Repeated episodes of yawning and penile
erection can be induced in rats and other
experimental animals by the systemic
administration of low doses of dopamine (DA)
agonists, such as apomorphine, by the central
administration of adrenocorticotropin (ACTH),
alpha-melanocyte-stimulating hormone (a-MSH) and
related peptides, and by the
intracerebroventricular (i.cv.) injection of
oxytocin. While the importance of penile
erection in reproduction does not need to be
further stressed, it is pertinent to recall
that yawning, alone or associated with
stretching, is considered to be an ancestral
vestige surviving throughout evolution, that
subserves the purpose of arousal. In
particular, the role of yawning could be that of
increasing attention when sleep is imminent due
to fatigue or boredom but cannot be engaged in,
as in face of a danger or social circumstances.
The capability of the above unrelated
substances to induce such similar symptomatology
raises the possibility that a neuronal link
exists among DA, oxytocin, and ACTH in the
central nervous system. The results of the
experiments presented below, which were
performed with the aim of demonstrating the
existence of such a link and to clarify the
neural mechanisms underlying the expression of
yawning and penile erection as well, provide
evidence for the first time that DA, oxytocin,
and ACTH act in the hypothalamus in a sequence
to induce these behavioral responses.
Effect of DA Antagonists on Yawning and
Penile Erection Induced by Apomorphine,
Oxytocin, and ACTH
The first group of experiments was aimed at
the identification of possible interactions
among DA agonists, oxytocin, and ACTH-MSH
peptides in the induction of yawning and penile
erection, namely to clarify whether oxytocin
and/or ACTH induce the above responses by
releasing DA in brain or vice versa. Male
Sprague-Dawley rats were used in all the
experiments. For icv. injections, chronic guide
cannulae aimed at one lateral ventricle were
stereotaxically implanted under chloral hydrate
anaesthesia 5 days before the experiments, as
previously described. Peptides were injected
into a lateral ventricle by means of an internal
cannula connected by a polyethylene tubing to a
10-µl Hamilton syringe driven by a
micrometric screw. TABLE 1 shows the effect of
haloperidol and (-)-sulpiride, two specific DA
receptor blockers, on yawning and penile
erection induced by apomorphine, oxytocin, and
ACTH. In agreement with previous
studies,apomorphine (80 µg/kg s.c.),
oxytocin (30 ng i.cv.), and ACTH (5 µg
i.cv.) induced repeated episodes of yawning and
penile erection. Haloperidol (0.2 mg/kg i.p.)
and (-)-sulpiride (10 mg/kg i.p.) administered
30 min beforehand completely suppressed
apomorphine-induced response; these receptor
blockers, however, were ineffective against
yawning and penile erection induced by oxytocin
and ACTH even at doses of 2 mg/kg and 50 mg/kg,
respectively.
The failure of haloperidol and sulpiride to
antagonize yawning and penile erection induced
by oxytocin and ACTH suggests that the two
peptides do not induce the above responses by
releasing DA in the central nervous system, but
act downstream front DA receptors.
Effect of the Oxytocin Antagonist
Vasotocin on Yawning and Penile Erection Induced
by Apomorphine, Oxytocin and ACTH
The availability of a potent oxytocin
antagonist vasotocin gave us the possibility to
verify if DA agonists and/or ACTH induce yawning
and penile erection by releasing oxytocin in
brain. The effect of the icv. administration of
vasotocin on yawning and penile erection induced
by apomorphine, oxytocin, and ACTH is shown in
FIGURE 1. The oxytocin antagonist injected icv.
15 min before the administration of the above
substances not only antagonized in a
dose-dependentmanner oxytocin-induced response,
as was expected, but also even more effectively
antagonized the response induced by apomorphine.
A 50% inhibition of the apomorphine and oxytocin
effect was already obtained with 10 ng and 50 ng
of the oxytocin antagonist, respectively. A
complete suppression of either the apomorphine
or oxytocin effect was obtained with 100 ng of
the peptide. On the contrary, a dose as high as
up to 10 gg of the oxytocin analogue was unable
to antagonize yawning and penile erection
induced by 5 gg of i.cv. ACTH. It is noteworthy
that doses of the oxytocin antagonist that
suppressed apomorphine-induced yawning and
penile erection were totally ineffective in
antagonizing stereotypy and hypermotility
induced by 1 mg/kg of apomorphine administered
subcutaneously (results not shown). The results
obtained with vasotocin suggest that apomorphine
and other DA agonists, but not ACTH-MSH
peptides, induce yawning and penile erection by
releasing oxytocin in some brain area, and that
ACTH-MSH peptides act downstream from DA
receptors as well as oxytocin to induce the
above responses.
