Male Sprague-Dawley rats (250-300 g) were
used. The animals were caged in groups of 4-6 at
22 °C with water and standard laboratory
food ad libitum. For electrolytic lesion, the
animals were anaesthetized with chloral hydrate
and positioned in a stereotaxic apparatus.
Following exposure of the skull, two small
bilateral holes were drilled using a dental
burr, at the PVN coordinates (0.4 mm lateral to
midline and 0.2 mm anterior to bregma) 16 . The
0.2 mm diameter tip of a tungsten electrode was
lowered to a depth of 7.3 mm into the brain. A
current of 2 mA was passed for 30 s using a
Grass D.C. constant current lesion maker. In
control animals (sham-operated rats) the
electrode was bilaterally lowered for 30 s at
the PVN coordinates but no current was passed.
After the appropriate lesion had been made, the
scalp was sutured and the animals were allowed
to recover. Fifteen days after lesion or
sham-lesion stainless-steel guide cannulas (22
gauge) aimed at one lateral ventricle were
stereotaxically implanted under chloral hydrate
anaesthesia 5 days before the experiments.
For i.c.v. injections, ACTH1-24 or oxytocin
dissolved in saline was injected into a lateral
ventricle via an internal cannula (28 gauge),
which extended 2 min below the tip of the guide
cannula and was connected by a polyethylene
tubing to a 10 ul Hamilton syringe driven by a
micrometric screw. Volumes injected into the
lateral ventricle were 10ul in 2 min. For
systemic injections, apomorphine-HCI was
dissolved in saline and subcutaneously
administered in the back of the neck in a volume
of 200 ul/rat. Sham-lesioned and lesioned rats
were treated either with saline, apomorphine,
oxytocin or ACTH1-24 at 4 day intervals between
two successive treatments. After treatment, the
animals were placed individually in Plexiglas
cages (30 X 30 X 30 cm) and observed for 60 min
(after apomorphine or oxytocin) or 90 min (after
ACTH1-24), during which the number of penile
erection and yawning episodes were counted. At
the end of the experiments, the animals were
killed by decapitation. Brains were rapidly
removed and stored in saline containing 2%
formaldehyde for 12-15 days. In order to
localize the i.c.v. injection site and/or to
evaluate the extent of the electrolytic lesion,
50 um transverse brain sections were made by
means of a freezing microtome, stained with
neutral red and inspected on a phase-contrast
microscope. The statistical evaluation of the
data was performed by the Student's mest or
Duncan's new multiplerange test.
In 20 out of 36 rats which underwent
lesioning, the whole region of the PVN was found
to be damaged in both lateral and rostrocaudal
directions. F ig shows a photograph of a 50um
Neutral red-stained transverse brain section
showing a representative bilateral electrolytic
lesion of the PVN region. The lesion extended
from the plane of the suprachiasmatic nucleus to
the dorsomedial hypothalamic nucleus and
involved parts of the anterior hypothalamic
nucleus and the dorsomedial nucleus. These
animals will be indicated as PVN-lesioned rats.
Lesions placed laterally or dorsally were found
in 9 and 7 rats, respectively. For statistical
analysis, these animals were considered as two
other experimental groups. Fig. 2 shows the
effect of i.c.v. oxytocin, i.c.v. ACTH1-24 and
systemic apomorphine in sham-lesioned rats
(controls), PVN-lesioned rats, and in rats with
the lesion placed dorsally or laterally to the
PVN. During the observation period, i.c.v.
oxytocin (30 tig) or ACTH 1-24 (10 lig) or
apomorphine (50 ug/kg s.c.) significantly
increased the number of penile erection and
yawning episodes in sham-operated rats and in
rats with the lesion placed dorsally or
laterally to the PVN. In contrast, the effect of
apomorphine and oxytocin, but not of ACTH1-24,
on yawning and penile erection was strongly
reduced in PVN-lesioned rats. Interestingly, all
PVN-lesioned rats showed hypermotility and
stereotypy similar to sham-lesioned controls
when treated with 1 mg/kg s.c. of
apomorphine.
