Corticotropin-releasing
factor neurons in the hypothalamic
paraventricular nucleus are involved in
arousal/yawning response of rats
Kita I, Seki Y, Nakatani Y, Fumoto M, Oguri
M, Sato-Suzuki I, Arita H.
Department of Physiology,
Toho University School of Medicine, Tokyo
Japan
-Kita I, Kubota N,
Yanagita S, Motoki C Intracerebroventricular
administration of corticotropin-releasing factor
antagonist attenuates arousal response
accompanied by yawning behavior in rats.
Neurosci.Letter 2008;433(3):205-208
-Kita I, Yoshida
Y, Nishino S. An activation of parvocellular
oxytocinergic neurons in the paraventricular
nucleus in oxytocin-induced yawning and penile
erection. Neurosci Res. 2006;54(4):269-275
-Kita I,
Sato-Suzuki et al.Yawning responses induced
by local hypoxia in the paraventricular nucleus
of the rat.Behavioural Brain Research
2000;117(1-2):119-126
-Kubota N, Amemiya
S, Motoki C, Otsuka T, Nishijima T, Kita I.
Corticotropin-releasing factor antagonist
reduces activation of noradrenalin and serotonin
neurons in the locus coeruleus and dorsal raphe
in the arousal response accompanied by yawning
behavior in rats. Neurosci Res.
2012;72(4):316-323
-Seki Y, Y
Nakatani, et al Light induces cortical
activation and yawning in rat Behav Brain Res
2003;140(1-2):65-73
-Seki Y,
Sato-Suzuki I, et al Yawning/cortical
activation induced by microinjection of
histamine into the paraventricular nucleus of
the rat. Behav Brain Res.
2002;134(1-2):75-82.
-Sato-Suzuki I,
Kita I, Oguri M, Arita H Stereotyped yawning
responses induced by electrical and chemical
stimulation of paraventricular nucleus of the
rat Journal of Neurophysiology,
1998;80(5)2765-2775
Our previous studies have suggested that
activation of the hypothalamic paraventricular
(PVN) descending oxytocinergic projections is
involved in the induction of yawning accompanied
by an arousal response, but the possibility that
neural systems other than the oxytocinergic
system in the PVN also mediate the
arousal/yawning response cannot be ruled out. We
assessed the activity of corticotropin-releasing
factor (CRF) neurons during yawning induced by
the PVN stimulation in anesthetized,
spontaneously breathing rats using
double-staining for c-Fos and CRF. Yawning
response was evaluated by monitoring an
intercostals electromyogram as an index of
inspiratory activity and a digastric
electromyogram as an indicator of mouth opening.
We also recorded the electrocorticogram (ECoG)
to determine the arousal response during
yawning. Microinjection of l-glutamate (2-5nmol)
into the PVN produced a frequent yawning
accompanied by an arousal shift in the ECoG, and
these behavioral effects were associated with a
significant increase of c-Fos positive CRF
neurons in the medial parvocellular subdivision
of the PVN. In addition, a marked enhancement in
the c-Fos expression was found in the both locus
coeruleus (LC) and global area in the cortex
when the frequency of yawning response was
increased by the PVN stimulation, suggesting
that the arousal response during yawning might
be mediated by the activation of LC neurons. The
present study suggests that an activation of CRF
neurons in the PVN is responsible for the
arousal response accompanied by yawning
behavior.
1. Introduction
We have reported that a stereotyped yawning
response can be evoked by several forms of
chemical stimulation of the paraventricular
nucleus (PVN) of the hypothalamus in
anesthetized, spontaneously breathing rats.
Yawning response in the hypothalamus is
typically associated with the induction of
arousal response, and in our previous studies,
we recorded the electrocorticogram (ECoG) to
evaluate arousal responses during yawning and
found that an arousal shift in the ECoG,
represented by lower voltage and faster rhythm,
occurred before the yawning behavior.