Effect of Neonatal Monosodium Glutamate
(MSG) treatment on Yawning and penile Erection
Induced by Apomorphine, Oxytocin, and
ACTH
The results obtained with DA and oxytocin
antagonists suggest that DA, oxytocin, and ACTH
act in sequence to induce yawning and penile
erection. However, the possibility that DA
and/or oxytocin induce the above responses by
releasing an ACTHderived peptide from the
recently discovered opiomelanotropinergic
neurons in the hypothalamus (for a review, see
ref. 6) remains to be verified. Since specific
antagonists of ACTH-MSH peptides capable of
antagonizing their central effects are not
available at present, we have attempted to
verify the above possibility by studying the
effect of apomorphine, oxytocin, and ACTH, on
yawning and penile erection in rats neonatally
treated with monosodiurn glutamate (MSG). Such
treatment bas been found to cause the almost
complete depletion of brain ACTH, MSH, and
endorphinlike peptides without altering their
pituitary and circulating concentrations. Under
our conditions, neonatal MSG treatment caused
both the expected marked reduction in growth,
secondary to the destruction of hypothalamic
growth-hormone-releasing hormone, and a decrease
of about 90% in the hypothalamic concentration
of ACTH and a-MSH as measured by specific
radioimmunoassays. The results obtained with
neonatally MSG-treated rats are shown in TABLE
2. Surprisingly, the depletion of ACTHMSH-like
peptides from the hypothalamus was completely
ineffective in modifying yawning and penile
erection induced not only by apomorphine and
oxytocin but also by ACTH.
The ineffectiveness of the hypothalamic
depletion of ACTH-MSH peptides to modify the
behavioral effects of the DA agonist and
oxytocin effect suggests that oxytocin and DA
agonists do not induce yawning and penile
erection by releasing an ACTHderived peptide in
brain, although it is possible that the small
amount of ACTH and a-MSH remaining in the
hypothalamus still might be sufficient to
mediate the DA and/or oxytocin effect. This
possibility is unlikely, however, since doses of
ACTH or a-MSH much higher than those of DA
agonists or oxytocin are needed to induce
yawning and penile erection, and no
supersensitivity to ACTH or a-MSH was found in
neonatally MSG-treated rats not only with regard
to yawning and penile erection (present results)
but also to other behavioral and biochemical
responses.
Another possibility that cannot be completely
ruled out by the results obtained with
MSG-treated rats is that ACTH or a-MSH are
released by DA agonists or oxytocin in some
brain area where they are not depleted by MSG
treatment, since opiomelanotropinergic neurons
have been identif ied also in extrahypothalamic
brain areas. However, previous studies have
shown that the most sensitive brain areas for
the induction of yawning and penile erection by
ACTH-MSH peptides are localized in the
hypothalamus, and are those surrounding the
third ventricle. Hence, it is unlikely that
ACTH-MSH peptides act to induce yawning and
penile erection in an area other than the
hypothalamus.
Oxytocin- and Apomorphine-induced Yawning
and Penile Erection: Site of Action in
Brain
Besides the studies cited above showing that
ACTH-MSH peptides induce yawning and pende
erection by acting in the hypothalamic regions
surrounding the third ventricle, no information
was available so far about the brain areas where
DA agonists and/or oxytocin act in order to
induce such responses. In an attempt to identify
these brain areas we have microinjected
apomorphine and/or oxytocin in discrete brain
regions through chronic guide cannulae,
stereotaxically implanted under chloral hydrate
anaesthesia 5 days before the experiments,
according to a stereotaxic atlas of the rat
brain. Apomorphine and oxytocin were dissolved
in saline and injected into the various nuclei
in a volume of 0.3 µl per site by means of
an internal cannula connected by polyethylene
tubing to a 10-gl Hamilton syringe driven by a
Stoelting microinfusion pump. The length of the
internal cannula was adjusted according to the
position nuclei to be injected. The correct
position of the cannula tip was verified at the
end of the experiments by histological analysis
as previously described. The brain areas that
were microinjected with saline, apomorphine (1
µg), and oxytocin (30 ng) are listed in
TABLE 3. The paraventricular nucleus of the
hypothalamus (PVN) was found to be the only
area where microinjections of apomorphine and
oxytocin induced yawning and penile erection.
