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Sleep and Sleep Disorders: A Neuropsychopharmacological Approach, edited by Malcolm Lader, Daniel P. Cardinali
and S.R. Pandi-Perumal. 2004 Landes Bioscience/Eurekah.com.
Role of Wakefulness Area in the Brainstem Reticular
Formation in Regulating Rapid Eye Movement Sleep
Birendra N. Mallick, Satvinder Kaur, Stephen Thankachan and Dinesh Pal
CHAPTER 3
Abstract
R
apid eye movement sleep is a unique paradoxical state
within sleep period. Normally it follows deep sleep, is
maintained for varying duration and may terminate in
either sleep or wake state. During REM sleep some neurons in-
crease firing, the REM-ON neurons, while some others cease
firing, the REM-OFF neurons. Although the mechanism is not
completely known, these REM sleep -related neurons are likely
to play a significant role in the initiation and maintenance of
REM sleep. It was proposed that GABA may be involved in the
cessation of REM-OFF neurons and the classical sleep and wak-
ing areas in the brain stem are likely to modulate the REM-ON
and REM-OFF neurons for the regulation of REM sleep.
Results from our single neuronal activity experiments in freely
behaving animals confirmed that the brain stem area, which in-
duce wakefulness inhibit the REM-ON neurons but stimulate
the REM-OFF neurons. Microinjection studies revealed that the
increase in REM sleep by the cholinergic input (possibly from
REM-ON neurons) to the locus coeruleus (where REM-OFF
neurons are located) is mediated through GABA. Thus, it is pro-
posed that during wakefulness the REM-ON neurons are inhib-
ited while the REM-OFF neurons are active. During sleep gradu-
ally the awake-related neurons slow down withdrawing their effects
on REM sleep related neurons. This causes an increase in the
REM-ON neuronal activity inducing release of acetylcholine on
the GABA-ergic neurons in the locus coeruleus. GABA then in-
hibits the REM-OFF neurons resulting in the initiation of REM
sleep. The presence of GABA in optimum concentration main-
tains the duration of REM sleep episode.
Introduction
Rest and activity are basic instinct behavioral phenomena of
living systems present across species. It is believed that in higher
species these phenomena further evolved into complex processes
like sleep and wakefulness. Sleep-wakefulness is a subjective be-
havioral phenomenon. Therefore, for its identification and quan-
tification in an objective manner some of the electrophysiologi-
cal signals are taken into consideration. The three primary
electrophysiological signals that are commonly considered for the
purpose are that from the brain, the electroencephalogram (EEG),
from the muscles, the electromyogram (EMG) and due to the
movements of the eyes, the electrooculogram (EOG). Based on
characteristic signals from these records, wakefulness has been
divided into two stages viz. active wakefulness (AW) and quiet
wakefulness (QW) while sleep has been divided into three stages
viz. slow wave sleep (SWS), deep sleep (DS) and rapid eye move-
ment (REM) sleep (Fig. 1). REM sleep is a unique phenomenon
that has been identified in its present form in the mid-twentieth
century.
1
The characteristic features of REM sleep are presence of
low voltage fast electrical waves in the cortical EEG, rapid bursts
of eye movements in the EOG and atonia in the EMG recorded
from antigravity muscles (usually recorded from the neck muscles).
REM sleep episodes repeat several times within sleep period and
its frequency of occurrence as well as duration per bout increases
with the progress in depth of sleep.
2
The mechanism of generation of REM sleep is not completely
understood. However, it is known that there are at least two types
of neurons related to REM sleep. During REM sleep one type of
neurons ceases firing - the REM-OFF neurons, while the second
type increases firing - the REM-ON neurons. Some of the
characterising features of REM sleep viz. desynchronization of
EEG and eye movements, resemble signs associated to wakeful-
ness, while some other signs viz. muscle atonia, resemble signs
associated to sleep. It may also be noted that normally REM sleep
appears at certain depth of sleep and it does not follow wakeful-
ness although it may terminate either into sleep or waking state
(Fig. 2A & B). However, some characteristic sign(s) associated to
REM sleep might be triggered and expressed during wakefulness
in some disorders e.g., narcolepsy.
