Download Hypocretinergic Neurons are Primarily involved in Activation

BASIC SCIENCE
Hypocretinergic Neurons are Primarily involved in Activation of the Somatomotor
System
Pablo Torterolo1,2 MD, Jack Yamuy1 MD, Sharon Sampogna1, Francisco R. Morales1,2 MD and Michael H. Chase1 PhD
1Department
of Physiology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90095. 2Departamento de Fisiología, Facultad de Medicina, General Flores 2125, 11800, Montevideo, Uruguay.
Abstract: The hypocretinergic system has been implicated in the generation and/or maintenance of wakefulness. Our results challenge this
hypothesis. Utilizing cats as an animal model and immunocytochemical
procedures for the simultaneous detection of hypocretin and Fos, we
determined that hypocretinergic neurons are activated during wakefulness
but only when somatomotor activity is present. These neurons are not
activated during alert or quiet wakefulness in the absence of motor activity or during quiet sleep. We conclude that the hypocretinergic system is
not responsible for the generation and/or maintenance of wakefulness,
per se; on the contrary, we suggest that hypocretinergic neurons are primarily involved in motor functions irrespective of the animal’s behavioral
state.
Keywords: Hypocretin, orexin, motor, hypothalamus, sleep, wakefulness,
REM, paradoxical sleep
Citation: Torterolo P, Yamuy J, Sampogna S, et al. Hypocretinergic neurons are primarily involved in activation of the somatomotor system.
SLEEP 2003;1: 25-8.
INTRODUCTION
METHODS
FOUR YEARS AGO, A NEW NEUROTRANSMITTER SYSTEM
WAS DISCOVERED IN THE DORSAL, LATERAL AND POSTERIOR HYPOTHALAMUS.1,2 The cell bodies of this system, which contain hypocretin (orexin), project diffusely to all levels of the central nervous system.3 Numerous publications have posited that the hypocretinergic system is involved primarily in the regulation of sleep and
wakefulness; disruption of this system results in the sleep disorder, narcolepsy (reviewed in 4-8).
It is currently believed that the hypocretinergic neurons are involved
in the regulation of the sleep states vis a vis their interaction with the
waking system.4-8 In support of this concept are reports demonstrating
that dense concentrations of hypocretin-containing axon terminals are
located in areas that initiate and/or maintain wakefulness, such as the
noradrenergic locus coeruleus and the histaminergic tuberomammilary
nucleus of the posterior hypothalamus. Hypocretin exerts an excitatory
effect on these and related populations of neurons.9-14 In addition, the
intraventricular injection of hypocretin increases the time spent in wakefulness.9,15,16
We have previously reported that hypocretinergic neurons are active
during wakefulness in conjunction with explorative and locomotor
behavior (as well as during carbachol-induced active [rapid eye movement (REM)] sleep).17 In the present study, in order to test the hypothesis that the hypocretinergic system is primarily involved in the promotion of somatomotor activity, we further explored the Fos immunoreactivity of the hypocretinergic neurons during active wakefulness when
movements are occurring, in comparison with aroused waking without
movements, quiet wakefulness, and quiet sleep.
Thirteen adult male cats were used in the present study. They were
obtained from and determined to be in good health by the UCLA Division of Laboratory Animal Medicine; the Chancellor’s Animal Research
Committee of the University of California Los Angeles approved all
experimental procedures.
The animals were implanted with electrodes for recording the electroencephalogram (EEG), electromyogram (EMG), electrooculogram
(EOG) and pontogeniculooccipital (PGO) waves for the determination
of behavioral states (see 17 for details of the surgical procedures). During experimental sessions (10 am to 2 pm), the animals spent 1 to 2
hours before euthanasia either in the state of quiet wakefulness (QW),
alert wakefulness with motor activity (AW-with M), alert wakefulness
without motor activity (AW-without M) or quiet sleep (QS). Within this
period of time, Fos protein (a marker of neuronal activity) reaches an
optimal degree of concentration.18-20 All the animals had free access to
water and food until 1 hour prior to the beginning of the recording sessions; the experimental sessions for all the conditions were held in the
same room with the presence of the same researcher.
The QW, AW-without M, and QS animals were maintained in a headrestraining device. The EEG of the QW animals was monitored continuously and if slow wave activity or spindles appeared, low noise and/or
mild somesthetic stimulation were applied. The AW-without M group
was maintained awake by continuous bilateral loud clicks of approximately 90 dB SPL (with a variable frequency of sound presentation
ranging from 1 to 50 Hz to avoid habituation). The QS group was sleepdeprived for 2 to 3 hours; during the following 1 hour period, in which
they exhibited sustained QS, active (REM) sleep was prevented from
occurring by the application of low noise and/or mild somesthetic stimulation at the moment that a QS-AS transition was detected polygraphically. The EEG activity (sampled at 250 Hz) was subjected to a Fast
Fourier Transform (FFT). Power values were obtained for consecutive
1second epochs in the delta (0.5 to 4 Hz), theta (4.5 to 8.5 Hz), sigma (9
to 14 Hz), beta (14.5 to 30 Hz), and gamma (30.5 to 50 Hz) frequency
bands. The percentage of each band power in the total power spectrum
was calculated for consecutive 1second epochs.
