Theme : Dreams
Changes in Anti-phosphoserine and Anti-phosphothreonine Antibody Binding
during the Sleep-Waking Cycle and after Lesions of the Locus Coeruleus
Chiara Cirelli and Giulio Tononi
The Neurosciences Institute, San Diego, CA 92121, USA
Abstract
Cellular responses to many extracellular signals occur through phosphorylation
or dephosphorylation of intracellular proteins. To determine whether changes
in protein phosphorylation accompany the electrophysiological changes
occurring during the sleep-waking cycle, immunocytochemical mapping of
cells labeled with anti-phosphoserine and anti-phosphothreonine antibodies
was performed on brain sections of sleeping and waking rats. Animals implanted
for chronic polysomnographic recordings were sacrificed after either 3h
of sleep or 3h of sleep deprivation by gentle handling. Anti-phosphoserine
and anti-phosphothreonine staining was mainly localized in neurons and
was high in some brain regions, such as cerebral cortex and hypothalamus,
and low in others, such as the thalamus. In all cases, the number of cells
labeled with either antibody in the cerebral cortex was markedly higher
in rats sacrificed after 3h of waking than in rats sacrificed after 3h
of sleep. Unilateral lesions of the locus coeruleus by local injection
of 6-hydroxydopamine were performed in other animals to determine whether
the increase in protein phosphorylation during waking was influenced by
the activity of the noradrenergic system, which is higher in waking than
in sleep. In animals sacrificed after 3h of spontaneous or forced waking,
the number of labeled neurons in the cerebral cortex was decreased on
the side in which noradrenergic fibers had been lesioned. These results
suggest that 1) neurons exist physiologically in different states of phosphorylation,
ranging from a state of very high phosphorylation (e.g., in the cerebral
cortex) to a state of very low phosphorylation (e.g., in many thalamic
nuclei); 2) the fraction of highly phosphorylated neurons in cerebral
cortex is higher in waking than in sleep and 3) part of the immunoreactive
phosphorylation present in highly labeled cortical neurons is controlled
by the locus coeruleus.
Current Claim: Anti-phosphoserine and anti-phosphothreonine antibody
staining in cerebral cortex is higher in waking than in sleep and is controlled
in part by the locus coeruleus.
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Sleep and waking are traditionally differentiated on the basis of behavioral
and electroencephalographic parameters. At the cellular level, sleep and
waking have been investigated electrophysiologically, revealing changes
in firing rate, firing pattern, membrane potential and channel conductance
in brainstem, thalamic and cortical cells (Steriade and McCarley, 1990;
Steriade et al., 1997). Recently, differences between sleep and waking
have been demonstrated at the level of gene expression in many brain regions.
In both cats and rats, immediate early genes such as c-fos and NGFI-A
are activated after short periods of spontaneous waking or sleep deprivation
(Pompeiano et al., 1992, 1994; Grassi-Zucconi et al., 1993; O'Hara et
al., 1993; Cirelli et al., 1995; Ledoux et al., 1996), and the expression
of other genes may be changing as well (Tononi and Cirelli, 1997).
The cellular events leading from changes in firing patterns and membrane
potential to changes in gene expression are complex, involving several
classes of receptors, G-proteins, second-messenger systems, and protein
kinases and phosphatases. Comparatively few studies have examined the
mechanisms of signal transduction in sleep and waking. Changes in receptor
affinity and number have been investigated during the sleep-waking cycle
(Pompeiano and Tononi, 1990) and after sleep deprivation (e.g., Nuñes
et al., 1994; Tsai et al., 1994; Jimenez-Anguiano et al., 1996). One study
has addressed the G-protein and second-messenger system mediating the
carbachol-induced increase in REM sleep (Shuman et al., 1995). Changes
in cAMP levels in relation to sleep and waking have been described in
cortex, hippocampus, brainstem, and preoptic hypothalamus (Ogasahara et
al., 1981; Perez et al., 1991). Intracellular calcium levels as measured
in synaptosomes decrease in several brain regions after REM sleep deprivation
(Mallick and Gulyani, 1996).