3
Therefore, it is most likely
that for proper regulation of REM sleep, the neurons in the wak-
ing area(s) in the brain maintain a communication with the neu-
rons related to REM sleep. In order to understand these issues,
interactions between the REM-ON and the REM-OFF neurons
and responses of these neurons to input from waking area(s) in
the brain will be discussed here.
Possible Interaction Between REM-ON and
REM-OFF Neurons for the Regulation of REM
Sleep
REM-ON neurons were initially identified to be cholinergic
and are located in the latero-dorsal and pedunculo-pontine teg-
mental (LDT/PPT) areas of the brain stem.
4,5
However, a later
report suggest that some noncholinergic neurons could also be-

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Sleep and Sleep Disorders: A Neurophychopharmacological Approach 2
have as REM-ON neurones.
6
REM-OFF neurons are reported
to be aminergic and are located in locus coeruleus
(nor-epinephrinergic neurons)
7,8
raphe region (serotonergic
neurones)
9
and in the posterior hypothalamus (histaminergic
neurons).
10,11
In addition to the presence of REM-OFF neurons
in locus coeruleus (LC) there are other evidence that support in-
volvement of LC neurons in REM sleep. It was found that the
firing rate of these neurons decrease during REM sleep depriva-
tion - possibly a compensatory phenomenon -,
12
continuous ac-
tivation (noncessation) of these neurons by mild electrical stimu-
lation reduced REM sleep
13
and cholinergic stimulation of
dorsolateral pons, including LC, increased REM sleep .
14,15,16,17
Both temporary as well as permanent inactivation of LC by local
cooling
18
and lesioning
19,20
respectively, increased REM sleep.
The cholinergic REM-ON neurons in LDT/PPT increase fir-
ing, while the nor-epinephrinergic REM-OFF neurons in LC cease
firing during spontaneous
5,8,12,21
as well as induced
22
REM sleep.
It was proposed that a reciprocal connection exists between these
two types of neurons for the regulation of REM sleep.
21,23
Such
interaction may be supported by the facts that LC receives
ACh-ergic inputs,
24
ACh-ergic receptors have been identified on
the LC neurons
25
and levels of ACh increased in and around LC
during REM sleep.
26
However, a closer and detailed study re-
vealed that acetylcholine or its agonist did not inhibit the
nor-epinephrinergic neurons in LC that are supposed to be
REM-OFF type.
27
Therefore, it was proposed that an inhibitory
neurotransmitter, may be GABA, could be inhibiting the
REM-OFF neurons in LC and that the ACh released from the
cholinergic REM-ON neurons could be mediating its effects
through GABA-ergic neurons for the regulation of REM sleep.
28,29
The possible role of GABA in LC region for the regulation of
REM sleep will be further discussed.
The involvement of GABA in LC for the regulation of REM
sleep may be supported by the fact that GABA-ergic interneu-
rons and terminals are present in LC,
30,31,32,33
GABA receptors
are present on the neurons in LC
34,35
and GABA levels increase
in LC during REM sleep.
36
The hypothesis was further supported
by our study that blocking of GABA-A receptors by microinjec-
tion of its antagonist, picrotoxin, into LC significantly reduced
REM sleep.
37
However, it was not known how the cholinergic
inputs from REM-ON neurons to the LC got translated into a
GABA-ergic input for the regulation of REM sleep. Besides, it
was also not known whether the cholinergic and the GABA-ergic
inputs to the LC had different roles to play for initiation and
maintenance of REM sleep. The investigation demanded a study
of micro-anatomical connections between the proposed neurons
and the role of respective neurotransmitters released from those
neurons in regulating bio-behavioral response, REM sleep in this
case. The complexity of the problem was such that just
micro-anatomical (histological) investigation could not have re-
solved the complex issue and provided an answer to such micro
Figure 1. Polygraphic traces showing simultaneous recording of EEG,
EOG and EMG in freely moving rat classifying five stages of sleep-
wakefulness.
Figure 2. Polygraphic traces showing transition from sleep to REM sleep
and then again to either (A) sleep or (B) wakefulness.

