Neuroscience and Biobehavioral Reviews 35 (2011) 939–955

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Review Central control of penile erection: A re-visitation of the role of oxytocin and its interaction with dopamine and glutamic acid in male rats

Maria Rosaria Melis ∗, Antonio Argiolas

Bernard B. Brodie Department of Neuroscience and Centre of Excellence for the Neurobiology of Addictions, University of Cagliari, and Institute of Neuroscience, National Research Council, Cagliari Section, Cittadella Universitaria, 09042 Monserrato, CA, Italy article info abstract

Article history: Oxytocin is a potent inducer of penile erection when injected into the central nervous system. In male Received 17 June 2010 rats, the most sensitive brain area for the pro-erectile effect of oxytocin is the paraventricular nucleus of Received in revised form 8 October 2010 the hypothalamus. This nucleus and surrounding regions contain the cell bodies of all oxytocinergic neu- Accepted 26 October 2010 rons projecting to extra-hypothalamic brain areas and the spinal cord. This review shows that oxytocin induces penile erection also when injected in some of these areas (e.g., ventral tegmental area, ven- Keywords: tral subiculum of the hippocampus, posteromedial cortical nucleus of the amygdala and thoraco-lumbar Penile erection spinal cord). Microinjection studies combined with intra-cerebral microdialysis and double immuno- Oxytocin Dopamine fluorescence studies suggest that oxytocin in these areas activates directly or indirectly (mainly through Glutamic acid glutamic acid) mesolimbic dopaminergic neurons. Dopamine released in the nucleus accumbens in turn Paraventricular nucleus activates neural pathways leading to the activation of incerto-hypothalamic dopaminergic neurons in Ventral tegmental area the paraventricular nucleus. This activates not only oxytocinergic neurons projecting to the spinal cord Ventral subiculum of the hippocampus and mediating penile erection, but also those projecting to the above extra-hypothalamic areas, mod- Amygdala ulating directly or indirectly (through glutamic acid) the activity of mesolimbic dopaminergic neurons Spinal cord controlling motivation and reward. Together these neural pathways may constitute a complex hypothet- ical circuit, which plays a role not only in the consummatory phase of sexual activity (erectile function and copulation), but also in the motivational and rewarding aspects of the anticipatory phase of sexual behaviour. © 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 940 2. Oxytocin influences penile erection by acting in different brain areas ...... 940 2.1. The paraventricular nucleus of the hypothalamus ...... 940 2.2. The ventral tegmental area ...... 942 2.3. The hippocampus ...... 943 2.4. The amygdala ...... 944 2.5. The spinal cord...... 944 3. Interactions between oxytocin, dopamine and glutamic acid in the central nervous system and penile erection ...... 945 3.1. Dopamine–oxytocin interaction in the paraventricular nucleus ...... 945 3.2. Glutamic acid–oxytocin interaction in the paraventricular nucleus...... 947 3.3. Oxytocin–dopamine interaction in the ventral tegmental area ...... 948 3.4. Oxytocin–glutamic acid interaction in the ventral subiculum of the hippocampus ...... 949 4. Concluding remarks ...... 949 Acknowledgements ...... 953 References...... 953

∗ Corresponding author at: University of Cagliari, Bernard B. Brodie Department of Neuroscience, Cittadella Universitaria, S.P. Sestu-Monserrato, km 0.700, 09042 Monserrato, CA, Italy. Tel.: +39 070 6754317; fax: +39 070 6754320. E-mail address: [email protected] (M.R. Melis).

0149-7634/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neubiorev.2010.10.014 940 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955

1. Introduction the induction of penile erection by oxytocin is the paraventricu- lar nucleus of the hypothalamus (Melis et al., 1986), from which Penile erection is a male sexual response that plays a key all extra-hypothalamic oxytocinergic projections originate (see role in reproduction of mammals including man, and that can above). Here, oxytocin was found to be able to induce penile erec- be also observed in contexts different from those strictly related tion (and yawning) when injected at doses as low as 3 pmol (see to reproduction. Depending on the context in which penile erec- Section 2.1 below). Oxytocin induced penile erection also when tion occurs, different central and peripheral neural and/or humoral injected bilaterally into the CA1 field of the hippocampus, but not mechanisms participate in its regulation (see Meisel and Sachs, in the dorsal subiculum (see Section 2.3 below), the lateral septum, 1994; Argiolas and Melis, 1995, 2004, 2005; Sachs, 2000, 2007; the caudate nucleus, the medial preoptic area, the ventromedial McKenna, 2000; Giuliano and Rampin, 2000, 2004; Andersson, nucleus of the hypothalamus and the supraoptic nucleus (Melis 2001; Melis and Argiolas, 1995a, 2003; Hull et al., 2002). Among et al., 1986). As to the mechanism by which oxytocin acts in the central neurotransmitters and neuropeptides that control penile paraventricular nucleus to induce this sexual response, numerous erection, the best known are dopamine, serotonin, excitatory amino studies suggest that oxytocin activates its own neurons. In line with acids, nitric oxide, adrenocorticotropin, oxytocin and opioid pep- this hypothesis, sexual interaction increases FOS, the gene product tides. They can facilitate or inhibit penile erection by acting in of the immediate early gene c-fos in paraventricular oxytocinergic several brain areas, i.e., the medial preoptic area, the paraventric- neurons projecting to the spinal cord, which are involved in the ular nucleus of the hypothalamus, the ventral tegmental area, the control of penile erection (see Witt and Insel, 1994 and references hippocampus, the amygdala, the bed nucleus of the stria termi- therein), and sexual impotence (e.g., the inability of an adult male nalis, the nucleus accumbens, the medulla oblongata and the spinal rat to copulate with an ovariectomized oestrogen–progesterone- cord (Table 1) (see Meisel and Sachs, 1994; Witt and Insel, 1994; primed receptive female) has been associated in the male rat with Stancampiano et al., 1994; Argiolas and Melis, 1995, 2005; Argiolas, low levels of oxytocin mRNA in the paraventricular nucleus of the 1999; Bancila et al., 2002; Giuliano and Rampin, 2000, McKenna, hypothalamus (Arletti et al., 1997). 