In most cases, the activation of second messengers systems leads to changes
in the phosphorylation state of intracellular proteins through the action
of kinases and phosphatases. These fall into two major categories: serine/threonine
kinases and phosphatases, that phosphorylate and dephosphorylate either
serines or threonines, and tyrosine kinases and phosphatases, that are
specific for tyrosyl residues. The brain contains a large number of serine/threonine
kinases and many of them, such as cAMP- and cGMP-dependent protein kinases,
Ca++/calmodulin-dependent protein kinases, and protein kinase C, are directly
regulated by second messengers (Scott and Soderling, 1992). Protein phosphorylation
is the most common post-translational modification by which protein properties
are regulated, and the effects of serine/threonine phosphorylation on
many neuronal proteins has been studied extensively (for review see Walaas
and Greengard, 1991).
Hardly anything is known, however, about the regulation of protein phosphorylation
in neural tissue in relation to sleep and waking. The present experiment
took advantage of the commercial availability of anti-phosphoserine and
anti-phosphothreonine antibodies to investigate the distribution of protein
phosphorylation in neural tissue by immunocytochemistry, and to ask whether
the amount or distribution of protein phosphorylation changes between
waking and sleep. We found that the proportion of neurons labeled with
anti-phosphoserine and anti-phosphothreonine antibodies varies in different
brain regions and that in many cortical areas the number of neurons heavily
stained by either antibody was much higher after 3 hours of waking induced
by gentle handling than after 3 hours of sleep.
Protein phosphorylation is strongly affected by neuromodulatory systems
with diffuse projections, such as the noradrenergic locus coeruleus (Duman
and Nestler, 1995) that fire at higher levels during waking than during
sleep (Aston-Jones and Bloom, 1981). For this reason, this study also
explored the possibility that the number of cells stained with anti-phosphoserine
and anti-phosphothreonine antibodies is controlled by the noradrenergic
system. We found that lesions of the locus coeruleus reduce the number
of labeled neurons in cortical regions. Part of this work has been presented
elsewhere (Cirelli and Tononi, 1996).
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Recording
Male WKY rats (N=12) under pentobarbital anesthesia (60-75 mg/kg, i.p.)
were implanted with screw electrodes in the skull to record the EEG and
silver electrodes in the neck muscles of both sides to record the electromyogram.
Immediately after recovery from anesthesia rats were housed individually
in soundproof recording cages where lighting and temperature were kept
constant (LD 12:12, light on at 10:00, 24 ± 1°C, food and drink
ad libitum). They were connected by means of a flexible cable and a commutator
(Airflyte) to a Grass electroencephalograph, and recorded continuously
for 1-2 weeks until the sleep-waking percentages were according to published
norms (Borbély and Neuhaus, 1978). Each day from 09:30 to 10:00
rats were gently stroked by using a small painter's brush to become familiar
with the sleep deprivation procedure. Rats were then sacrificed at the
same circadian time (13:00) either after 3 hours of sleep (N=6) or 3 hours
of total sleep deprivation (N=6). Sleep deprivation was achieved by gentle
handling, eliciting an orienting reaction whenever a slowing of the EEG
was noted. Sleep-deprived rats never showed slow-wave activity for longer
than 10 sec. Sleeping rats were sacrificed after spending at least 75%
of the previous 3 hours asleep. Sacrifice occurred after a long period
of sleep (at least 30 min with periods of waking not longer than 5 min)
during an NREM episode (at least 5 min).
A second group of rats (same as in Cirelli et al., 1996) implanted with
electrodes for polysomnographic recordings was infused with 6-hydroxydopamine
(6-OHDA, RBI) unilaterally into the left or right locus coeruleus by way
of a 24G stainless steel needle connected to a 5µl Hamilton syringe.