It is widely believed that an activation of
the descending oxytocinergic system in the PVN
is one of the most important mechanisms for
mediating the induction of yawning. However, the
PVN contains various neuropeptides besides
oxytocin, such as corticotropin releasing factor
(CRF), vasopressin, and dynorphine. Thus, the
possibility that not only the oxytocinergic
system but also the other neural systems in the
PVN could be involved in the arousal/yawning
responses cannot be ruled out. In the present
study, we focused on CRF neurons as a candidate
for the induction of arousal response
accompanied by yawning behavior.
CRF neurons in the PVN are generally known
to play a critical role in arousal, autonomic,
and behavioral responses associated with various
stressors. Several studies have reported that
mild stresses such as foot shock, forced
swimming and REM sleep deprivation modify the
induction of yawning response. We have also
indicated that the yawning response occurs by
stress-like stimulation such as local hypoxia of
the PVN and light stimulation , and these
manipulations induce not only the yawning
response but also an arousal shift in the ECoG,
suggesting that the cortical activation
accompanied by yawning behavior may be related
to stress. Taken together, it is possible that
CRF neurons in the PVN are involved in the
induction of arousal/yawning response.
Although the mechanism behind the arousal
response accompanied by yawning evoked by PVN
stimulation or stress is still unknown, the
arousal response may be mediated by the
widespread projecting neurons in the brainstem
reticular formation. We have previously proposed
a hypothesis that the arousal response
accompanied by yawning evoked by PVN stimulation
could be accounted by the projection of CRF
neurons from the PVN to the locus coeruleus
(LC), which is one of the neuronal aggregates
within the brainstem reticular formation. In the
present study, we first investigated the
involvement of CRF neurons in the PVN in the
arousal/yawning responses with double-staining
for c-Fos and CRF. Then, we observed c-Fos
expression to evaluate neuronal activity in the
LC and the cortical area in the arousal/yawning
response evoked by chemical stimulation of the
PVN. The present study suggested that activation
of CRF neurons in the PVN is involved in the
arousal response during yawning behavior.
4. Discussion
This is the first study determining the
involvement of CRF neurons of the PVN in the
arousal/yawning response in anesthetized rats. A
marked increase in double-labeled neurons for
c-Fos and CRF was found in the PVN when frequent
spontaneous yawning accompanied with an arousal
shift in ECoG was produced by the PVN
stimulation. These results suggest that an
activation of CRF neurons in the PVN is involved
in the arousal/yawning response. In addition,
the PVN stimulation induced significant
increases in c-Fos expression in the LC and over
the cortical area. Since it is indicated that
the LC receives CRF afferents from the PVN, and
contains widespread projecting neurons to
various brain areas, CRF neurons in the PVN may
preferentially mediate the arousal response
during yawning behavior.
A series of animal studies has suggested
that the PVN of the hypothalamus is one of the
most important brain structures for the
induction of arousal/yawning responses. We
previously indicated that microinjection of
L-glutamate or nitric oxide (NO)-releasing
compound (NOC-7) into the mp subdivision of the
PVN increased the occurrence of stereotyped
yawning response which is characterized by an
initial depressor response and an arousal shift
in ECoG followed by a single large inspiration
with mouth opening, and suggested that the mp of
the PVN is a crucial site for the induction of
yawning response. In the present study,
microinjection of L-glutamate into the PVN,
which produced frequent spontaneous yawning
accompanied with an arousal shift in the ECoG,
enhanced c-Fos expression in all PVN portions,
especially in the mp subdivision of the PVN,
supporting that the neuronal structure in the mp
subdivision of the PVN might be responsible for
triggering induction of frequent yawning.
The mechanism by which the frequent yawning
is produced after L-glutamate injection is still
unknown, but NO released by activation ofNMDA
receptor might cause the frequent yawning.
NO is a diffusible
neurotransmitter/neuromodulator and acts as a
paracrine agent. It is, thus, possible that
diffusible NO activates various neurons within
the yawning-triggering structure in the PVN and
facilitates the induction of yawning response.
