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Fetal yawning assessed by 3D and 4D sonography
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28 février 2013
Pharmacol Biochem Behav.
2013;103(4):750-755
Clavulanic acid induces penile erection and yawning in male rats: comparison with apomorphine.
Sanna F, Melis MR, Angioni L, Argiolas A.
 
Department of Biomedical Sciences, Neuroscience and Clinical Pharmacology Section, University of Cagliari, Italy   

Chat-logomini

Tous les travaux de MR Melis & A Argiolas 
Tous les travaux de M Eguibar & G Holmgren
 
 
Abstract
The beta-lactamase inhibitor clavulanic acid induced penile erection and yawning in a dose dependent manner when given intraperitoneally (IP, 0.05-5mg/kg), perorally (OS, 0.1-5mg/kg) and intracereboventricularly (ICV, 0.01-5?g/rat) to male rats. The effect resembles that of the dopamine receptor agonist apomorphine given subcutaneously (SC) (0.02-0.25mg/kg), although the responses of the latter followed a U inverted dose-response curve, disappearing at doses higher than 0.1mg/kg. Clavulanic acid responses were reduced by about 55% by haloperidol, a dopamine D2 receptor antagonist (0.1mg/kg IP), and by d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin, an oxytocin receptor antagonist (2?g/rat ICV), both given 15min before clavulanic acid. A higher reduction of clavulanic acid responses (more than 80%) was also found with morphine, an opioid receptor agonist (5mg/kg IP), and with mianserin, a serotonin 5HT(2c) receptor antagonist (0.2mg/kg SC). In contrast, no reduction was found with naloxone, an opioid receptor antagonist (1mg/kg IP). The ability of haloperidol, d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin and morphine to reduce clavulanic acid induced penile erection and yawning suggests that clavulanic acid induces these responses, at least in part, by increasing central dopaminergic neurotransmission.
 
Dopamine in turn activates oxytocinergic neurotransmission and centrally released oxytocin induces penile erection and yawning. However, since both penile erection and yawning episodes were reduced not only by the blockade of central dopamine and oxytocin receptors and by the stimulation of opioid receptors, which inhibits oxytocinergic neurotransmission, but also by mianserin, an increase of central serotonin neurotransmission is also likely to participate in these clavulanic acid responses.
 
 
1. Introduction
Clavulanic acid is a beta-lactamase inhibitor produced by Streptomyces clavuligenus, with a chemical structure similar to that of penicillin. The drug is used clinically and veterinary often in combination with penicillins, e.g. amoxicillin, in order to potentiate the antibacterial action of the latter. The compound crosses readily the blood brain barrier and has been recently reported to possess anxiolytic properties in non human primates (e.g., cotton-top tamarins, Saguinus oedipus) (Kim et al., 2009). Clavulanic acid was also found capable of exerting neuroprotective effects in animal models of Parkinson's disease (Huh et al., 2010; Kost et al., 2012). These central effects may be secondary at least in part to the ability of clavulanic acid to increase dopamine and serotonin release from dopamine and serotonin neurons (Kim et al., 2009). Accordingly, the drug was recently found to be able to increase dopamine release from two dopaminergic neuronal cell lines, PC12 cells (derived from adrenal gland pheochromocytoma) and SH-SY5Y cells (derived fromhuman neuroblastoma) in vitro by amechanism involving vesicle trafficking and fusion through the binding and regulation of Munc18-1 and Rab4 proteins (Kost et al., 2011).
 
In one of the above experiments with cotton-top tamarins, it was also found that the drug increased sexual arousal as indicated by the increased rate of penile erections (see Kim et al., 2009). This sexual effect was confirmed in male rats by experiments showing that chronic treatment with clavulanic acid given orally at doses ranging from 0.01 to 1.0 mg/kg for 7&endash;14 days decreases the latency to the first ejaculation and increases the number of ejaculations in a classic test of male rat copulatory activity (Chan et al., 2009). Interestingly, at central level dopamine is one of the most studied neurotransmitters involved in the control of penile erection and sexual behaviour. Accordingly, dopamine receptor agonists increase penile erection and facilitate copulatory behaviour in rodents (rats, mice and others) and also in other mammals including non human primates (seeMelis and Argiolas, 1995;McKenna, 2000; Andersson, 2001; Giuliano and Rampin, 2001; Hull et al., 2002). Dopamine receptor agonist-induced penile erection usually (but not always) occurs concomitantly with yawning (see Argiolas and Melis, 1998; Melis and Argiolas, 1995 and references therein). Both these effects are secondary to the stimulation of dopamine receptors of the D2 family, mainly D2 and D4 receptor subtypes, although a role for the D3 receptor subtype cannot be completely ruled out (see Brioni et al., 2004; Collins et al., 2007, 2009; Depoortère et al., 2009; Sanna et al., 2011, 2012a and references therein). Different brain areas, such as the medial preoptic area, the paraventricular nucleus of the hypothalamus, the ventral tegmental area, the nucleus accumbens and the hippocampus are involved in these responses (see Melis and Argiolas, 1995; Hull et al., 1995; Melis et al., 2003; Andersen et al., 2003; Andersen and Tufik, 2005; Succu et al., 2007; Richards et al., 2009).
 