The stereotaxic coordinates according to the atlas of Paxinos and Watson
(1986) were 0.74 mm posterior to the interaural line, 7.5 mm below the
dura and 1.2 mm lateral to the midline. Rats were pretreated with the
selective serotonin uptake inhibitor fluoxetine (Sigma, 10mg/kg, i.p.),
to prevent possible effects of 6-OHDA on serotoninergic terminals. The
volume of 6-OHDA injected ranged between 0.5 and 4 µl of a solution
of 2.5 µg/µl 6-OHDA in saline containing 1mg/ml ascorbic acid,
delivered over 5 minutes. The needle was left in place an additional 5
minutes to avoid back-diffusion. Animals were recorded continuously for
2-3 weeks after surgery, and then sacrificed at 13:00 either after 3 hours
of sleep or 3 hours of sleep deprivation by gentle handling, or at 1:00
after 3 hours of spontaneous waking. Animal care was in accordance with
institutional guidelines.
Immunocytochemistry
All rats were anesthetized with pentobarbital and transcardially perfused
with 0.9% cold saline (50 ml) followed by 4% cold paraformaldehyde in
0.1M phosphate buffer (350ml; pH 7.4). In two animals (one sleep and one
sleep-deprived), the calcium chelator BAPTA AM (Calbiochem, 20mg/kg) was
added to saline during the perfusion in order to block phosphatase 2B
activity (Tymianski et al., 1993). The interval occurring between pentobarbital
injection and the starting of perfusion ranged between 2 and 10 min. Brains
were removed, postfixed for 5 hours at 4°C, cryoprotected (20% sucrose)
and frozen. Frontal sections (40µm) of the entire brain were cut
on a cryostat and stored at 4°C in 0.1M PBS with 0.01% sodium azide.
To detect phosphoproteins, 2 commercial polyclonal antibodies were used:
anti-phosphoserine (1:1000, Zymed), and anti-phosphothreonine (1:2500,
Zymed). Free-floating sections were incubated with primary antibodies
for 24 hours at room temperature, followed by secondary antibodies for
2 hours. The immunoreactivity was finally detected with the avidin-biotin
immunoperoxidase system (Vector) and nickel-enhanced diaminobenzidine.
When the primary or the secondary antibody was omitted, no staining was
detected. Double-labeling experiments with antibodies against phosphoproteins
and glial fibrillary acidic protein (GFAP, 1:500, Sigma) or microtubule-associated
protein 2 (MAP-2, 1:250, Sigma) were performed on a subset of sections.
In 6-OHDA treated rats, noradrenergic cell bodies and fibers were identified
by incubating free-floating sections of the entire brain with a monoclonal
antibody against tyrosine hydroxylase (1:1000, Boehringer) or, to ensure
specificity, with a polyclonal antibody against dopamine B hydroxylase
(1:1000, Eugene). To reduce variability due to incubation procedures and
to facilitate comparisons, corresponding sections from each pair of animals
(one sleep and one sleep-deprived rat) were always processed together
for immunocytochemistry. Animals were paired depending on the time necessary
to start the perfusion after the anesthesia (from 2 to 10 min). Later
inter-pair comparison showed no difference in staining among animals with
different perfusion time. In the pair of animals that received BAPTA-AM
during perfusion, the level of phosphostaining was comparable to that
in the other pairs. Cell counting was performed with the Image-1/Metamorph
imaging system (Universal Imaging) by observers blind to the origin of
the sections. Cell densities (cells/µm2) were calculated over a
450 µm-wide area centered over the frontal (B-0.3 according to the
atlas of Paxinos and Watson), parietal (B-0.3), or perirhynal (B-3.8)
cortex. The background level was set such that only cells with unequivocally
stained cytoplasm and/or nucleus were considered. In most cases, the distinction
between background and stained cells was clear-cut. Cells were counted
as positive whether the staining was present in the nucleus, in the cytoplasm,
or in both. Wilcoxon signed-rank test for matched pairs was used for statistical
analysis.
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Figure 1
Figure 2
Figure 3
Figure 4
Before sacrifice, all rats showed percentages of waking, NREM sleep,
and REM sleep in agreement with published values (Borbély and Neuhaus,
1978). In sleep-deprived rats, NREM and REM sleep were completely suppressed.