2000; Andersson, 2001; Hull et al., 2002; Coolen et al., 2004). Whether oxytocin influences the anticipatory phase or the con- Oxytocin, the neurohypophyseal peptide well known for its summatory phase of sexual behaviour is unclear at present. As hormonal role in lactation and parturition, is present in females oxytocin induces penile erection and the main effect of oxytocin on and males, not only in neurons with cell bodies located in the copulatory behaviour is a decrease in the post-ejaculatory interval paraventricular and supraoptic nuclei of the hypothalamus project- in male rats (Arletti et al., 1985), it is reasonable to assume that the ing to the neurohypophysis, but also in neurons projecting from peptide improves sexual performance. However, as oxytocin also the paraventricular nucleus and surrounding structures to extra- increases socio-sexual interaction (see Pedersen et al., 1992; Carter hypothalamic brain areas (i.e., the septum, the ventral tegmental et al., 1997; Ivell and Russel, 1995), and oxytocin receptor antago- area, the hippocampus, the amygdala, the medulla oblongata and nists prevent noncontact erections (Melis et al., 1999a), which are the spinal cord). These neurons are thought to be involved in considered as an index of sexual arousal (see Sachs, 1997, 2000, numerous central functions, such as memory, learning, affiliative 2007; Melis et al., 1998, 1999b and references therein), a possi- and socio-sexual behaviours, including penile erection and copu- ble role of oxytocin in sexual arousal and sexual motivation cannot latory behaviour (see Buijs, 1978; Sofroniew, 1983; Argiolas and be ruled out. This review summarizes published and unpublished Gessa, 1991; Pedersen et al., 1992; Carter, 1992; Wagner and results of recent studies, which show that oxytocin induces penile Clemens, 1993; Ivell and Russel, 1995; Carter et al., 1997; Tang erection not only when injected into the paraventricular nucleus et al., 1998; Veronneau-Longueville et al., 1999). Indeed, oxytocin of the hypothalamus, but also in other extra-hypothalamic brain facilitates erectile function and male sexual behaviour in mice, rats, areas, such as the ventral tegmental area (Melis et al., 2007, 2009a; rabbits and monkeys (see Argiolas and Gessa, 1991; Carter, 1992; Succu et al., 2008), the ventral subiculum of the hippocampus and Pedersen et al., 1992; Argiolas and Melis, 1995, 2004; Argiolas, the posterior nucleus of the amygdala (Melis et al., 2009b, 2010), 1999). This may occur also in humans, since plasma oxytocin is which are important constituents of the limbic system and are increased by sexual stimuli, especially at ejaculation (Carmichael thought to play a key role in motivation and reward processes. et al., 1987; Murphy et al., 1987) and by the manipulation of These studies reveal that oxytocin participates in neural circuits, breast and of the genitalia, which usually occur during sexual inter- which include other neurotransmitters, such as dopamine and glu- course (Tindall, 1974). A facilitative effect of oxytocin on male tamic acid, and other brain areas other than the paraventricular sexual behaviour was first demonstrated by the ability of intra- nucleus, e.g., the ventral tegmental area, the nucleus accumbens, venous oxytocin to decrease the latency to the first ejaculation the hippocampus and areas yet to be identified. These circuits are and to retard sexual exhaustion of male rabbits paired with recep- likely to mediate an interaction between the mesolimbic and the tive females (Melin and Kihlstrom, 1963). However, the sexual incerto-hypothalamic dopaminergic system, and to play a role not effects of oxytocin were definitively recognized only in the eight- only in the consummatory phase of male sexual behaviour (e.g., ies. Oxytocin given centrally in nanogram amounts was then found penile erection and copulation), but also in sexual motivation and able to induce penile erection (Argiolas et al., 1985, 1986) and to sexual arousal, hence providing a neural substrate for explaining improve copulatory behaviour (Arletti et al., 1985) in male rats, the motivational and rewarding properties of sexual activity. and to increase lordosis in female rats (Arletti and Bertolini, 1985; Caldwell et al., 1986), apparently by acting on uterine-type oxy- 2. Oxytocin influences penile erection by acting in different tocinergic receptors (see Argiolas and Melis, 1995, 2004; Argiolas, brain areas 1999; Melis and Argiolas, 2003; and references therein). Oxytocin improves sexual behaviour not only in sexually potent male rats 2.1. The paraventricular nucleus of the hypothalamus (Arletti et al., 1985) but also in aged male rats (Arletti et al., 1990), and in dominant, but not in subordinate, male squirrel monkeys As recalled above the paraventricular nucleus of the hypotha- (Winslow and Insel, 1991). The pro-erectile effect of oxytocin is lamus was soon identified as the brain area most sensitive for the testosterone-dependent, since it is abolished by hypophysectomy pro-erectile effect of oxytocin. When injected unilaterally in this and castration, and restored by supplementation with testosterone nucleus, oxytocin was found active at doses as low as 3 ng (3 pmol) or its metabolites, estradiol and 5␣-dihydro-testosterone given (Melis et al., 1986). Structure–activity relationship studies revealed together (Melis et al., 1994a). The most sensitive brain area for M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 941

Table 1 Neurotransmitters and neuropeptides that influence penile erection in the central nervous system: interaction with oxytocin.

