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Brain Research Bulletin, Vol. 24, pp. 853-856. 0 Pergamon Press plc, 1990. Printed in the U.S.A
RAPID COMMUNICATION
Role of Calcium in the Expression of ACTH-Induced Stretching, Yawning and Penile Erection A. ARGIOLAS, B. B. Brodie Department
M. R. MELIS,
of Neurosciences,
R. STANCAMPIANO
University of Cagliari,
Received
AND G. L. GESSA Via Porcell 4, 09124
Cagliari,
Italy
28 March 1990
ARGIOLAS, A., M. R. MELIS, R. STANCAMPIANO AND G. L. GESSA. Role of calcium in the expression of ACTH-induced stretching, yawning and penile erection. BRAIN RBS BULL 24(6) 853-856, 19!90.-The effect of w-conotoxin GVIA, a potent and selective inhibitor of N-type calcium channels and of the organic calcium channel inhibitors nimodipine, verapamil and flunarizine, on stretching, yawning and penile erection induced by ACTH l-24 was studied in male rats. w-Conotoxin (l-10 ng ICV 15 min before ACTH, 10 pg ICV), but not carboxymethylated o-conotoxin, induced a dose-dependent prevention of all ACTH effects. In contrast,
organic calcium channel inhibitors (20 mg/kg II’ 30-60 min before ACl’H) failed to modify ACTH-induced stretching and yawning but induced a 25% decrease in the number of penile erection episodes induced by the peptide, and prevented, like ICV w-conotoxin, oxytocin- and apomorphine-induced yawning and penile erection. When injected in the paraventricular nucleus of the hypothalamus, w-conotoxin prevented the above behavioral responses induced by apomorphine and oxytocin but not by ACTH l-24. The present results suggest that ACTH induces stretching, yawning and penile erection by mobilizing calcium through central w-conotoxinsensitive calcium channels in brain sites different from those sensitive to oxytocin and apomorphine. ACTH l-24
w-Conotoxin
Organic
calcium channel inhibitors
Stretching
Yawning
oxytocin in the expression investigated.
THE intracerebroventricular (ICV) administration of adrenocorticotropin (ACTH) and related peptides induces a peculiar symptomatology characterized by recurrent episodes of stretching, yawning, penile erection and ejaculation in different experimental animals [for a review see (10) and (1 l)]. In rats, the symptomatology begins 25-30 min after the treatment and lasts for several hours. The reason for the delay in the onset of the effect and the mechanism by which ACTH acts in the central nervous system to induce these behavioral responses are unknown, although the hypothalamus is believed to be the brain site where the peptide acts to induce the above effects (13) and pharmacological studies suggest that central cholinergic and opioid transmission are involved (12). As to the physiological significance of these behavioral responses, while the importance of penile erection in reproduction does not need to be stressed, it is pertinent to recall that stretching and yawning have been considered to have the role of increasing attention when sleep is pressing in front of a danger or social circumstances [see (12)]. With the aim to clarify the mechanism by which ACTH and related peptides induce the above behavioral responses, we studied the effect of calcium channel blockade on ACTH-induced stretching, yawning and penile erection. Furthermore, since penile erection and yawning can be also induced by the dopaminergic agonist apomorphine and oxytocin [see (1) and (20)], the relationship among ACTH, dopamine and
Penile erection
of these behavioral
responses
was
METHOD
Male Sprague-Dawley rats (200-250 g) were used in all the experiments. The animals were caged in groups of 4-6 at 22°C with water and standard laboratory food ad lib. ICV Injections and Microinjections in the Paraventricular Nucleus of the Hypothalamus (PVN) Stainless steel chronic guide cannulas (22 gauge) aimed at one lateral ventricle or monolaterally to the PVN were stereotaxically implanted under chloral hydrate anaesthesia 5 days before the experiments (coordinates: lateral ventricle, 1 mm anterior to bregma, 1.5 mm lateral to midline and 22 mm ventral to dura; PVN, 0.2 mm anterior to bregma, 0.4 mm lateral to midline and 2 mm ventral to dura) (24). Peptides dissolved in saline or saline alone were injected into the lateral ventricle via an internal cannula (28 gauge) which extended 2 mm below the tip of the guide cannula, and connected by polyethylene tubing to a lo-p1 Hamilton syringe driven by a micrometric screw. Volumes injected ICV were 5-10 p,l in 0.5-l min. In those experiments in which w-conotoxin was used to inhibit brain calcium channels, for ICV
853
ARGIOLAS , MELIS, STANCAMPIANO
854
injections o-conotoxin was dissolved in saline and injected in a volume of 5 l.~l 15 min before ACTH l-24 through the same guide cannula used for ACTH l-24 injection. For PVN microinjections, o-conotoxin was injected by means of an internal cannula which extended 5.3 mm below the tip of the guide cannula aimed to me PVN and connected by polyethylene tubing to a Stoelting microinfusion pump in a volume of 0.3 pl of saline in 2 min. ACTH l-24 or oxytocin was injected in a volume of 10 l.t,lof saline into the ventricle 10 min after w-conotoxin through the same guide cannula by lowering the internal cannula 2 mm below the tip of the guide cannula (24).