Anti-phosphoserine staining in sleep and waking
The rabbit anti-phosphoserine antibody gave robust staining throughout
the entire brain localized in the gray but not in the white matter. However,
the levels of staining differed among brain regions. In sleep-deprived
rats, they were high in all cortical areas, hypothalamus, amygdala, hippocampal
formation, superior colliculus, and ventral tegmental area, and very low
in lateral septum, striatum, and most thalamic nuclei. In all regions,
staining was restricted to cell bodies and/or cell nuclei, while the neuropil
did not show appreciable levels of staining. Cells with cytoplasmic, nuclear
or both cytoplasmic and nuclear staining were intermingled together, but
the percentage of the three types of staining varied in different areas.
In most cases, anti-phosphoserine staining was present in cells that were
positive for the microtubule-associated protein MAP2. On the other hand,
anti-phosphoserine staining was generally not present in cells that were
positive for the glial fibrillary acidic protein GFAP. Therefore, the
majority of cells that were stained were neurons.
In neocortical areas, staining was uniform in layers II to VI in all
anatomical subdivisions (Fig. 1A). Staining was present either in the
cytoplasm or in both the cytoplasm and the nucleus of the cells. Large
pyramidal cells with faint cytoplasmic staining were easily recognizable.
In the allocortex, cells in layers 2 and 3 of the piriform cortex showed
strong cytoplasmic staining. In the septum, many positive cells were present
in the medial but not in the lateral septal division. In the thalamus,
moderate to strong staining was only present in the anterodorsal nucleus
and in the intralaminar and midline nuclear groups, while it was almost
absent in the ventral and lateral nuclear complex and in the ventropostero-medial
and lateral nuclei. In the habenular complex, the medial habenula was
much more stained than the lateral habenula. All hypothalamic nuclei were
heavily stained, with the highest levels of staining in the supraoptic
nucleus and in the tubero-mammillary region. In the hippocampal formation,
strong staining was present in the pyramidal cell layer of all fields
of Ammon's Horn and in the granule cell layer of the dentate gyrus.
In rats sacrificed after 3 hours of sleep, the pattern of staining over
the entire brain was similar to that described above. In particular, immunostaining
was present mainly in neuronal cells, and the highest levels were observed
in cerebral cortex and hypothalamus. In many brain regions, however, the
number of anti-phosphoserine stained cells was greatly decreased with
respect to sleep-deprived rats. This decrease was most apparent in the
cerebral cortex. An example of anti-phosphoserine staining in the parietal
cortex in waking and sleep is shown in Fig. 1 (A, A'). Cell counting performed
on frontal, parietal and perirhynal cortices confirmed that in all these
regions the level of anti-phosphoserine staining in sleep was reduced
to 30-50% of the level in waking (mean ± SEM: frontal cortex: 38.2
± 11.3%; parietal cortex: 32.8 ± 13.4%; perirhynal cortex:
49.4 ± 15.4%; Fig. 2A). On the other hand, as determined by visual
inspection, there was no region in which the level of anti-phosphoserine
staining was higher after sleep than after waking.
Anti-phosphothreonine staining in sleep and waking
The staining with the rabbit anti-phosphothreonine antibody was also robust
and present in specific anatomical locations in the gray but not in the
white matter. As for the anti-phosphoserine staining, double-labeling
experiments with MAP2 and GFAP demonstrated that it was mainly present
in neurons, while the majority of glial cells had little or no detectable
staining. In sleep-deprived rats phosphothreonine staining was mainly
neuronal and it was cytoplasmic, nuclear, or both. It was diffusely present
in all neocortical areas in layers II to VI, but much less in layer I
(Fig. 1B). In the hippocampal formation most of the staining was evenly
distributed in the pyramidal cell layer of all CA fields and in the granule
cell layer of the dentate gyrus. High to moderate levels of staining were
also present in the septum, amygdala, intralaminar and midline thalamic
nuclei. In all the regions cited above the overall level of staining was
higher than that obtained with the anti-phosphoserine antibody. In addition,
phosphothreonine positive cells were numerous in some regions, such as
the caudate-putamen and the ventral and lateral thalamic groups, that
were not labeled with the anti-phosphoserine antibody.