Compound Effect on erection Effect on penile reflexes Brain areas Main mechanisms involved and of action identified identified so far so far

Dopamine Facilitatory Facilitatory PVN, MPOA, NAs Activation of PVN oxytocinergic neurons Serotonin Inhibitory, facilitatorya Inhibitory SpC, MPOA Inhibition of oxytocinergic neurotransmission in the SpC Glutamic acid Facilitatory NA PVN, VTA, VS Activation of PVN oxytocinergic neurons; activation of mesolimbic dopaminergic neurons in the VTA and of glutamatergic neurons in the VS GABA Inhibitory Inhibitory PVN, SpC Inhibition of PVN oxytocinergic neurons Nitric oxide Facilitatory NA PVN, MPOA, VTA, Activation of PVN VS oxytocinergic neurons; activation of mesolimbic dopaminergic neurons in the VTA; activation of glutamatergic neurons in the VS Acetylcholine Facilitatory NA Hippocampus NA Noradrenaline Inhibitory NA Hypothalamus NA Oxytocin Facilitatory NA PVN, SpC, VTA, Activation of PVN hippocampus, oxytocinergic amygdala neurons; activation of mesolimbic dopaminergic neurons in the VTA; activation of glutamatergic neurons in the VS, activation of pro-erectile neurons in the thoraco-lumbar SpC ACTH-MSH peptides Facilitatory NA APVH NA (but see Argiolas and Melis (2005) Opioid peptides Inhibitory NA PVN, MPOA Inhibition of PVN oxytocinergic neurons Hexarelin peptides Facilitatory NA PVN Activation of PVN oxytocinergic neurons Pro-VGF peptides Facilitatory NA PVN Activation of PVN oxytocinergic neurons Endocannabinoids Inhibitory NA PVN Inhibition of PVN oxytocinergic neurons secondary to the inhibition of glutamic acid neurotransmission

Further information on the mechanism of action of the above compounds can be found in the cited References (see Argiolas and Melis, 2005) and in Fig. 1. Abbreviations: APVH: periventricular hypothalamic area; MPOA: medial preoptic area; NAs: nucleus accumbens; PVN: paraventricular nucleus of the hypothalamus; SpC: spinal cord; VS: ventral subiculum of the hippocampus; VTA: ventral tegmental area; NA: not available. a Depending on the receptor subtype involved. that oxytocin-induced penile erection was mediated by uterine- mechanism (not involving guanylate cyclase) leads to the activa- type oxytocin receptors, coupled to a Ca2+ influx into the cell bodies tion of oxytocinergic neurons projecting to the spinal cord and of oxytocinergic neurons projecting to extra-hypothalamic brain extra-hypothalamic brain areas, inducing penile erection (Fig. 1) areas and to the activation of nitric oxide-synthase. Nitric oxide in (see below and Argiolas and Melis, 1995, 2004, 2005 and refer- turn by acting as an intracellular messenger with a yet unknown ences therein). The ability of oxytocin to activate its own neurons 942 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955

Fig. 1. Schematic representation of oxytocinergic neurons, which originate in the paraventricular nucleus of the hypothalamus and project to extra-hypothalamic brain areas, such as the spinal cord, the VTA, the hippocampus, the amygdala, etc. The activation of these neurons by dopamine, excitatory amino acids, oxytocin itself, hexarelin analogue peptides and VGF-derived peptides leads to penile erection, which can be reduced and/or abolished by the stimulation of GABAergic, opioid and cannabinoid CB1 receptors. The activation of oxytocinergic neurons is secondary to the activation of nitric oxide-synthase present in these neurons. Indeed endogenous nitric oxide formed by the stimulation of dopamine, excitatory amino acid or oxytocin receptors or exogenous nitric oxide, as that derived from nitric oxide donors given directly into the paraventricular nucleus, activates oxytocinergic neurons by a yet unidentified mechanism, apparently not related to the stimulation of guanylate cyclase. This causes in turn the release of oxytocin in the spinal cord and in extra-hypothalamic brain areas. Some details on the mechanisms by which oxytocin induces penile erection when released in these areas, e.g., the VTA, the ventral subiculum and the amygdala are described in the respective brain area sections. Here, oxytocin acts on its own receptors and increases NO production, which leads to penile erection as found in the PVN. However, at variance with the PVN, in the caudal VTA NO activates guanylate cyclase. This causes an increase in cGMP concentration leading to the activation of mesolimbic dopaminergic neurons and to penile erection. In the VS NO activates glutamatergic neurons projecting to extra-hippocampal areas, including the VTA. Glutamic acid in the VTA activates in turn mesolimbic dopaminergic neurons as found with oxytocin. Mechanisms similar to those described above are likely to operate also when penile erection occurs in physiological contexts, namely as when male rats are placed in the presence of an inaccessible receptive female (e.g., noncontact erections) or during copulation. in the paraventricular nucleus was supported by studies show- 1995; Arletti et al., 1997) (for an extensive review of these studies ing that: (1) oxytocin receptors are present in this hypothalamic see Argiolas, 1999; Argiolas and Melis, 2004, 2005). nucleus (Freund-Mercier et al., 1987; Freund-Mercier and Stoeckel, 1995); (2) oxytocin