Bernard B. Brodie Department
of Neuroscience, University of Cagliari,
Italy
Penile erection and yawning are two
différent behavioral responses that often
occur concomitantly in experimental animals.
Among agents that induce both penile erection
and yawning in male rats the best known are
dopaminergic agonists ), oxytocin and
ACTH-related peptides. Recently,
N-methyl-D-aspartic acid (NMDA) also was found
to induce penile erection, yawning and grooming
when injected in the paraventricular nucleus of
the hypothalamus (PVN). NMDA-induced penile
erection and yawning are apparently mediated by
the stimulation of excitatory amino acid
receptors of the NMDA subtype, that in turn
activates oxytocinergic transmission, being
prevented by MK-801, a non-competitive
antagonist of this receptor subtype and by the
oxytocin receptor antagonist
d(CH2),Tyr(Me)-Orn'-vasotocin. Interestingly,
NMDA receptor stimulation induces a calcium
influx that has been associated with the
activation of nitric oxide (NO) synthase to
produce NO in several tissues, including brain
neurons, which in turn causes the stimulation of
guanylate cyclase. This raises the possibility
that NMDA induces penile erection and yawning
by activating NO synthase in the PVN. In
order to test this hypothesis, the effect of
NO-synthase inhibitors, methylene blue, an
inhibitor of guanylate cyclase and
methemoglobin, a NO scavenge, on NMDA-induced
penile erection and yawning was studied in male
rats.
Male Sprague Dawley rats (200-220 g) were
used in all the experiments. Animals were caged
in groups of 4-6 at 24°C, humidity 60%,
with water and standard laboratory food ad
libitum. Stainless-steel guide cannulas (22
gauge) aimed unilaterally at the PVN were
stereotaxically implanted under chloral hydrate
anaesthesia 5 days befère the experiments
(coordinates: 0.2 mm anterior to bregma, 0.4
lateral to midline and 2.0 mm ventral to dura).
The animals were given 5 days to recover from
surgery; each rat was used only once. The same
guide cannula was used for
intracerebroventricular (i.c.v.) and PVN
injections. Drugs were dissolved in saline and
injected i.c.v. in 10 jul in 1 min, and into the
PVN in 0.3 ul in 2 min via an internal cannula
(28 gauge), which extended 1.0 mm and 5.3 mm
below the tip of the guide cannula for i.c.v.
and PVN injections, respectively. The internai
cannula was connected by polyethylene tubing to
a 10µl Hamilton syringe driven by a
Stoelting,microinfusion pump. After injection,
the tip of the cannula was left in the injection
site for 30 s in order to allow the spread of
the injected solution. N-NitrO-L-arginine
methylester (NAME),
N-monomethyl-L-arginine (NMMA),
N-monomethyl-Darginine (D-NMMA), Methylene blue
and bovine methemoglobin (ail from Sigma, St.
Louis, MOI USA), (5R,IOS)-(+)-5-methyl-10, 1
1-dihydro-5H-dibenzo[a,d]
cyclo-hepten-5,10-imine hydrogen maleate
«+)-MK801) or saline were given i.c.v. or
in the PVN 15 min before NMDA. NMDA was given in
the PVN. Shortly after NMDA treatment, animais
were placed individually into Plexiglas cages
(30 x 30 x 30 cm) and observed for 60 min,
during which penile erection and yawning
episodes were counted. Only those animais that
were found to have the cannula tip positioned
correctly in the lateral ventricles or in the
PVN by histological examination (see [
18,19] for details) were considered for the
statistical evaluation of the results by means
of Oneway ANOVA, followed by Duncan's multiple
range test. A P < 0.05 was considered
significant.
Fig. 1 shows that penile erection and yawning
induced by NMDA (50 ng into the PVN) were
prevented dosedepentlently by NAME (75-150,ug),
NMMA (250-500 mg) and Methylene blue (200-400
ug) but not by DNMMA (250-500 /ig), the inactive
isomer of NMMA, or by methemoglobin (50-100,ug)
ail given i.c.v. 15 min before NMDA. As shown in
Fig. 2, NAME (10-20,ug), like MK-801 (50-100
ng), prevented NMDA-induced penile erection and
yawning when injected into the PVN 15 min
befère NMDA. In contrast, both Methylene
blue (10-20,ug) and methemoglobin (10-20,ug)
injected into the PVN were ineffective.