This possibility could be supported by previous
studies showing that stimulation of NMDA
receptors by N-methyl-D-aspartic acid in the PVN
activates NO synthesis, which in turn activates
oxytocinergic neurons projecting to the pons and
medulla oblongata to induce yawning in awake
rats. Another possibility is that positive
feedback control through a linkage between
L-glutamate and NO may be one of the mechanisms
in the induction of frequent yawning. Because it
has been suggested that NO is produced by
neuronal activation through the excitatory
neurotransmitter, L-glutamate, and diffuses to
the presynaptic terminal where it amplifies the
release of the L-glutamate, this effectively
creates a positive feedback system.
In the current study, we demonstrated that
microinjection of L-glutamate into the PVN,
which produces frequent spontaneous yawning
accompanied by the arousal response,
significantly increases the percentage of c-Fos
positive CRF neurons in the mp subdivision of
the PVN. This result suggests that an activation
of CRF neurons in the mp subdivision of the PVN
could be responsible for the induction of
arousal/yawning response.
The magnitude of the increase in c-Fos
positive CRF neurons after injection of
L-glutamate was approximately 25% of CRF neurons
in this structure (40.7% after saline injection,
64.8% after L-glutamate injection). It is,
however, noted that only a small portion of
c-Fos positive neurons are identified as CRF
positive (7.9 ± 3.4% after saline
injection, 10.5 ± 3.4% after L-glutamate
injection, respectively). These results suggest
that non-CRF neurons such as oxytocin neurons
are also activated during the arousal/yawning
response. However, it is not known whether all
CRF neurons are stained with the antibody we
used and whether a large number of non-CRF
neurons are indeed significantly activated.
What is the main role of CRF neurons in the
arousal/yawning response? CRF in the PVN is
generally known to play an important role as the
hypothalamic neurohormone that initiates
activation of the hypothalamic-pituitary-adrenal
axis (HPA axis) during stress. In addition, CRF
can serve in extrahypophyseal brain regions as
one of the most putative neurotransmitters that
mediate arousal response as well as autonomic
and behavioral responses to various stressors.
Several studies have suggested that stressful
manipulations could modify the induction of
yawning response. We previously reported that
stress-like manipulation such as light
stimulation and local hypoxia of the PVN induces
cortical arousal response accompanied by yawning
behavior. These suggest one possibility that CRF
neurons in the PVN, which could be activated by
various stressors, might mediate the arousal
response accompanied by yawning behavior.
Although the CRF-containing pathway involved
in an arousal response accompanied by yawning
evoked by PVN stimulation is still unknown, a
series of animal studies has suggested that CRF
serves as an excitatory neurotransmitter in the
LC, which is one of the regions within the
brainstem reticular formation responsible for
cortical activation, and its actions on LC
neurons elicit a cortical activation. It is
identified that the LC, which is one of the
major sources of noradrenalin (NA) in the
central nervous system, receives CRF afferents
from the PVN, and projects to various brain
areas containing the cortical regions. Several
studies have shown that CRF terminals
synaptically contact catecholaminergic dendrites
of the LC, and local administration of CRF into
the LC increases LC neuronal discharge. Curtis
et al. [5] indicated that activation of
the LC neurons by CRF administration into the LC
increases cortical NA release and ECoG activity,
whereas local microinfusion of a CRF antagonist
directly into the LC greatly decreases the
magnitude of LC activation produced by
intracerebroventricular administration of CRF.
These studies suggest that CRF serves as an
excitatory neurotransmitter to activate the
noradrenergic neurons in the LC, thereby
increasing cortical activity. In the present
study, we found that PVN stimulation
significantly enhances c-Fos expression in the
LC as well as throughout the cortical area,
together with increases in the percentage of
c-Fos positive CRF neurons in the PVN and the
frequency of yawning. Taken together, it is
suggested that an activation of the
noradrenergic system by the PVN CRF neurons
projecting to the LC may serve as a mechanism
for the arousal response during yawning induced
by the PVN stimulation or stressful
manipulations. However, several anatomical
studies have indicated multiple sources of CRF
innervation to the LC region, including the
nucleus paragigantocellularis, Barrington's
nucleus, central nucleus of the amygdala, bed
nucleus of the stria terminalis as well as the
PVN. Although the PVN CRF neuron is one of the
most possible sources of CRF innervations to the
LC, which can mediate the arousal response, we
cannot rule out the possibility that the other
sources of CRF neurons projecting to the LC may
contribute to the arousal response during
yawning.