facilitates its own release in vitro and in vivo 2.2. The ventral tegmental area (Freund-Mercier and Richard, 1981, 1984; Moos et al., 1984); and (3) oxytocin excites its own neurons by acting in the paraven- The ventral tegmental area was discovered only recently as a tricular nucleus (Yamashita et al., 1987). Moreover, oxytocinergic brain site in which oxytocin induces penile erection. This area con- synapses impinging on the cell bodies of magnocellular oxytocin- tains oxytocinergic nerve endings originating in the paraventricular ergic neurons have also been identified in the paraventricular and nucleus and oxytocin receptors (Freund-Mercier et al., 1987; supraoptic nucleus of the hypothalamus (Theodosis, 1985). Finally, Vaccari et al., 1998). More precisely, oxytocin was found capable of destruction of central oxytocinergic neurons by electrolytic or inducing penile erection when injected unilaterally into the caudal, chemical excitotoxic lesions of the paraventricular nucleus, which but not in the rostral ventral tegmental area in a dose-dependent completely depletes oxytocin content across the central nervous manner (Melis et al., 2007). The active doses were higher than those system and the spinal cord, abolishes not only the pro-erectile required when injected into the paraventricular nucleus and simi- effect of oxytocin, but also impairs drug-induced penile erection lar to those inducing penile erection when injected into the ventral and noncontact erections (see below and Argiolas et al., 1987a,b; subiculum of the hippocampus or into the posteromedial cortical Liu et al., 1997 and references therein). Results similar to those nucleus of the amygdala (see below). Apparently, the pro-erectile found with lesions of the paraventricular nucleus are found with effect is mediated by the activation of mesolimbic dopaminer- potent and selective oxytocin receptor antagonists. Indeed, these gic neurons projecting to the shell of the nucleus accumbens, compounds injected into the paraventricular nucleus in nanogram which in turn activates yet unknown neural pathways projecting amounts prevented completely oxytocin-induced penile erection, to the incerto-hypothalamic dopaminergic neurons impinging on while when given into the lateral ventricles prevented not only paraventricular oxytocinergic neurons mediating penile erection penile erections induced by oxytocin itself, but also drug-induced (Melis et al., 2007, 2009a). As to the mechanism by which oxytocin penile erection (see the Section 3 below and Argiolas and Melis, activates dopaminergic neurotransmission in the ventral tegmental 1995, 2004, 2005 and references therein) and noncontact erec- area, the available data suggest that oxytocin stimulates oxytocin- tions (Melis et al., 1999a), and were moreover extremely effective ergic receptors located in the cell bodies of mesolimbic dopamin- 2+ in impairing copulatory behaviour of sexually potent male rats ergic neurons. This increases Ca influx inside the cell bodies of (Argiolas et al., 1988). Furthermore, sexual interaction increases dopaminergic neurons, thereby activating nitric oxide-synthase FOS, the gene product of the immediate early gene c-fos in par- (Succu et al., 2008). At variance with the paraventricular nucleus aventricular oxytocinergic neurons projecting to the spinal cord (see the Section 3 below), nitric oxide in turn activates guanylate involved in the control of penile erection (see Witt and Insel, 1994 cyclase, hence increasing the concentration of cyclic GMP. In line 2 8 and references therein). Finally, sexual impotence (e.g., the inability with this mechanism, either d(CH2)5Tyr(Me) -Orn -vasotocin, a l of an adult male rat to copulate with an oestrogen–progesterone- potent oxytocin antagonist, or S-methyl-thio- -citrulline, a potent primed receptive female) has also been associated in the male rat inhibitor of neuronal nitric oxide-synthase, injected into the caudal with low levels of oxytocin mRNA and of nitric-oxide synthase in ventral tegmental area before oxytocin, abolished penile erection the paraventricular nucleus of the hypothalamus (Benelli et al., and the increase in extra-cellular dopamine concentration in the shell of the nucleus accumbens induced by oxytocin. Moreover, M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 943

Fig. 2. Effect of 8-bromo-cyclic GMP injected into the caudal ventral tegmental area (A) and into the paraventricular nucleus of the hypothalamus (B) on penile erection and on extra-cellular dopamine concentration in the nucleus accumbens dialysate. 8-bromo-cyclic GMP (3 ␮g) dissolved in saline or saline alone was injected into the caudal ventral tegmental area or into the paraventricular nucleus of male rats implanted with intra-cerebral microdialysis cannulas aimed at the shell of the nucleus accumbens and chronic guide cannulas for microinjections into the caudal ventral tegmental area or into the paraventricular nucleus. Intra-cerebral microdialysis conditions and other experimental conditions were identical to those reported in Melis et al. (2004b, 2007, 2009a). Values are means ± SEM of 9 rats per group. *P < 0.001 with respect to saline-treated rats values (Mann–Whitney U test).

