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.