In rats sacrificed after 3 hours of sleep, the general pattern of staining
was similar but globally reduced. As with phosphoserine immunostaining,
no region was more stained after sleep than after waking as determined
by visual inspection. In the cerebral cortex there was a significant reduction
in the number of highly stained cells in all layers following sleep. As
shown in Fig. 1B', the nuclear staining in particular was decreased after
sleep, and many cortical cells that were labeled in both the nucleus and
the cytoplasm after waking were labeled only in the cytoplasm after sleep.
Cell counting showed that in frontal, parietal and perirhynal areas the
number of labeled neurons in sleep was reduced to 30-50% of the values
in waking (frontal cortex: 46.7 ± 15.4%; parietal cortex: 31.7
± 13.1%; perirhynal cortex: 42.0 ± 10.4%; Fig. 2B).
Anti-phosphoserine and anti-phosphothreonine staining in 6-OHDA-treated
rats
In rats in which the locus coeruleus of one side was effectively lesioned
by 6-OHDA injection, the noradrenergic innervation was significantly reduced
ipsilaterally in many cortical areas, as determined by tyrosine hydroxylase
and dopamine B hydroxylase immunocytochemistry (see Cirelli et al., 1996
for details). The cortical regions in which noradrenergic fiber depletion
was strongest varied slightly from one animal to the other, depending
on the exact location of the 6-OHDA injection inside the locus coeruleus.
In 6-OHDA-treated rats that had been sleep deprived, anti-phosphoserine
and anti-phosphothreonine staining on the non-lesioned side of the brain
were comparable to those described above in normal rats sleep-deprived
for 3 hours. Staining levels were also comparable on the intact side of
the brain of 6-OHDA-treated rats whether they had been sacrificed after
3 hours of sleep deprivation by gentle handling or after 3 hours of spontaneous
waking. Fig. 3 (panels A and A') shows a section at the level of the parietal
cortex from a rat in which the left locus coeruleus was lesioned. A marked
reduction of staining is evident on the lesioned side. Anti-phosphothreonine
staining also showed lower levels in the cerebral cortex on the lesioned
side (Fig. 3B, B'). Anti-phosphoserine and anti-phosphothreonine labeled
cells were counted on those cortical areas in which there was a reduction
of the noradrenergic innervation by at least 80%. In these areas, the
number of neurons stained with the anti-phosphoserine antibody was lower
on the lesioned side with respect to the intact side by 26.0 ±
8.1% (Fig. 4). With the anti-phosphothreonine antibody the mean decrease
was 19.5 ± 2.8%, although in this case it did not reach statistical
significance.
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Anti-phosphoserine and anti-phosphothreonine positive cells were mapped
immunocytochemically in the rat brain after short periods of waking and
sleep and after lesions of the locus coeruleus. Cells labeled with either
antibody were mainly neurons and the staining was present in the cell
body and/or the nucleus, while the neuropil was poorly stained. The number
of highly stained neurons was greatest in cerebral cortex and hypothalamus
and lowest in the lateral septum and thalamus. In many cortical areas,
these heavily labeled cells were more numerous after 3 hours of waking
than after 3 hours of sleep and their number during waking was partly
modulated by the noradrenergic system.
Neurons contain high levels of protein kinases and phosphatases by which
they finely regulate the phosphorylation state of intracellular proteins,
leading to short- and long-term effects within the cell. In previous studies,
immunocytochemistry was employed to map the location of phosphotyrosine-containing
proteins in the rat brain (e.g.,Tillotson and Wood, 1989; Moss et al.,
1990). In preliminary studies using anti-phosphotyrosine commercial antibodies,
we observed that both neurons and glial cells were stained, and the high
level of glial staining made it difficult to determine whether there was
a difference in the neuronal anti-phosphotyrosine staining between sleep
and waking. To our knowledge, no immunocytochemical mapping of brain tissue
using anti-phosphoserine and anti-phosphothreonine antibodies has so far
been reported.
The anti-phosphoserine and anti-phosphothreonine antibodies used in this
study bind to phosphoserine and phosphothreonine, respectively, and not
to unphosphorylated serine or threonine peptides, as demonstrated by Western
blots and ELISAs. The anti-phosphoserine antibody shows, however, a 5-10%
cross-reactivity with phosphotyrosine, so it cannot be excluded that some
of the neuronal phosphoserine staining reported here was due to phosphotyrosine.