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Role of Wakefulness Area in the Brainstem Reticular Formation in Regulating Rapid Eye Movement Sleep 3
chemo-anatomicobehavioral question. The study needed to be
conducted in freely behaving animals combined with microin-
jection. It was proposed that in LC the input from the cholin-
ergic REM-ON neurons possibly acted on the GABA-ergic neu-
rons for the regulation of REM sleep. The working model of the
hypothesis was that if the cholinergic input was mediated through
the GABA-ergic neurons, the agonist of the former would be in-
effective when the latter was blocked by antagonist, while the
agonist of the latter would be effective even in presence of an-
tagonist of the former. Therefore, experiments were conducted
to locally microinject in LC the agonist or the antagonist of these
neurotransmitters, either alone or in selected combinations. The
experiments were carried out in chronically prepared freely mov-
ing rats and the effects on total REM sleep, rate of generation of
REM sleep and duration of REM sleep per episode were studied.
Experiments were conducted on male Wistar rats (250-300
g), maintained in 12:12 L: D cycle with food and water ad libi-
tum. Under surgical anaesthesia (Nembutal, pentobarbital sodium,
35mg/kg, ip) rats were implanted with bilateral EEG, EOG and
EMG electrodes for chronic sleep-wakefulness recording.
38
A pair
of guide cannulae with blockers were implanted bilaterally in LC.
After recovery from surgical trauma and acclimatisation to the
recording environment, rats were connected to a polygraph to
record the electrophysiological correlates of sleep-wakefulness.
Baseline sleep-wakefulness recording was done for 8 hours (be-
tween 9am-6pm). In the experimental group, 250nl of either sa-
line or 1% (0.25 mg in 250 nl) carbachol (cholinergic agonist) or
scopolamine (cholinergic antagonist) or 0.1% (250 ng in 250 nl)
GABA or picrotoxin (GABA A-antagonist) was injected bilater-
ally into the LC either alone or in a sequential combination. The
injection was performed using an injector cannula connected to
a 2l Hamilton syringe by polyethylene tubing. The combina-
tion injections involving cholinergic and GABA-ergic agonists/
antagonists were done such that injection of antagonist always
preceded the agonist. After the experiment, 2% pontamine sky
blue was injected through the same guide cannulae using the same
injector. Thereafter, under deep Nembutal anaesthesia (45 mg/
kg, i.p.) the brain was perfused intracardially and site as well as
spread of injection were histologically identified. The polygraphic
records were analysed into active wakefulness (AW), quiet wake-
fulness (QW), slow wave sleep (SWS), deep sleep (DS) and REM
sleep as per the criteria followed earlier.
39,13
Total REM sleep,
frequency of generation of REM sleep per hour and mean dura-
tion of REM sleep per episode were calculated. The effects of
injection on frequency of REM sleep as well as duration of REM
sleep per episode were statistically compared with that of baseline
and saline values.
Percent change in REM sleep during baseline (BSNL) and
after microinjection of saline and other agonists and antagonists
are shown in (Fig. 3). It was observed that cholinergic agonist/
antagonist affected the frequency of REM sleep initiation; the
agonist increased while the antagonist decreased the frequency of
generation of REM sleep. On the other hand, GABA and picro-
toxin affected the mean duration of REM sleep per episode; the
former increased while the latter decreased the duration of REM
sleep. Representative polygraphic tracings through REM sleep
episode under baseline and after microinjection of saline are shown
in (Fig. 4A&B); while that after microinjection of carbachol and
GABA are shown in (Fig. 5A&B). These results suggest that
GABA-ergic input to the LC modulated the duration of REM
sleep per episode, while that of the cholinergic modulated the
frequency of generation of REM sleep. These results also indi-
cated that GABA acted after the cholinergic system had initiated
the action, because maintenance of any process would be required
only after the process has been initiated. Thus, individual injec-
tion studies brought forward the possibility that cholinergic in-
puts, presumably from the REM-ON neurons, acted on
GABA-ergic neurons for initiation of REM sleep and that GABA
acted on a system that has been primed by ACh to maintain the
duration of REM sleep. Therefore, to confirm, LC was
microinfused with any of the following three combinations a)
GABA-ergic antagonist followed by cholinergic agonist; or b)
cholinergic antagonist followed by GABA; or c) cholinergic an-
tagonist followed by GABA-ergic antagonist. These were done
with the assumption that if cholinergic input acted on the
GABA-ergic neurons, carbachol in presence of picrotoxin would
induce an effect similar to that of picrotoxin alone, while GABA
in presence of scopolamine would show an effect similar to that
of the GABA alone.
