors have been reported to occur after different types of stress. Interestingly, however, autoradiographic studies focusing on the muscarinic subtypes have consistently shown increased, rather than decreased, muscarinic binding after acute or chronic stress. In this light, the generalized decrease in [3H]AF-DX 384 binding observed in the present study would seem to relate to REM sleep-specific effects rather than generalized stress effects.
In summary, paradoxical sleep deprivation resulted in a generalized down regulation of M2-type muscarinic receptors labelled by [3H]AF-DX 384 in rat brain. This does not seem to be attributable to the stress component of the procedure, and may be causally related to previously reported decreases in cholinergically induced yawning behavior after PSD. Our results are consistent with the notion that pontine M2-type receptors may participate, but not necessarily play a special role, in effects associated with PSD. Further work is needed to distinguish pre- vs. post-synaptic nature of muscarinic cholinergic effects as well as the relative contributions of other subtypes of muscarinic receptors in functional effects observed after paradoxical sleep deprivation.