In Western blot analysis performed in rat brain tissue using the same
antibodies, 10-15 distinct major bands were detected with each antibody.
These bands were not present after preincubation of the antibodies with
phosphoserine or phosphothreonine (data not shown). It is likely that
these antibodies, while relatively specific, do not detect every single
protein present in brain tissue that is phosphorylated on serine or threonine
residues. In some brain regions, for example, cells phosphorylated on
Ser133 of the transcription factor CREB were recognized by a specific
anti-phospho-CREB antibody (Cirelli et al., 1996) but not by the anti-phosphoserine
antibody. The strong staining of cell bodies and nuclei and the poor staining
of the neuropil suggest that the antibodies may have preferentially labeled
a subset of phosphorylated proteins. For example, some cellular proteins
may be more accessible than others to the antibodies or more resistant
to phosphatases during the short interval between sacrifice and tissue
processing. Despite these limitations, the antibodies used in this study
demonstrate sufficient sensitivity for detecting, with cellular resolution,
changes in the degree of labeling among different brain regions, over
the sleep-waking cycle, and after lesions of the locus coeruleus.
The first finding of this study is that, although phosphorylation reactions
occur in every living cell, the level of anti-phosphostaining varied greatly
from neuron to neuron and was almost absent in glial cells. This is quite
surprising and suggests that individual neurons can exist physiologically
in different states of phosphorylation. The proportion of neurons in a
highly stained state was found to vary throughout the brain, being higher
in cerebral cortex and hypothalamus, and lower, for example, in many thalamic
nuclei.
The second finding of this study is the marked increase in the number
of highly stained neurons, as detected by immunocytochemistry in cerebral
cortex, in rats that had been awake with respect to animals that had been
asleep. Since the rats were sacrificed at the same circadian time, this
increase cannot be due to circadian factors. Although the animals were
kept awake by gentle handling, this limited experimental intervention
is unlikely to have significantly contributed to a higher number of stained
cells. In the rats with lesions of the locus coeruleus, the levels of
anti-phosphoserine and anti-phosphothreonine staining in the intact cortex
were comparable whether they had been sacrificed after sleep deprivation
or after spontaneous waking. A previous study has shown that the phosphorylation
of CREB and the expression of c-fos and NGFI-A were similar after 3 hours
of sleep deprivation induced by gentle handling during the day and after
3 hours of spontaneous waking at night (Cirelli et al., 1996). Finally,
short periods of sleep deprivation by gentle handling do not increase
corticosterone levels or other indicators of stress (Tobler et al., 1983),
and the animals used in this study had been accustomed to gentle handling
for weeks before the experiment. Thus, the changes observed in this study
were presumably due to waking and sleep per se, and they point to important
differences at the cellular level between these behavioral states.
Since brain kinases and phosphatases are believed to function in a state
of dynamic equilibrium, and their number is estimated to lie between one
and three thousands (Hunter, 1995), the finding of a net increase of phosphoimmunostaining
in waking vs. sleep is intriguing. A net effect could be due to a change
in the ratio of kinase/phosphatase activity correlated to behavioral state,
which would lead to a generalized change of the phosphorylation state
of many proteins. Alternatively, the phosphorylation/dephosphorylation
of a few abundant or readily stained proteins may be responsible for the
observed effect. It is difficult to distinguish between these two possibilities
based on the immunocytochemical mapping employed in the present study.
Many kinds of proteins present in cortical neurons are regulated by serine/threonine
phosphorylation (see e.g., Girault, 1993 for a review). They include ion
channels (e.g., Na+ channels, Ca++ channels, nicotinic receptors), ion
transporters (Na+/K+ ATPase), neurotransmitter receptors (beta-adrenoceptors,
glutamate receptors), signal transduction enzymes (e.g., nitric oxide
synthase), proteins involved in neurotransmitter release (e.g., synapsins),
and transcription factors (e.g., CREB). The pattern of staining reported
in this study cannot point to specific phosphoproteins as responsible
for the changes that were observed between sleep and waking, but it suggests
that proteins present in the cell body and/or the nucleus, as opposed
to those concentrated in the neuropil, are more likely to be involved.