AND GESSA
5 --
@=e 2 1:
-\;\I
Systemic Treatments ( k )Verapamil, nimodipine-HCl and flunarizine-HCl (all from Sigma) were dissolved in distilled water:ethylene glycol:ethanol (70:20: 10, V:V:V) and injected intraperitoneally (IP) in a volume of 1 ml/rat. Controls received 1 ml of vehicle. Apomorphine-HCl (Sigma, MO) was injected subcutaneously (SC) in the back of the neck in a volume of 0.1 ml of saline/100 g body weight. Behavioral Studies Soon after treatment, rats were individually placed in Plexiglas cages (30 x 30 X 30 cm) and observed for 2 or 1 hr after ACTH or oxytocin and apomorphine, respectively, during which stretching, yawning and penile erection episodes were counted. At the end of the experiments, rats were killed by decapitation, the brains were removed and visually inspected to ascertain me correct position of the cannula tip in the lateral ventricle. In those experiments in which PVN microinjections were made, brains were stored in saline containing 2% formaldehyde for 10-12 days. Transverse 50 pm brain sections were prepared by means of a freezing microtome, stained with Neutral Red and inspected on a phase-contrast microscope. Only those animals that were found to have the tip of the cannula positioned correctly into the lateral ventricle or in the PVN were used for statistical evaluation of the data (Duncan multiple
range
Oo -8
2
6
(I 10
4
8
Cd-conotoxin
(ng I C V)
Prevention of ACTH I-24-induced stretching, yawning and penile erection by ICV o-conotoxin. w-Conotoxin (0) or carboxymethylated w-conotoxin (0) were dissolved in saline and injected ICV in a volume of 5 pl 10 min before ACTH l-24 (10 p,g/rat ICV in 5 pl of saline). After treatment, rats were placed individually into Plexiglas cages and observed for 2 hours, during which stretching, yawning and penile erection episodes were counted. Values are means * S.E.M. of 3 experiments (12 rats per group). *p
Brain Research Bulletin, Vol. 24, pp. 853-856. 0 Pergamon Press plc, 1990. Printed in the U.S.A
RAPID COMMUNICATION
Role of Calcium in the Expression of ACTH-Induced Stretching, Yawning and Penile Erection A. ARGIOLAS, B. B. Brodie Department
M. R. MELIS,
of Neurosciences,
R. STANCAMPIANO
University of Cagliari,
Received
AND G. L. GESSA Via Porcell 4, 09124
Cagliari,
Italy
28 March 1990
ARGIOLAS, A., M. R. MELIS, R. STANCAMPIANO AND G. L. GESSA. Role of calcium in the expression of ACTH-induced stretching, yawning and penile erection. BRAIN RBS BULL 24(6) 853-856, 19!90.-The effect of w-conotoxin GVIA, a potent and selective inhibitor of N-type calcium channels and of the organic calcium channel inhibitors nimodipine, verapamil and flunarizine, on stretching, yawning and penile erection induced by ACTH l-24 was studied in male rats. w-Conotoxin (l-10 ng ICV 15 min before ACTH, 10 pg ICV), but not carboxymethylated o-conotoxin, induced a dose-dependent prevention of all ACTH effects. In contrast,
organic calcium channel inhibitors (20 mg/kg II’ 30-60 min before ACl’H) failed to modify ACTH-induced stretching and yawning but induced a 25% decrease in the number of penile erection episodes induced by the peptide, and prevented, like ICV w-conotoxin, oxytocin- and apomorphine-induced yawning and penile erection. When injected in the paraventricular nucleus of the hypothalamus, w-conotoxin prevented the above behavioral responses induced by apomorphine and oxytocin but not by ACTH l-24. The present results suggest that ACTH induces stretching, yawning and penile erection by mobilizing calcium through central w-conotoxinsensitive calcium channels in brain sites different from those sensitive to oxytocin and apomorphine. ACTH l-24
w-Conotoxin
Organic
calcium channel inhibitors
Stretching
Yawning
oxytocin in the expression investigated.