Surprisingly, no effect was observed when
apomorphine was microinjected in areas very rich
in DA and DA receptors, such as the striatum,
the nucleus accumbens, or the substantia nigra.
Microinjections of apomorphine or oxytocin in
other hypothalamic nuclei very close to the PVN,
such as the ventromedial and dorsomedial nuclei
and the preoptic area were also ineffective. The
effect of apomorphine and oxytocin
microinjections into the PVN was then studied in
detail. As shown in FiGURE 2, yawning and penile
erection were induced in a dose-dependent manner
by both substances. The minimal effective dose
of oxytocin and apomorphine was 3 ng and 5 ng,
respectively, which induced the response in
about 60% of the treated animals. The yawning
and penile erections induced by intracranial
microinjection of apomorphine or oxytocin
microinjection was similar to that observed
after systemic apomorphine or ic-v oxytocin,
except the response started within 5 min after
the microinjections. Even at the highest dose
tested (1 µg), apomorphine failed to induce
stereotypy and hypermotility.
The above results show that both
apomorphine and oxytocin act in the hypothalamic
PVN to induce yawning and penile erection.
The potency of the two substances and the fact
that both are present in the PVN suggest that DA
and oxytocin might have a physiological role in
the control of the nucleus responses. Indeed,
PVN contains the cell bodies of at least two
types of oxytocinergic neurons: the
magnocellular neurons, projecting to the
neurohypophysis, from which oxytocin is released
into the circulation to exert its hormonal role
in parturition and lactation, and the
parvocellular neurons, many of which send their
projections to several extrahypothalamic brain
areas. In addition to oxytocinergic cell bodies,
the PVN also contains the cell bodies of
dopaminergic neurons of the group A14, which
together with those of the groups A11 and A13
constitute the so-called incertohypothalamic
system. Our results suggest that DA agonists
interact with DA receptors in the PVN or
surrounding structures to stimulate the activity
of oxytocinergic neurons, which in turn mediate
the appearance of yawning and penile erection.
In support of this hypothesis,
immunocytochernical studies have shown that DA
neurons in the PVN are mainly located in the
proximity of oxytocinergic neurons. As for the
kind of DA receptors mediating yawning and
penile erection, previous studies have shown
that they belong to the D2 type, although
whether they are DA autoreceptors (a special
kind of inhibitory receptor located in the nerve
terminal and cell body of the neuron itself) or
postsynaptic DA receptors is still
controversial.
As to the mechanism by which oxytocin acts in
the PVN to induce yawning and penile erection,
only some speculation is possible at present. A
possible explanation is that oxytocin activates
oxytocinergic neurons. Supporting this
hypothesis, oxytocinergic receptors have been
identified in the rat PVN and exogenous oxytocin
has been found to increase in vivo the activity
of oxytocinergic neurons and to stimulate in
vitro the release of endogenous oxytocin.
Furthermore, oxytocinergic synapses have been
found to impinge on oxytocinergic neurons in
hypothalamic nuclei.
As previously mentioned the hypothalamic
regions surrounding the third ventricle,
comprising the PVN, were found to be the most
sensitive for the induction of yawning,
stretching. and penile erection by ACTH-MSH
peptides. ACTH fibers have been identified in
the PVN, but they seem not to contact
oxytocinergic or DA neurons.
Conclusions
These results show for the first time that DA
agonists induce yawning and penile erection by
releasing oxytocin in the central nervous
system. The brain area where DA agonists
apparently act in order to induce the release of
oxytocin release seems to be the hypothalamic
PVN. This finding suggests a direct involvement
of the incertohypothalamic dopaminergic system
in the expression of yawning and penile
erection.
On the other hand, DA agonists and oxytocin
do not appear to induce yawning and pende
erection by releasing an ACTH-derived peptide
from hypothalamic opiomelanotropinergic neurons.
However, our results suggest that ACTH-MSH
peptides induce their effect by acting at sites
localized downstream from DA receptors and
oxytocin. Finally, the finding that oxytocin is
implicated in the expression of pende erection
opens new clinical perspectives, raising the
possibility that abnormalities in the central
oxytocinergic function might be responsible for
penile erection disturbances.