8-bromo-cyclic GMP, an active phosphodiesterase-resistant cyclic Bujis, 1978; Sofroniew, 1983). However, at variance with the par- GMP analogue, induces penile erection when injected into the cau- aventricular nucleus, here oxytocin was found capable of inducing dal ventral tegmental area and increases extra-cellular dopamine penile erection only when injected bilaterally and at higher doses concentration in the shell of the nucleus accumbens, as found with than those found active in the paraventricular nucleus (Melis et al., oxytocin injected into the caudal ventral tegmental area (Succu 1986; Chen et al., 1992). Injections of oxytocin into the subiculum et al., 2008; Melis et al., 2009a) (see also Fig. 2). Always in line were found inactive in these earlier studies. However, recent and with this mechanism, haloperidol, a potent dopamine D2 recep- more careful microinjection studies lead to the identification of tor antagonist, injected into the shell of the nucleus accumbens a region of the ventral subiculum in which the injection of oxy- reduces penile erection induced by oxytocin injected into the ven- tocin was capable of inducing penile erection in a dose-dependent tral tegmental area (Melis et al., 2007). The above mechanism is manner (Melis et al., 2009b). The pro-erectile effect of oxytocin also supported by double immuno-fluorescence studies, showing injected into this brain area was observed at doses similar to those that oxytocin fibres impinge on the cell bodies of dopaminergic found active in the ventral tegmental area after unilateral injec- neurons in the caudal ventral tegmental area, which were previ- tion (Melis et al., 2007), as found in the paraventricular nucleus. ously labeled with the retrograde tracer Fluorogold injected into Apparently, oxytocin injected into the ventral subiculum induces the shell of the nucleus accumbens (Melis et al., 2007; Succu penile erection by activating oxytocinergic receptors in neurons et al., 2008). The activations of these dopaminergic neurons and of containing nitric oxide-synthase, causing an increase in nitric oxide dopamine receptors in the nucleus accumbens lead in turn to the production. Nitric oxide in turn by acting as intercellular messenger activation of neural pathways yet to be identified, which stimulate activates glutamic acid neurotransmission, leading to penile erec- incerto-hypothalamic dopaminergic neurons to release dopamine tion, possibly through neural (glutamatergic) efferent projections in the paraventricular nucleus, thereby activating oxytocinergic from the ventral subiculum to extra-hippocampal brain areas mod- neurons projecting to the spinal cord and mediating penile erec- ulating the activity of mesolimbic dopaminergic neurons (e.g., the tion (see above and Melis et al., 2007; Succu et al., 2007, 2008). ventral tegmental area, the prefrontal cortex, the paraventricular Indeed, oxytocin injected into the caudal ventral tegmental area nucleus) (see below and Melis, 2007, 2009b; Succu et al., 2008). at a dose that induced penile erection, increased extra-cellular This mechanism of action is supported by intra-cerebral dopamine concentration in the dialysate obtained not only from microdialysis experiments, which show that oxytocin the nucleus accumbens, but also from the paraventricular nucleus injected into the ventral subiculum at doses that induce (Succu et al., 2007). penile erection, increases nitric oxide production and extra- cellular glutamic acid concentration in the dialysate from the ventral subiculum (Melis et al., 2010) and of extra- 2.3. The hippocampus cellular dopamine in the nucleus accumbens (Melis et al., 2007). These responses were antagonized not only by the oxytocin The CA1 field of the hippocampus was the other brain area rich receptor antagonist d(CH ) Tyr(Me)2-Orn8-vasotocin, but also in oxytocinergic fibres and receptors identified by the earlier stud- 2 5 by the neuronal nitric oxide-synthase inhibitor S-methyl-thio-l- ies in which the injection of oxytocin induced penile erection (see 944 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 citrulline and by the nitric oxide scavenger haemoglobin given nucleus accumbens, as found for penile erection induced by oxy- into the ventral subiculum few minutes before oxytocin (Melis tocin injected into the ventral subiculum (Melis et al., 2009b). This et al., 2010). Moreover, in line with this mechanism of action, suggests that oxytocin injected into the posteromedial nucleus of activation of glutamatergic neurotransmission by NMDA injected the amygdala activates glutamic acid neurotransmission in the ven- into the ventral subiculum induces penile erection (Melis et al., tral tegmental area. This causes in turn the activation of mesolimbic 2010). The phenotype of efferent projections from the ventral dopaminergic neurons, leading to penile erection. In view of stud- subiculum, which cause the activation of mesolimbic dopamin- ies showing neural pathways interconnecting this nucleus of the ergic neurons and the increase of extra-cellular dopamine in the amygdala with the ventral subiculum (Canteras et al., 1995; French nucleus accumbens, is unknown at present. However, since penile and Totterdell, 2003), these findings raise the possibility that an erection induced by oxytocin injected into the