THE intracerebroventricular (ICV) administration of adrenocorticotropin (ACTH) and related peptides induces a peculiar symptomatology characterized by recurrent episodes of stretching, yawning, penile erection and ejaculation in different experimental animals [for a review see (10) and (1 l)]. In rats, the symptomatology begins 25-30 min after the treatment and lasts for several hours. The reason for the delay in the onset of the effect and the mechanism by which ACTH acts in the central nervous system to induce these behavioral responses are unknown, although the hypothalamus is believed to be the brain site where the peptide acts to induce the above effects (13) and pharmacological studies suggest that central cholinergic and opioid transmission are involved (12). As to the physiological significance of these behavioral responses, while the importance of penile erection in reproduction does not need to be stressed, it is pertinent to recall that stretching and yawning have been considered to have the role of increasing attention when sleep is pressing in front of a danger or social circumstances [see (12)]. With the aim to clarify the mechanism by which ACTH and related peptides induce the above behavioral responses, we studied the effect of calcium channel blockade on ACTH-induced stretching, yawning and penile erection. Furthermore, since penile erection and yawning can be also induced by the dopaminergic agonist apomorphine and oxytocin [see (1) and (20)], the relationship among ACTH, dopamine and
Penile erection
of these behavioral
responses
was
METHOD
Male Sprague-Dawley rats (200-250 g) were used in all the experiments. The animals were caged in groups of 4-6 at 22°C with water and standard laboratory food ad lib. ICV Injections and Microinjections in the Paraventricular Nucleus of the Hypothalamus (PVN) Stainless steel chronic guide cannulas (22 gauge) aimed at one lateral ventricle or monolaterally to the PVN were stereotaxically implanted under chloral hydrate anaesthesia 5 days before the experiments (coordinates: lateral ventricle, 1 mm anterior to bregma, 1.5 mm lateral to midline and 22 mm ventral to dura; PVN, 0.2 mm anterior to bregma, 0.4 mm lateral to midline and 2 mm ventral to dura) (24). Peptides dissolved in saline or saline alone were injected into the lateral ventricle via an internal cannula (28 gauge) which extended 2 mm below the tip of the guide cannula, and connected by polyethylene tubing to a lo-p1 Hamilton syringe driven by a micrometric screw. Volumes injected ICV were 5-10 p,l in 0.5-l min. In those experiments in which w-conotoxin was used to inhibit brain calcium channels, for ICV
853
ARGIOLAS , MELIS, STANCAMPIANO
854
injections o-conotoxin was dissolved in saline and injected in a volume of 5 l.~l 15 min before ACTH l-24 through the same guide cannula used for ACTH l-24 injection. For PVN microinjections, o-conotoxin was injected by means of an internal cannula which extended 5.3 mm below the tip of the guide cannula aimed to me PVN and connected by polyethylene tubing to a Stoelting microinfusion pump in a volume of 0.3 pl of saline in 2 min. ACTH l-24 or oxytocin was injected in a volume of 10 l.t,lof saline into the ventricle 10 min after w-conotoxin through the same guide cannula by lowering the internal cannula 2 mm below the tip of the guide cannula (24).
AND GESSA
5 --
@=e 2 1:
-\;\I
Systemic Treatments ( k )Verapamil, nimodipine-HCl and flunarizine-HCl (all from Sigma) were dissolved in distilled water:ethylene glycol:ethanol (70:20: 10, V:V:V) and injected intraperitoneally (IP) in a volume of 1 ml/rat. Controls received 1 ml of vehicle. Apomorphine-HCl (Sigma, MO) was injected subcutaneously (SC) in the back of the neck in a volume of 0.1 ml of saline/100 g body weight. Behavioral Studies Soon after treatment, rats were individually placed in Plexiglas cages (30 x 30 X 30 cm) and observed for 2 or 1 hr after ACTH or oxytocin and apomorphine, respectively, during which stretching, yawning and penile erection episodes were counted. At the end of the experiments, rats were killed by decapitation, the brains were removed and visually inspected to ascertain me correct position of the cannula tip in the lateral ventricle. In those experiments in which PVN microinjections were made, brains were stored in saline containing 2% formaldehyde for 10-12 days. Transverse 50 pm brain sections were prepared by means of a freezing microtome, stained with Neutral Red and inspected on a phase-contrast microscope. Only those animals that were found to have the tip of the cannula positioned correctly into the lateral ventricle or in the PVN were used for statistical evaluation of the data (Duncan multiple
range
Oo -8
2
6
(I 10
4
8
Cd-conotoxin
(ng I C V)
Prevention of ACTH I-24-induced stretching, yawning and penile erection by ICV o-conotoxin. w-Conotoxin (0) or carboxymethylated w-conotoxin (0) were dissolved in saline and injected ICV in a volume of 5 pl 10 min before ACTH l-24 (10 p,g/rat ICV in 5 pl of saline). After treatment, rats were placed individually into Plexiglas cages and observed for 2 hours, during which stretching, yawning and penile erection episodes were counted. Values are means * S.E.M. of 3 experiments (12 rats per group). *p