The results from the combination injection studies showed
that picrotoxin followed by carbachol in LC significantly decreased
REM sleep due to a reduction in REM sleep duration per epi-
sode, while scopolamine followed by GABA microinjection sig-
nificantly increased REM sleep due to an increase in mean dura-
tion per episode of REM sleep. Thus, when cholinergic and
GABA-ergic agonist or antagonist (as the case may be) were in-
jected into the LC in any combination, the effect of the GABAergic
agonist/antagonist prevailed over that of the cholinergic. The most
likely explanation for getting such a result is that the cholinergic
input in the LC was acting on the GABA-ergic neurons.
40
Thus,
Figure 3. The frequency (Mean + SEM) of REM sleep per hour for the
entire recording period in normal (BSLN) and after bilateral microin-
jection of saline, carbachol (CARB), scopolamine (SCOP), GABA and
picrotoxin (PICRO) into the locus coeruleus are shown in this figure.
**Significant as compared to baseline, $ significant as compared to sa-
line; **, $, p<0.01

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Sleep and Sleep Disorders: A Neurophychopharmacological Approach 4
the observation supported our contention that cholinergic influ-
ence in LC was mediated through GABA. The
cholinergic-sensitive GABA-ergic neurons are present in and im-
mediately around LC.
6
Although a total loss of REM sleep was
not observed after blocking either the cholinergic receptor or
GABA-ergic receptor, there was an almost complete suppression
of REM sleep when scopolamine and picrotoxin were injected
together. This supports mutually permissive and cooperative role
between cholinergic and GABA-ergic systems in LC. However,
possibility of additional GABA-ergic input to LC from any other
source could not be ruled out. Subsequently, it has been shown
that GABA-ergic input from prepositus hypoglossus to LC may
also modulate REM sleep.
41
It is known that the PrH receives
input from PPT,
42
the site of REM-ON neurons. Thus REM-ON
neurons may excite the GABAergic neurons in PrH, which in
turn inhibit the REM-OFF neurons in LC and generate REM
sleep. Based on these results the connection between the neurons
are shown in (Fig. 6).
Does Waking Area in the Brain Stem Have a Role
to Play in REM Sleep Regulation
It is well known that normally REM sleep appears at certain
depth of sleep period i.e., it does not normally follow wakeful-
ness, although it may terminate into either sleep or wakefulness
(Fig. 2A&B). Some of the electrophysiological signals associated
to REM sleep viz. EEG desynchronization and rapid bursts of
eye movements are apparently similar to those observed during
wakefulness.
43,44
However, wakefulness and REM sleep are sepa-
rated by a distinctly different behavioral state viz., the nonREM
sleep. None of the models proposed for the generation of REM
sleep
21,23,29
could explain that why does not REM sleep or its
signs appear during wakefulness. Recently, we have suggested that
there are likely to be separate groups of neurons in the brain stem
responsible for EEG desynchronization during wakefulness and
REM sleep states.
45
Hence, it is likely that the neurons respon-
sible for expression of REM sleep associated signs remain sup-
pressed during wakefulness and that as and when they get trig-
gered at certain depth of sleep, REM sleep is expressed. Therefore,
it was proposed that REM sleep related neurons i.e., REM-ON
and REM-OFF neurons in the brain stem may be differentially
modulated by the awake inducing area in the brainstem. It was
hypothesised that the wakefulness inducing area in the brain stem
reticular formation i.e., the midbrain reticular formation (MRF)
Figure4. Representative polygraphic traces of EEG, EOG and EMG
associated to REM sleep episode (marked by arrows), recorded before
(baseline) (A) and after bilateral microinjection of saline into the locus
coeruleus in a freely moving rat (B).