ventral subiculum interaction may exist between these two brain areas, although occurs concomitantly to an increase of extra-cellular glutamic acid direct pathways from the amygdala either to the nucleus accum- in the dialysate from the ventral tegmental area, but not from the bens or to the ventral tegmental area have been described (Kelley nucleus accumbens and is antagonized by (+)MK-801, a potent and Domesick, 1982; Witter, 2006). non-competitive antagonist of excitatory amino acid receptors of the NMDA subtype (Woodruff et al., 1987), injected into the 2.5. The spinal cord ventral tegmental area, but not in the nucleus accumbens (see Fig. 2 and Melis et al., 2009b), it is likely that these projections The spinal cord is another area of the central nervous system lead to the activation of glutamatergic neurotransmission in that contains oxytocinergic fibres and receptors (Freund-Mercier the ventral tegmental area, which in turn activates mesolimbic et al., 1987; Uhl-Bronner et al., 2005), in which oxytocin induces dopaminergic neurons projecting to the nucleus accumbens. penile erection (Tang et al., 1998; Veronneau-Longueville et al., Whether the increased concentration of glutamic acid found in 1999; Giuliano and Rampin, 2000; Giuliano et al., 2001). As recalled the ventral tegmental area after oxytocin injection into the ventral above, these oxytocinergic fibres originate in the paraventricu- subiculum is released from neurons originating in the subiculum lar nucleus of the hypothalamus and contribute to descending or in other brain areas (e.g., the prefrontal cortex) is unknown pathways controlling spinal autonomic neurons mediating penile at present. Nonetheless, this causes the activation of mesolimbic erection. Indeed these fibres make synaptic contacts in the dorsal dopaminergic neurons and an increased release of dopamine in horn preganglionic sympathetic and parasympathetic cell columns the nucleus accumbens. Here the activation of dopamine receptors in the thoraco-lumbar and lumbo-sacral tract with spinal neu- leads to the activation of incerto-hypothalamic dopaminergic neu- rons innervating penile cavernous corpora (Marson and McKenna, rons, releasing dopamine in the paraventricular nucleus, thereby 1996; Giuliano and Rampin, 2000; Giuliano et al., 2001). These activating oxytocinergic neurons projecting to the spinal cord and synaptic contacts were demonstrated by labeling of spinal neurons mediating penile erection (see above and Melis et al., 2007, 2009a; originating in the penis and reaching the spinal cord with spe- Succu et al., 2008). cific retrograde tracers injected into cavernous corpora, combined with double immuno-fluorescence and confocal laser microscopy 2.4. The amygdala studies (Tang et al., 1998; Veronneau-Longueville et al., 1999). In line with these studies, in anaesthetized male rats intrathecal The amygdala is another brain area rich in oxytocin fibres injection of cumulative doses of oxytocin at the lumbo-sacral, but and receptors (see Freund-Mercier et al., 1987; Vaccari et al., not at the thoraco-lumbar level, elicited intracavernous pressure 1998; Uhl-Bronner et al., 2005). Oxytocin here is thought to be rises in a dose dependent manner. These effects were abolished 2 involved in different functions, from anxiolysis, social memory by the blockade of oxytocinergic receptor with d(CH2)5Tyr(Me) - and cognition, socially reinforced learning, emotional empathy, Orn8-vasotocin and by section of the pelvic nerves (Giuliano and emotional face processing and fear in humans to erectile func- Rampin, 2000; Giuliano et al., 2001). These results demonstrate tion and sexual behaviour (see Kondo et al., 1998; Dominguez that oxytocin, acting at the lumbo-sacral spinal cord, increases et al., 2001; Ebner et al., 2005; Huber et al., 2005; Domes et al., intracavernous pressure, and suggest that oxytocin, released dur- 2007; Petrovic et al., 2008; Lee et al., 2009; Donaldson and Young, ing physiological activation of the paraventricular nucleus is a 2009; Hurlemann et al., 2010). However, the ability of oxytocin potent activator of spinal pro-erectile neurons projecting to the to induce penile erection in male rats when induced in the pos- cavernous corpora. Interestingly, these pro-erectile spinal neu- teromedial cortical nucleus of the amygdala was discovered only rons on which oxytocin acts to exert its pro-erectile effect, also recently (Melis et al., 2009b). This response occurred concomi- receive synaptic contacts from serotoninergic neurons originat- tantly with an increase in extra-cellular dopamine concentration ing in the nucleus paragigantocellularis of the reticular formation in the dialysate obtained from the shell of the nucleus accum- of the medulla oblongata (Marson and McKenna, 1992; Tang bens, as found after oxytocin injection into the ventral subiculum et al., 1998). Destruction of these serotoninergic neurons facil- (Melis et al., 2009b). The mechanism by which oxytocin injected itates ejaculation and penile reflexes in male rats (Marson and into the posteromedial cortical nucleus of the amygdala induces McKenna, 1992; Yells et al., 1992). Since drugs that stimulate 5HT2C penile erection is unknown at the moment. The available data receptors facilitate penile erection when given intracerebroven- show that