A role of dopamine receptors recently identified in cavernous tissue on penile erection has been also suggested, although the available data are still very scarce (Senbel, 2011). Interestingly, a dopamine&endash;oxytocin interaction is thought to occur in a hypothetical complex neural circuit interconnecting many of the above brain areas, which controls not only the consummatory phase of sexual behaviour (e.g., penile erection and copulation), but also sexual motivation, sexual arousal and sexual reward (see Melis and Argiolas, 2011). Moreover, since yawning inmammalsmay be an unconscious signal that communicates one's own physiological/psychological state to othermembers of a social group, e.g., it may represent a primitive, unconscious form of empathy (see Palagi et al., 2009), this neural circuitmay be also involved in other central functions in which dopamine and oxytocin play a role, mainly social and emotional behaviours (Hurlemann et al., 2010; Lee et al., 2009; Neumann, 2008; Palagi et al., 2009; Sanna et al., 2012b). In order to provide direct support for a role of dopamine in the facilitatory effect of clavulanic acid on male sexual response and the other central functions on which this drug is supposed to exert an effect (i.e., anxiety, neuroprotection) (see above), we compared the effects of clavulanic acid on penile erection and yawning in male rats with those of apomorphine, a classic dopamine receptor agonist known for its ability to induce both these responses. The effects of drugs known to interfere with spontaneous or drug-induced penile erection and yawning are also reported 4. Discussion
 
The present results show that the potassium salt of clavulanic acid given IP induced penile erection and yawning in male rats. The drug was already active at doses as low as 0.1 mg/kg, with the maximal responses found at the dose of 2.0 mg/kg. The drug induced penile erection and yawning alsowhen given per OS or into the cerebral ventricles, suggesting that clavulanic acid induced these behavioural responses by acting in the central nervous system. The effect of clavulanic acid given IP resembles the effect of apomorphine, a mixed dopamine D1/D2 agonist that is extremely effective in inducing penile erection and yawning. However, while the efficacy of clavulanic acid in inducing penile erection was comparable to that of the dopamine receptor agonist, its efficacy in inducing yawning was much lower than that of apomorphine, especially when the drug was given per OS or ICV. Indeed, yawning tended to disappear when clavulanic acid was given by these routes of administration. The reason for the tendency of yawning to disappear is unknown, although it is likely that this is related to the route of administration. Indeed, it may be that the drug given IP reaches brain areas which are involved in yawning at a concentration that makes this response likely to occur, while this does not happen when the drug is given per OS or ICV. Another difference between clavulanic acid and apomorphine is that no U inverted dose response curve was found with clavulanic acid at the doses used in this study. This is at variance from apomorphine, for which it is well established that doses up to 0.08 mg/kg SC increase penile erection and yawning in a dose-dependent manner, and that these responses tend back to basal values with doses higher than 0.1 mg/kg SC, as these induce marked hyper-motility and stereotyped behaviour that masks yawning and penile erection, as found also in this study. Although it is possible that doses of clavulanic acid higher than 5 mg/kg may induce effects that mask the appearance of penile erection and yawning, this is unlikely, since this dose is already two&endash;three-fold the dose found to induce the maximal response in the present experiments, irrespective of the route of administration, and 5-fold the dose found capable of inducing penile erection in cotton top tamarins (Kimet al., 2009) and of facilitating male rat sexual behaviour (Chan et al., 2009).
 
The mechanism by which clavulanic acid induces penile erection and yawning by acting in the central nervous system is unknown at the moment. However, some explanation may be suggested by the results showing that clavulanic acid induced responses were markedly reduced by drugs which are known for their ability to reduce or abolish these responses when induced by apomorphine. First, clavulanic acid-induced penile erection and yawning were reduced by more than 50% by haloperidol, a classic non selective dopamine D2-like receptor antagonist, which is also well known for its ability to reduce these behavioural responses when induced by apomorphine and other dopamine receptor agonists (see Melis et al., 1987, 1996). Second, clavulanic acid responses were also reduced by more than 50% by d(CH2)5Tyr(Me)2-Orn8-vasotocin, an oxytocin receptor antagonist (Bankowski et al., 1980), given ICV at doses that reduced penile erection and yawning induced also by apomorphine, which induces these responses by increasing central oxytocinergic neurotransmission (see Melis and Argiolas, 2011; Sanna et al., 2012b).
 
