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 Brain Research
1987; 421; 349-352
Paraventricular nucleus lesion prevents yawning and penile erection induced by apomorphine and oxytocin but not by ACTH in rats 
Argiolas A, Melis MR, Mauri A, Gessa GL
Department of neuroscience Cagliari University Italy
Tous les travaux de MR Melis & A Argiolas 
Tous les travaux de M Eguibar & G Holmgren


Recurrent episodes of yawning and penile erection can be induced in experimental animals either by intracerebroventricular (i.c.v.) injection of adrenocorticotropin (ACTH) and derived peptides, or by the i.c.v. injection of nanogram amounts of oxytocin, or by the systemic administration of dopamine (DA) agonists such as apomorphine.
While the importance of penile erection in reproduction does not need to be further stressed, it is pertinent to recall that yawning is considered to be a mechanism of arousal. Previous studies showed that the hypothalamus is the brain area most sensitive for the induction of the above effects by ACTH-derived peptides, suggesting a role of this brain area in the expression of yawning and penile erection. Moreover recent results from our laboratory have shown that the paraventricular nucleus of the hypothalamus (PVN) is a brain area where oxytocin and apomorphine apparently act to induce yawning and penile erection.
To further elucidate the role of the PVN on yawning and penile erection induced by the above substances, we have studied the effect of PVN electrolytic lesion on yawning and penile erection induced by apomorphine, oxytocin and ACTH,-24.
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.