both penile erection and the increase in extra-cellular tricularly, but not into the paraventricular nucleus, and drugs dopamine concentration in the dialysate obtained from the nucleus that that block 5HT2C receptors reduce also dopamine agonist- accumbens are mediated by the activation of oxytocinergic recep- and oxytocin-induced penile erection, while dopamine antago- tors, as both responses were abolished by the oxytocin receptor nists do not reduce 5HT2C agonist-induced penile erection (see 2 8 antagonist d(CH2)5Tyr(Me) -Orn -vasotocin injected in the amyg- Stancampiano et al., 1994 and references therein), it has been dala nucleus few minutes before oxytocin (Melis et al., 2009b). also suggested that oxytocin facilitates the action of pro-erectile Whatever mechanism oxytocin activates in the posteromedial cor- 5HT2C receptors at the level of the lumbo-sacral spinal cord tical nucleus of the amygdala, the sexual response induced by the (Stancampiano et al., 1994). Alternatively, oxytocin might influ- peptide is abolished by the blockade of all dopaminergic recep- ence the activity of spinal descending serotoninergic neurons by tors with cis-flupenthixol injected into the shell of the nucleus acting directly in the nucleus paragigantocellularis, where these accumbens and by the blockade of NMDA receptors with (+)MK- neurons originate (see Stancampiano et al., 1994). In line with this 801 injected into the ventral tegmental area, but not into the possibility, an oxytocinergic pathway originating in the paraven- M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 945 tricular nucleus and reaching the nucleus paragigantocellularis of 3.1. Dopamine–oxytocin interaction in the paraventricular the reticular formation in the medulla oblongata has been described nucleus (Bancila et al., 2002), and the activation of 5HT2C receptors located in the spinal cord down-stream to dopamine and oxytocin recep- The ability of dopamine agonists to induce penile erection by tors is considered a common mechanism of the pro-erectile effect activating central oxytocinergic neurons was suggested immedi- of these compounds and even of ACTH-MSH peptides (Kimura et al., ately after the discovery that apomorphine induces penile erection 2008). when injected into the paraventricular nucleus (Melis et al., 1987) 2 8 when the oxytocin receptor antagonist d(CH2)5Tyr(Me) -Orn - vasotocin injected intracerebroventricularly (i.c.v.) was found 3. Interactions between oxytocin, dopamine and glutamic able to reduce almost completely penile erection induced not acid in the central nervous system and penile erection only by oxytocin given i.c.v., but also by apomorphine, given subcutaneously (Argiolas et al., 1987b). These results were fol- As recalled in Section 1, all oxytocinergic neurons present lowed by those of other studies showing similar results when 2 8 in the central nervous system originate in the paraventricular d(CH2)5Tyr(Me) -Orn -vasotocin was given i.c.v. and apomor- nucleus and surrounding structures. The activity of these neu- phine was given directly into the paraventricular nucleus (Melis rons is under the control of different neurotransmitters and/or et al., 1989b), leading to suggest that dopamine agonists induce neuropeptides. Among the most studied at the paraventricu- penile erection by activating paraventricular oxytocinergic neu- lar level are dopamine, glutamic acid, gamma-aminobutyric acid rons projecting to extra-hypothalamic brain areas and in particular (GABA), nitric oxide, endocannabinoids, opioid peptides, growth to the spinal cord (see Argiolas and Melis, 1995, 2004, 2005). hormone-releasing peptides, VGF-related peptides and oxytocin In line with this hypothesis, in anaesthetized rats, blockade of itself. Dopamine, glutamic acid, growth hormone releasing pep- lumbo-sacral oxytocinergic receptors by a non-peptide oxytocin tides, VGF-derived peptides and oxytocin are stimulatory, e.g., receptor antagonist has been recently found capable of abolish- these compounds and their agonists facilitate penile erection when ing apomorphine-induced rises in intracavernous pressure induced injected into the paraventricular nucleus, while GABA, opioid pep- by the dopamine agonist apomorphine, providing evidence for a tides and endocannabinoids are inhibitory, e.g., these compounds paraventriculo-spinal oxytocinergic pathway involved in penile or their agonists inhibit penile erection (see Meisel and Sachs, 1994; erection (Baskerville et al., 2009). Witt and Insel, 1994; Argiolas and Melis, 1995, 2004, 2005; Giuliano Studies aimed at the identification of the dopamine recep- and Rampin, 2000, 2004; McKenna, 2000; Andersson, 2001; Hull tor responsible for the induction of penile erection, revealed that et al., 2002). Several lines of experimental evidence suggest that also in the paraventricular nucleus dopamine receptor agonists these oxytocinergic neurons and the above neurotransmitters and induce penile erection by acting on dopamine receptors of the neuropeptides are involved in the control of erectile function and D2 family, as found with dopamine receptor agonists given sys- sexual behaviour in different physiological contexts. Moreover, temically (see Melis et al., 1987; Eaton et al., 1991; Melis and oxytocin released in extra-hypothalamic brain areas, such