The AW-with M animals, which were not restrained, were introduced
to the experimental room for the first time; this room contained a variety of objects to attract their attention and induce motor (locomotor-
Disclosure Statement
This study was supported by USPHS grants MH43362, NS09999, NS23426,
HL60269 and AG04307.
Submitted for publication June 2002
Accepted for publication November 2002
Address correspondence to: Michael H. Chase, Ph. D., Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095, TEL: (310) 825-3348;
FAX: (310) 206-3499, E-mail: [email protected]
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Activation of the Somatomotor System—Torterolo et al
RESULTS
explorative) activity; they were allowed to explore this novel environment for 90-120 minutes while being observed continuously. If the animals ceased moving, other objects were presented in order to maintain
constant motor activity and arousal. Actigraph recordings were used to
monitor motor activity (collar-mounted actiwatch, Minimitter, twominute bins).
At the end of the experimental sessions, each animal was deeply anesthetized with sodium pentobarbital (60 mg/kg) and perfused for
immunocytochemistry. Thereafter, the brain was frozen and serially sectioned at 20 µm; hypothalamic sections were then immunostained for
Fos and hypocretin-2 (see 17 for details of the immunocytochemical
methods). Because hypocretin-1 is colocalized with hypocretin-2, only
hypocretin-2 was examined.21
Hypothalamic sections were analyzed by light microscopy; photomicrographs were obtained using a SPOT digital camera attached to an
Olympus BX60 microscope. Images were analyzed using Adobe PhotoShop software with a Power Macintosh G3 computer. The distribution
of immunolabeled neurons was determined from drawings using a camera lucida attachment.
The mean number of double-labeled hypocretinergic neurons that
expressed c-fos (Hcrt+ Fos+) and of the total number of single-labeled
hypocretinergic neurons (Hcrt+) were determined for the following
behavioral states: AW-with M (3 cats), AW-without M (3 cats), QW (3
cats), and QS (4 cats). In order to perform the cell counts, two representative coronal sections were selected for each cat at the tuberal level,
where hypocretinergic neurons are highly concentrated22 (approximately
AP 1023). Values are indicated as the mean ± SEM. The statistical significance for the different behavioral conditions was evaluated utilizing
the individual means for each animal and the analysis of variance
(ANOVA) and Fisher posthoc tests. The criterion chosen to discard the
null hypothesis was P < 0.05.
Representative motor activity of cats during AW-with M is shown by
the horizontal bar A1 in Figure 1A. In the QS group of animals, QS consumed on average 90% of the animals’ behavioral state in the hour prior
to euthanasia. Figure 1B presents an example of delta, sigma, and
gamma EEG frequency bands during sleep deprivation and quiet sleep.
During QS, delta and sigma (spindle frequencies) power of the frontal
EEG increased, while the gamma band decreased. During AW-without
M, although the animals did not move, they exhibited a high level of
electroencephalographic activation (desynchronization of the EEG).
Figure 1C illustrates the fact that the EEG gamma power band, which
reflects the degree of behavioral arousal,24 was significantly higher during AW-without M compared to either QW or QS.
The photomicrographs in Figure 2 are examples of hypocretin and Fos
immunoreactivity in the lateral hypothalamic area during AW-with M,
AW-without M, QW, and QS. Figure 2A-C presents typical sections in
which all hypocretinergic neurons expressed c-fos during AW-with M
(arrows; also exhibited in A’). During AW-without M (Fig. 2D), QW
(Fig. 2E), and QS (Fig. 2F), nonhypocretinergic Fos+ neurons (filled
arrowheads) were intermingled with hypocretin-immunoreactive neurons that did not express c-fos (empty arrowheads). The number of
hypocretinergic neurons that expressed c-fos was larger during AW-with
M compared with all of the other behavioral states that were studied (P
< 0.0001, Fig. 3). No significant differences were found between AWwithout M, QW, and QS. During AW-with M, 79.4% of the total number of hypocretinergic neurons were immunoreactive for Fos (Hcrt+
Fos+); very few cells were Hcrt+ Fos+ during AW-without M (2.3%), QW
(1.7%), or QS (0.9%).
DISCUSSION
The present data demonstrate that hypocretinergic neurons become
active during aroused (alert) wakefulness, but only when the animal is
moving. In the absence of motor activity during aroused wakefulness,
quiet wakefulness or quiet sleep, the hypocretinergic system is not activated to any significant extent.