The present results show that NMDA-induced
penile erection and yawning is prevented by the
NO-synthase inhibitors given i.c.v. with a
potency that is parallel to their potency in
inhibiting NO-synthase The most potent, NAME,
prevented NMDA-induced responses also when
injected in the PVN. Since these NMDA responses
are prevented also by the selective antagonist
of NMDA receptors MK-801 and by the nonapeptide
oxytocin receptor antagonist d(CH,),-Tyr(Me)2
-Orn'vasotocin the findings suggest that NMDA
induces penile erection and yawning by
activating PVN NOsynthase, thereby
increasing the content of NO, which would in
turn activate oxytocinergic transmission. In
agreement with this hypothesis, NO-synthase
inhibitors prevent oxytocin-induced penile
erection and yawning; the NO donor nitroglycerin
induces penile erection and yawning when
injected in the PVN by activating oxytocinergic
transmission, the PVN is one of the brain
areas that contains the highest number of
NOsynthase-immunostaining neurons and
NO-synthase is localized in paraventricular
oxytocinergic neurons. It is likely that
NMDA-induced NO-synthase activation is mediated
by an increased calcium influx through NMDA
receptor-coupled calcium channels, as proposed
to explain the NMDA-induced increase of cyclic
guanosine monophosphate (cGMP) in cerebellar
granular cells in vitro and from rat cerebral
cortex in vivo.
The mechanism(s) by means of which NO,
formed by the stimulation of PVN NMDA receptors,
would activate oxytocinergic transmission to
induce penile erection and yawning is unknown.
The prevention by i.c.v. Methylene blue, an
inhibitor of guanylate cyclase, of the NMDA
responses suggests that NMDA activates the
NO-cGMP pathway.
In agreement with this hypothesis, NO would
be released from those neurons in which is
formed and activate guanylate cyclase in target
cells as a neurotransmitter. However, a role of
guanylate cyclase in these NMDA behavioral
responses must be considered with caution for at
least two reasons. The first is that both
Methylene blue and methemoglobin, a NO
scavenger, did not prevent NMDA responses when
injected directly in the PVN. This raises the
possibility that NMDA-induced penile erection
and yawning are mediated by a mechanism not
related to the stimulation of guanylate cyclase,
that is by a cGMP-indepenilent mechanims.
Accordingly, NO might interact with other
enzymes that, like guanylate cyclase, bind metal
ions such as iron, as described for instance in
fibroblasts.
The second reason is that Methylene blue
might inhibit directly NO-synthase. However,
this is unlikely in our experiments because
Methylene blue, unlike NAME, did not prevent
penile erection and yawning when injected in the
PVN. Whatever mechanism is activated by NO, the
failure of methemoglobin, that does not cross
cellular membranes [231, to prevent
NMDA-induced penile erection and yawning when
injected i.c.v. or in the PVN, suggests also
that NO acts intracellularly in the neurons
where it is formed, rather than as a
neurotransmitter, in contrast to what suggested
above. One possible unifying explanation for
this discrepancy is that NO activates the
neurons in which it is formed, thereby releasing
a neurotransmitter, e.g. oxytocin (see above),
which in turn activates a Methylene
blue-sensitive guanylate cyclase in a brain
area(s) distant from the PVN. The identification
of this brain area(s) will allow to clarify the
role of guanylate cyclase in NMDA-induced penile
erection and yawning, for instance by studying
the effect of stable analogs of cGMP injected in
this area on these responses.
In conclusion NMDA induces penile erection
and yawning in male rats apparently by
increasing NO synthesis in the PVN. This
provides further evidence that NO plays a key
role in the control of penile erection not only
in the corpus cavernosum, but also in the
central nervous system.