The present results show that bilateral
electrolytic lesion of the hypothalamic PVN
prevents yawning and penile erection induced by
apomorphine and oxytocin but not by ACTH1-24.
The finding suggests that the PVN is the
brain area where DA agonists and oxytocin, but
not ACTH-derived peptides, act for inducing
yawning and penile erection in rats. This is
in agreement with our previous studies showing
that this hypothalamic nucleus is the most
sensitive brain area for the induction of the
above responses by oxytocin and apomorphine.
Indeed, both penile erection and yawning can be
induced by the unilateral microinjection of
nanograrn amounts of either apomorphine or
oxytocin in the PVN. As to the possible
mechanisrn by which DA and oxytocin act on the
PVN for inducing yawning and penile erection, it
is noteworthy that this hypothalamic nucleus
contains the cell bodies of at least two kinds
of oxytocinergic neurons: the magnocellular
neurons that send their projections mainly to
the neurohypophysis, and the parvocellular
neurons, many of which send their projections to
extrahypothalamic brain areas; and the cell
bodies of DA neurons of the A14 group that
constitute, together with those of Al l and A13
groups, the so called incertohypothalamic DA
system. Furthermore both DA and oxytocinergic
receptors have been identified in this nucleus.
While the location of DA receptors mediating
yawning and penile erection is still obscure
(i.e.postsynaptic DA receptors vs DA
autoreceptors),they apparently are of the D-2
type, being the response induced by the
microinjection into the PVN of the selective D-2
agonist LY 171555, but not by the D-1 agonist
SKF 38393.
The prevention of apomorphine- and
oxytocin-induced yawning and penile erection by
PVN lesion provides further support to the
hypothesis that apomorphine and other DA
agonists induce the above responses by releasing
oxytocin in this brain nucleus. Accordingly,
like PVN electrolytic lesion that inhibits
central oxytocinergic transmission by depleting
almost completely oxytocin across the brain
blockade of oxytocinergic receptors by the
potent oxytocin antagonist D-(CH2)5Tyr(Me)-Orn
8-vasotocin was found to be capable of
antagonizing not only oxytocin- but also
apornorphine-induced yawning and penile erection
as well. Conversely, in agreement with the above
hypothesis, blockade of DA receptors by
neuroleptics, such as haloperidol or sulpiride,
was found to be able to antagonize yawning and
penile erection induced by apomorphine, but not
by oxytocin. As to the mechanism by which
oxytocin, either exogenous or released by DA
agonists, induces yawning and penile erection,
only some speculation is possible at present.
One possibility is that oxytocin activates its
own neurons. Accordingly, exogenous oxytocin bas
been found to activate the neuronal activity of
magnocellular neurons in vivo and to release
endogenous oxytocin in vitro. Further more,
oxytocinergic synapses have been found to
impinge on oxytocinergic cell bodies in
hypothalamic nuclei.
The failure of PVN electrolytic lesion to
modify yawning and penile erection induced by
ACTH1-24 is in agreement with previous studies
showing that ACTH1-24 induces yawning and
stretching when injected in several hypothalamic
nuclei. This suggests a site of action of
ACTH-derived peptides more diffuse than that of
oxytocin and DA agonists. Taken together
with the finding that ACTH-induced yawning and
penile erection are not antagonized either by
neuroleptics or by the oxytocin antagonist
D(CH2)5Tyr(Me)-Orn 8-vasotocin the
ineffectiveness of PVN lesion to modify ACTH
effect suggests that ACTH-derived peptides
induce yawning and penile election by a
mechanism not involving PVN hypothalamic DA or
oxytocin, or vice versa. Accordingly, oxytocin
and DA agonists apparently do not induce yawning
and penile erection by releasing an ACTH-like
peptide in the hypothalamus, since the depletion
of hypothalamic ACTH-melanocyte-stimulating
hormone peptides by neonatal monosodium
glutamate treatment was found to be unable to
modify oxytocin- and apomorphine-induced
responses.
In conclusion, the
present results provide further evidence for the
existence in the PVN of a DA-oxytocin link that
plays a physiological role in the control of
yawning and penile erection.