However, it must be noted that the reduction of clavulanic acid responses by haloperidol and d(CH2)5Tyr(Me)2-Orn8-vasotocin (about 55%) is lower than that of apomorphine responses (about 85%). In spite of this difference, morphine, an opioid receptor agonist, but not naloxone, an opioid receptor antagonist, reduced almost completely penile erection and yawning induced by clavulanic acid when given at doses that reduced almost completely also apomorphine- and oxytocin-induced responses. These results may be explained by the ability of opioid receptor agonists to markedly decrease oxytocin neurotransmission at central and posterior pituitary level (see Melis et al., 1999 and references therein). All together, the above findings support the hypothesis that clavulanic acid induces penile erection and yawning, at least in part, by increasing dopamine neurotransmission in the central nervous system. Dopamine in turn increases central oxytocinergic neurotransmission, leading to penile erection and yawning, as already suggested for apomorphine and other dopamine receptor agonists (see Melis et al., 1996; Melis and Argiolas, 2011). This interpretation is in line with previous studies showing that clavulanic acid is able to increase extracellular dopamine concentration in the dialysate obtained fromthe nucleus accumbens ofmale rats by intracerebral microdialysis (Kim et al., 2009). As to the mechanism by which clavulanic acid facilitates central dopamine release, it is noteworthy that the compound was found ineffective in binding assays for 63 different signalling pathways and mediators that included the classical neurotransmitters, their receptors and transporters, ion channels, hormones, brain/gut peptides, prostaglandins, and enzymes (Kim et al., 2009).
 
Nonetheless, it is tempting to speculate that this mechanism may be similar to that suggested to occur in neuronal cell lines, e.g., PC12 cells (derived from adrenal pheochromocitoma), and SH-SY5Y cells (derived from neuroblastoma) exposed to clavulanic acid. In these cells, the drug was recently reported to be able to increase dopamine release apparently by a mechanism involving vesicle trafficking and fusion through the binding and regulation of Munc18-1 and Rab4 proteins (Kost et al., 2011), although other mechanisms cannot be ruled out. The above interpretation is complicated by two findings of this study. First, the reduction of clavulanic acid responses by haloperidol and d(CH2)5Tyr(Me)2-Orn8-vasotocin (about 55%) was lower than that of apomorphine responses (about 85%), as if clavulanic acid responses were not completely due to an increased dopaminergic neurotransmission. Second, clavulanic acid-induced penile erection and yawning were markedly reduced by mianserin, a putative 5-HT2c receptor antagonist (Berendsen and Gower, 1986; Berendsen et al., 1990; Harvey et al., 1999), at doses that reduced apomorphine-induced penile erection, but not yawning. Indeed, these results support a possible role of serotonin in clavulanic acid responses, in addition to that of dopamine. In particular this finding suggests that clavulanic acid increases not only the release of dopamine but also that of serotonin (see also Kim et al., 2009), which in turn acts on 5HT2c receptors to induce both penile erection and yawning. Accordingly, 5HT2c receptor agonists such as m-CPP [1-(3-chlorophenyl)-piperazine] and TFMPP [N-(3-trifluoromethylphenyl)-piperazine], were found capable of inducing penile erection and yawning episodes in male rats, which were indistinguishable from those induced by apomorphine and which were antagonized by mianserin given at doses that antagonized also penile erection, but not yawning, when induced by apomorphine and oxytocin (see Berendsen and Gower, 1986; Berendsen et al., 1990; Stancampiano et al., 1995).
 
These results led to suggest that 5HT2c receptors located downstream to dopamine and oxytocin are involved in the control of penile erection (Kimura et al., 2008; Stancampiano et al., 1995),while such a link between dopamine, oxytocin and serotonin does not occur in yawning (see Stancampiano et al., 1995). In view of these findings, it is likely that centrally released serotonin participates in clavulanic acid-induced penile erection and yawning, respectively, by acting either at 5HT2c receptors located in sites downstreamto dopamine and oxytocin for the former response, and by a mechanism not involving dopamine or oxytocin for the latter. Studies aimed at characterizing further the role of dopamine and serotonin in clavulanic acid-induced penile erection and yawning (for instance through selective monoamine depletion studies or with the direct microinjection of dopamine and/or serotonin receptor antagonists into brain areas relevant for penile erection and yawning) are necessary to clarify this point.
 
5. Conclusions
 
In conclusion, clavulanic acid given IP, ICV or per OS induces penile erection and yawning in male rats, apparently by activating central dopaminergic neurotransmission, which leads to an increase in central oxytocinergic neurotransmission (Argiolas and Melis, 1998; Melis and Argiolas, 2011). In line with this hypothesis, clavulanic acid-induced penile erection and yawning are impaired by dopamine receptor antagonists, oxytocin receptor antagonists and by other drugs such asmorphine, which reduces oxytocin release. However, since clavulanic acid induced penile erection and yawning are also reduced bymianserin, which interferes with these behavioural responses by blocking 5HT2c receptors (Kimura et al., 2008; Stancampiano et al., 1995), an increase of central serotonin neurotransmission is also likely to participate in these effects of clavulanic acid. Whatever mechanism is responsible for clavulanic acid-induced penile erection, the results of this study are in line with the recently reported facilitatory effect of this compound on erectile function in nonhuman primates (Kim et al., 2009) and on copulatory behaviour in male rats (Chan et al., 2009).