While the level of antibody labeling changes markedly, its overall immunocytochemical
distribution in terms of brain areas and cell types appears relatively
preserved between sleep and waking. The degree of such modulation suggests,
however, that it may be possible to determine whether the change in phosphorylation
levels is generalized or it is limited to a few specific proteins by employing
more specific approaches such as front phosphorylation assays and two-dimensional
gel electrophoresis (e.g., Scheetz and Constantine-Paton, 1996).
All major serine/threonine kinases, such as cAMP- and cGMP-dependent
protein kinases, Ca++/calmodulin-dependent protein kinase, and protein
kinase C, are directly or indirectly regulated by second messenger systems.
Thus, many different mechanisms could be responsible for the higher phosphorylation
of serine and threonine residues during waking than during sleep. A likely
mechanism is the action of neuromodulatory systems with diffuse projections.
The release of serotonin, acetylcholine, and histamine is higher during
waking than during slow-wave sleep (Jasper and Tessier, 1968; Mochizuki
et al., 1992; Portas and McCarley, 1994). Norepinephrine release correlates
with locus coeruleus discharge (Berridge et al., 1996; Florin-Lechner
et al., 1996), which is higher during waking than during sleep (Aston-Jones
and Bloom, 1981). Furthermore, the influence of many of these neuromodulators
on protein phosphorylation in the brain is well-documented (Duman and
Nestler, 1995).
The results of locus coeruleus lesions in this study lend support to
this hypothesis: in cortical regions in which the noradrenergic innervation
had been destroyed unilaterally, the level of serine phosphorylation in
waking animals was reduced with respect to the contralateral side, and
threonine phosphorylation showed a similar trend. Since the destruction
of up to 80% of the noradrenergic fibers decreased serine phosphorylation
levels in the cortex by 15-20%, while the decrease in phosphorylation
between waking and sleep was of the order of 50-70%, norepinephrine is
not the only factor responsible for the changes in phosphorylation between
sleep and waking. It remains to be seen whether the cumulative action
of other neuromodulators that are released at higher levels during waking,
such as serotonin and acetylcholine, may be sufficient to account for
this effect.
Norepinephrine regulates many kinases and phosphatases including ion
channels and pumps in nerve terminals, proteins associated with synaptic
vesicles such as synapsins, receptors such as beta-adrenergic receptors,
and synthetic enzymes such as tyrosine hydroxylase (cf. Duman and Nestler,
1995). Changes in the activity of the Na+/K+ ATPase observed after periods
of REM sleep deprivation may be mediated in this way (Gulyani and Mallick,
1995). Long-term actions of norepinephrine such as the regulation of protein
synthesis in target neurons are also mediated by protein phosphorylation.
A well-documented example is the effect of norepinephrine on the activity
of CREB, a transcription factor that regulates the expression of many
genes. CREB is activated by phosphorylation on a serine residue (Ser133)
through a pathway that goes from norepinephrine, to beta-adrenoceptors,
to protein kinase A (see e.g., Schulman, 1995). In a previous study, we
showed that CREB phosphorylation on Ser133 is high during waking in cortical
areas equipped with noradrenergic fibers and low in areas in which such
fibers had been destroyed. This study also showed that changes in CREB
phosphorylation were associated with changes in the expression of immediate
early genes (Cirelli et al., 1996). Depending on the protein involved,
changes in phosphorylation during the sleep waking-cycle may represent
more than accompanying phenomena, and may actually play a role in regulating
neural function through the activation of gene expression.
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We thank G.A. Davis for his contribution to surgery, immunocytochemistry,
and Western blot analysis and J.A. Gally for his comments and suggestions.
This work was carried out as part of the experimental neurobiology program
at the Institute, which is supported by the Neurosciences Research Foundation.
The Foundation receives major support for this program from Novartis Pharmaceutical
Corporation. C. Cirelli is a Joseph Drown Foundation Fellow.
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