Figure5. Representative polygraphic traces of EEG, EOG and EMG
showing REM sleep episode (marked by arrows) after bilateral microin-
jection of carbachol (A) and GABA (B) in the locus coeruleus of freely
moving rat.

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Role of Wakefulness Area in the Brainstem Reticular Formation in Regulating Rapid Eye Movement Sleep 5
would inhibit REM-ON neurons and excite REM-OFF neurons
during wakefulness.
Experiments were conducted on freely moving normally be-
having cats (2.5-3.4 kg). Under surgical anaesthesia cats were pre-
pared for chronic recording of sleep-wakefulness (S-W) and single
neuronal activity.
12,45
In brief, under surgical anaesthesia bilat-
eral EEG, EOG, EMG and PGO electrodes and a stainless steel
tripolar epoxy coated stimulating electrode in the MRF were
implanted. Single neuronal activity was recorded with the help of
microwires introduced into the brain stem through guide can-
nula mounted on a mechanical microdrive fixed to the skull. Af-
ter recovery from surgical trauma, the cats were habituated to the
recording chamber and were attached via shielded cable to a Grass
polygraph to record electrophysiological signals viz., EEG, EOG,
EMG and PGO under unrestrained and freely moving condi-
tions. The physiological wakefulness-inducing region in the MRF
was confirmed by delivering for a few seconds high frequency
stimulation (100Hz, 200-300 A, 300 sec) that resulted in in-
duction of desynchronization of the EEG that outlasted the pe-
riod of stimulation.
Thereafter, once a well isolated signal (S:N 3:1) from single
neuron was encountered it was simultaneously recorded along
with EEG, EOG, EMG and PGO in separate channels of a poly-
graph. At least three spontaneous sleep-wake-REM sleep cycles
were recorded along with single neuronal activities. In order to
ascertain the behavior of each of these neurons their mean firing
rates during QW were statistically compared with the mean fir-
ing rates during other states by applying analysis of variance
(ANOVA) coupled with Newman-keuls test. Accordingly, the
neurons were classified into REM sleep related or nonrelated types.
Among REM sleep related neurons there were REM- ON and
REM- OFF neurons. The former increased firing only during
REM sleep, while the latter ceased firing only during REM sleep.
Thereafter, to study the influence of wakefulness inducing area
on each of those neurons, the MRF was stimulated with 1Hz
rectangular pulses of 500-600 A, 300 sec. The effect of stimu-
lation was recorded by overlapping 10-15 stimulus bound re-
sponses on a digitizing oscilloscope. Ten such responses were ob-
served and a consistent response (7 out of 10) was noted. The
difference in the concentration of neuronal spikes (activities) be-
fore and after the stimulus artefact on the overlapped figure was
compared. An increase in the cluster of spikes was taken as excita-
tion and a decrease, as inhibition while a comparable spike con-
centration as no change. The time delay between the artefact and
the onset of response i.e., the excitation or the inhibition was
taken as latency of response of that particular neuron. The dura-
tion of the response (excitation or inhibition) was also estimated.
After completion of the recording sessions, under deep anaesthe-
sia (sodium pentobarbital 45 mg/Kg), the recording and stimula-
tion sites were marked by electrolytic lesion (50-100A for 10sec)
by passing anodal current (D.C. lesion maker, Grass, USA). Later,
under overdose of anaesthesia the cats were euthanasized by
intracardial perfusion. The stimulating and the recording sites
were histochemically identified in 40m coronal sections stained
with cresyl violet or haematoxylin and eosin. The sites of the re-
corded neurons were also reconstructed.
The effect of MRF stimulation was studied on a total of 63
neurons including 10 REM-ON and 7 REM-OFF neurons
(Table1). Most of the REM-ON neurons (9 out of 10) were in-
hibited (Fig. 7A) while one neuron remained unaffected. All the
7 REM-OFF neurons were excited (Fig. 7B) by MRF stimula-
tion. Thus, the MRF excited the REM-OFF while inhibited the
REM-ON neurons. Among the rest of the 46 neurons studied,
the MRF excited about 50% of the neurons whose firing rate
increased during spontaneous waking period. Thus, the results
showed that a majority of the neurons whose firing rate increased
during spontaneous wakefulness, including the REM-OFF neu-
rons, were excited by the MRF while the REM-ON neurons were
inhibited.