Lesions, electrical stimulation, microinjections of excitatory and
inhibitory drugs, as well as unit recordings studies, have shown that the
lateral hypothalamus is an important area in the control of motor func-
Figure 1—A. Motor activity of cats while awake and moving (AW-with M); average of 4
days of actigraph recording from a representative cat. Between 10 and 12 am, this representative animal explored its new environment and exhibited a high level of motor activity,
as indicated by the bar labeled A1. Animals in this group (AW-with M) were euthanized
immediately after similar periods of motor activation. B. Quiet sleep (QS) after total sleep
deprivation. B1 shows the high levels of slow wave and spindle activity (corresponding to
delta and sigma EEG frequency bands, respectively) and a low level of gamma band power
of the frontal EEG during QS. In this representative animal, QS comprised 90% of the one
hour period immediately preceding euthanasia (arrow); active (REM) sleep was not present
during this time. C. The bar-chart presents the mean values of the gamma band power
(frontal EEG, expressed as the percentage of the total spectrum power) of a cat during a one
hour period in alert wakefulness without motor activity (AW-without M), quiet wakefulness
(QW) and quiet sleep (QS). There was a significant difference in the gamma band power
between AW-without M compared to both QW and QS (P <0.0001, ANOVA and Fisher
tests). The gamma band power was also significantly higher during QW compared to QS
(P <0.0001).
SLEEP, Vol. 26, No. 1, 2003
Figure 2—Photomicrographs containing hypocretin and Fos immunoreactive neurons in
the lateral hypothalamus during AW-with M (A, A’, B and C), AW-without M (D), QW (E)
and QS (F). Hypocretinergic neurons are stained brown; Fos immunoreactivity, that is
restricted to nuclei, is shown in black. Arrows indicate Hcrt+ Fos+ cells; these neurons are
exhibited with higher magnification in A’. Significant numbers of Hcrt+ Fos+ neurons were
observed only during AW-with M. Filled arrowheads point to non-hypocretinergic Fos+
neurons, these neurons were observed in all of these behavioral states. Empty arrowheads
indicate hypocretinergic neurons that did not express c-fos. Calibration bars: A-F, 50 µm;
A’, 20 µm.
26
Activation of the Somatomotor System—Torterolo et al
tions.25 Intraventricular microinjections of hypocretin produce an
increase in locomotor activity that is suppressed by D1 and D2 dopamine
antagonists.9,16,26 When hypocretin is microinjected in the trigeminal
motor nucleus, there is an increase in muscle tone;27 hypocretinergic terminals have been shown to lie in close apposition to hypoglossal and
trigeminal motoneurons.28 Hypocretinergic terminals have also been
found in the ventral horn where motoneuron cell bodies are located;29
moreover, the direct application of hypocretin onto intracellularly
recorded lumbar motoneurons results in depolarization of their membrane potential, a decrease in input resistance, and sustained discharge.30
Therefore, the hypocretinergic system is well positioned to initiate,
maintain and facilitate motor activity by operating directly on motoneurons and/or by modifying the activity of supraspinal systems that are
involved in motor functions. A recent study in rats suggested that the
activity of the hypocretinergic neurons is related to wakefulness;31 however, this study did not differentiate between wakefulness, per se, and
wakefulness that was present in conjunction with motor activity.
There is additional evidence that supports the hypothesis that the
hypocretinergic system is primarily involved in somatomotor functions.
First, levels of hypocretin in the CSF and in areas such as the perifornical hypothalamus are positively correlated with motor activation.32-34
Second, the discharge rate of units in the perifornical hypothalamic area
(where there is a high density of hypocretinergic cells) increases in association with heightened muscle activity.35 Third, in conjunction with the
restless legs syndrome, which is characterized by irresistible leg movements, there is an increase in CSF levels of hypocretin.36 In addition, as
we have shown in the present report, during QS, which is a quiescent
motor state, there is practically no activation of hypocretinergic neurons.
This latter finding agrees with the results in rats of a negative correlation
between the number of hypocretinergic Fos immunoreactive neurons
and the time spent in QS.31 On the other hand, during active (REM)
sleep, although motor output is inhibited at the motoneuron level,37,38
there is no question that motor systems are activated, as evidenced by
intracellular studies as well as by the presence of ocular movements and
twitches and jerks of the somatic musculature.37,39 Importantly, we have
shown that hypocretinergic neurons are active during carbachol-induced
active (REM) sleep;17 this is in agreement with an increase in the
hypocretin-1 release detected in the hypothalamus and basal forebrain
during this behavioral state.34
In conclusion, the activation of hypocretinergic neurons during wakefulness when the animal is moving, and the absence of Fos immunoreactivity in these cells either during quiet sleep or wakefulness when phasic motor activity is not present, suggests that this system is not directly
involved with the generation and/or maintenance of wakefulness, per se.
On the other hand, based upon the results of the present and a preceding
study,17 we present the hypothesis that hypocretinergic neurons are
involved primarily in promoting motor activities, irrespective of the animal’s ongoing behavioral state.
ACKNOWLEDGMENTS
We thank Dr. S. Fung for his critical comments regarding the manuscript
and Nia Wedhas for her excellent assistance with the immunocytochemical procedures. This work was supported by USPHS grants MH43362,
NS09999, NS23426, HL60269 and AG04307.
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Figure 3—Bar chart of the mean number of Hcrt+ Fos+ neurons during alert wakefulness
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