Another possibility of the CRF-containing
pathway involved in the arousal/yawning response
evoked by PVN stimulation might be indirect
signaling pathway via orexinergic neurons
projecting to the LC. Recent studies show that
CRFimmunoreactive terminal make direct contact
with orexin neurons in the lateral hypothalamic
area and that CRF excites orexin neurons through
CRF-RI receptors. Thus, it is possible that the
PVN CRF neurons may mediate an arousal response
during yawning through the activation of
orexinergic neurons projecting to the LC.
The results of this study suggest that an
activation of CRF neurons in the PVN may induce
the arousal response during yawning behavior,
but we do not exclude the involvement of
oxytocin neurons in the yawning response.
Sawchenko and Swanson [28] demonstrated
that parvocellular oxytocinergic neurons in the
caudal part, containing the mp subdivision, of
the PVN send descending axons to the lower
brainstem involved in respiratory,
cardiovascular, and other autonomic functions.
In the present study, we showed that the
percentage of c-Fos positive oxytocin neurons
was significantly increased in the caudal part
of parvocellular subdivisions of the PVN after
L-glutamate injection. This result is compatible
with previous anatomical and functional findings
that suggest the importance of parvocellular
oxytocinergic neurons in the PVN for the
induction of yawning behavior characterized by a
single large inspiration with mouth opening and
a depressor response. Therefore, the
parvocellular oxytocinergic neurons in the PVN
might be responsible for behavioral responses
rather than the arousal response during yawning
behavior.
In summary, we observed that microinjection
of L-glutamate into the PVN produced frequent
spontaneous yawning accompanied by an arousal
shift in the ECoG, and these behavioral effects
are associated with increases in c-Fos positive
CRF neurons in the mp subdivision of the PVN and
c-Fos positive oxytocin neurons in the
parvocellular division of the PVN. These results
suggest that activation of CRF neurons as well
as parvocellular oxytocinergic neurons in the
PVN is involved in the arousal/yawning response.
In addition, a marked enhancement of c-Fos
expression was found in the LC and global area
in the cortex when frequent spontaneous yawning
was produced by the PVN stimulation. These
results suggest that the PVN stimulation induces
an arousal response during yawning behavior
through the activation of LC neurons, which is
one of the regions in the brainstem reticular
formation responsible for cortical activation,
and which receives CRF afferents from the PVN.
Taken together, CRF neurons in the PVN may play
the preferential role for mediating the arousal
response during yawning behavior.
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administration of corticotropin-releasing factor
antagonist attenuates arousal response
accompanied by yawning behavior in rats.
Neurosci.Letter 2008;433(3):205-208
-Kita I, Yoshida
Y, Nishino S. An activation of parvocellular
oxytocinergic neurons in the paraventricular
nucleus in oxytocin-induced yawning and penile
erection. Neurosci Res. 2006;54(4):269-275
-Kita I,
Sato-Suzuki et al.Yawning responses induced
by local hypoxia in the paraventricular nucleus
of the rat.Beh Brain Res
2000;117(1-2):119-126
-Kubota N, Amemiya
S, Motoki C, Otsuka T, Nishijima T, Kita I.
Corticotropin-releasing factor antagonist
reduces activation of noradrenalin and serotonin
neurons in the locus coeruleus and dorsal raphe
in the arousal response accompanied by yawning
behavior in rats. Neurosci Res. 2012
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stimulation of paraventricular nucleus of the
rat Journal of Neurophysiology,
1998;80(5):2765-2775
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Nakatani, et al Light induces cortical
activation and yawning in rat Behav Brain Res
2003;140(1-2):65-73
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Sato-Suzuki I, et al Yawning/cortical
activation induced by microinjection of
histamine into the paraventricular nucleus of
the rat. Behav Brain Res.
2002;134(1-2):75-82.
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