Physiological Significance
It may be noted that wake-inducing area, the MRF, exerts an
opposite influence on REM-OFF and REM-ON neurons - the
former were excited while the latter inhibited. Based on these
results, we hypothesise and propose that during wakefulness when
the wake related neurons in MRF are active,
46,47,48
they excite
the norepinephrinergic REM-OFF neurons in LC, which then
remain active through waking period. This view may be supported
by the fact that activation of REM-OFF neurons is reported to
prevent REM sleep
13
and is likely to increase the level of norepi-
nephrine in the brain causing cortical activation and
desynchronization of the EEG
49,50,51
Therefore, it is possible that
activation of REM-OFF neurons may contribute to EEG
Figure6. Neural connections between REM-ON and REM-OFF neu-
rons for the regulation of REM sleep are shown. The numbers show the
Reference number and abbreviations are as in the text.
Table 1. Effect of 1 Hz stimulation of MRF on the activity of REM-ON and REM-OFF neurons
Stimulation REM - ON REM - OFF
Excitation Inhibition No Change Excitation Inhibition No Change
MRF 0 9 1 7 0 0

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Sleep and Sleep Disorders: A Neurophychopharmacological Approach 6
desynchronization associated with wakefulness, but not to that
of REM sleep.
The REM-ON neurons that usually remain inhibited during
normal wakefulness were inhibited by the MRF. It may be sup-
ported by the fact that activation of the site containing the
REM-ON neurons increased REM sleep.
52
It is reported that the
neurons in the peri-LC that contains the REM-ON neurons are
responsible for muscle atonia during REM sleep.
53,54,55,56
All the
results considered together provide possible explanation for neu-
ral mechanism as to why does not muscle atonia, associated with
REM sleep, appear during wakefulness although the EEG be-
comes desynchronized. Further, the information may be extended
that in case of narcolepsy possibly there occurs an error in this
pathway resulting in muscle atonia during wakefulness.
As a mechanism of action at the neuronal level it may be said
that during wakefulness the MRF neurons are active causing
stimulation of REM-OFF neurons and inhibition of REM-ON
neurons resulting in absence of REM sleep. This may be sup-
Figure7. Ten 1 Hz stimulus bound overlapped responses of a REM-ON
neuron (A) and a REM-OFF neuron (B) to stimulation of midbrain
reticular formation (MRF) are shown here. The REM-ON neuron was
inhibited, while the REM-OFF neuron was excited.
ported by the fact that the REM-OFF neurons are normally ac-
tive during all the stages except during REM sleep while the
REM-ON neurons behave in an opposite manner. As a mecha-
nism of action one or more or all three of the following possibili-
ties may exist. One, that MRF neurons exert an independent in-
hibitory and excitatory effects on the REM-ON and the
REM-OFF neurons, respectively; two, that the MRF exerts an
excitatory effect on the REM-OFF neurons that in turn (through
GABAergic neurons) then inhibit the REM-ON neurons;
57
and
three, that the MRF exerts an inhibitory effect on the REM-ON
neurons and that in turn (through GABAergic neurons) exert an
excitatory effect on the REM-OFF neurons.
57
At the onset of
sleep the activity of the MRF neurons is reduced
47
that gradually
withdraws the excitatory and the inhibitory effects from the
REM-OFF and the REM-ON neurons, respectively. Gradually
sleep is induced when the sleep inducing neurons further increase
firing and the wake active neurons further reduce or cease firing.
The reduction or cessation of the wake active neurons withdraws
the inhibition from REM-ON neurons, which in turn also with-
draws the GABA mediated inhibition from REM-OFF
neurones.
40
Finally, the activation of REM-ON and the inhibi-
tion of REM-OFF neurons initiate REM sleep. The neural con-
nections as proposed above have been shown in (Fig. 8).
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Figure8. Composite neuronal connections showing modulation of
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on which the model is based on.RAS - Reticular Activating System.

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Role of Wakefulness Area in the Brainstem Reticular Formation in Regulating Rapid Eye Movement Sleep 7
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