as the Argiolas, 1995a). Accordingly, apomorphine, a potent mixed D1/D2 ventral tegmental area, the hippocampus and its regions, the amyg- receptor agonist, and quinpirole, a potent selective D2 receptor ago- dala and the spinal cord may influence the activity of those neurons nist, but not SKF 38393, a selective D1 receptor agonist, injected on which oxytocinergic synapses impinge. At the moment, the into this hypothalamic nucleus were found able to induce penile only neurons important for penile erection on which oxytocinergic erection in a dose-dependent manner, and the sexual response synapses impinge, identified with certainty, are the cell bodies of induced by these D2 receptor agonists was abolished by D2 recep- mesolimbic dopaminergic neurons of the caudal ventral tegmental tor antagonists, such as haloperidol and l-sulpiride, but not by SCH area projecting to the shell of the nucleus accumbens (Melis et al., 23390, a selective D1 receptor antagonist (Melis et al., 1987). The 2007; Succu et al., 2008), and the pro-erectile spinal neurons pro- ability of apomorphine to induce penile erection when injected jecting from the lumbo-sacral tract to the cavernous corpora (see into the paraventricular nucleus was also confirmed by telemetry Giuliano and Rampin, 2000; Giuliano et al., 2001) (see also Sections studies showing that the dopamine agonist given into the par- 2.2 and 2.5). Indeed, although oxytocinergic synapses and receptors aventricular nucleus is able to increase intracavernous pressure have been also identified in the ventral subiculum, the amygdala in awake male rats without modifying systemic blood pressure and the spinal cord, areas which are all important for penile erection (Chen et al., 1999; Giuliano and Allard, 2001), as found after (see above), in these areas the type of neurotransmitter/s present systemic injection (Bernabè et al., 1999). These studies also con- in the neurons on which oxytocinergic nerve endings impinge, are firmed a main role of D2 receptors, as D1 receptor agonists still unknown. This section of the review summarizes briefly the were usually found to be unable to increase intracavernous pres- recent literature on the mechanisms underlying the pro-erectile sure when injected into the paraventricular nucleus (Chen et al., effect of oxytocin injected into the caudal ventral tegmental area, 1999). the ventral subiculum of the hippocampus and in the spinal cord. Several lines of experimental evidence were then available Particular attention is given to the interaction of the peptide with suggesting that paraventricular D2 receptors, whose stimulation dopamine and glutamic acid in these areas and on the role this induces penile erection, are located on the cell bodies of oxy- interaction may play in the central control of erectile function. A tocinergic neurons. First, the paraventricular nucleus contains brief summary of the effects of dopamine and glutamic acid on dopaminergic nerve terminals that belong to the so called incerto- oxytocinergic neurons in the paraventricular nucleus, which also hypothalamic dopaminergic neurons. The cell bodies of these play a key role in erectile function is provided first, in order to neurons are situated in the A13 and A14 group of Dahlstrom and make the reader aware of the early state of research in this field, as Fuxe (1964), arborize extensively and innervate several hypotha- these studies have been already reviewed extensively (see Argiolas lamic structures, including paraventricular oxytocinergic neurons and Melis, 1995, 2004, 2005; Melis and Argiolas, 2003). Also in projecting to the neurohypophysis and/or to extra-hypothalamic this case, particular attention is given to the most recent results, brain areas (Buijs et al., 1984; Lindvall et al., 1984). The involve- which suggest an important role for both a dopamine–oxytocin ment of these dopaminergic neurons at the paraventricular level link and a glutamic acid–oxytocin link not only in sexual perfor- in the control of penile erection and copulation is supported by mance (penile erection and copulation), but also in sexual arousal microdialysis studies showing that the concentrations of extra- and sexual motivation. cellular dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), 946 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 its main metabolite, are increased in the dialysate obtained from ular nucleus (Fig. 2) (see Melis and Argiolas, 1995b and references the paraventricular nucleus of sexually potent male rats showing therein). noncontact erections when put in the presence of an inacces- The above interpretation has been often considered not con- sible ovariectomized oestrogen + progesterone-primed receptive vincing, mainly because the stimulation of dopamine D2 receptors female (Melis et al., 2003). The increase of the dopamine and is usually coupled to inhibition rather than excitation of the cell DOPAC concentrations was even higher when copulation with the bodies of the neurons containing these receptors through differ- receptive female was allowed (Melis et al., 2003), as found in ent G protein coupled mechanisms (see Sokoloff and Schwartz, the medial preoptic area (Hull et al., 1995) and in the nucleus 1995). However, a possible explanation for this discrepancy, accumbens (Pfaus and Everitt, 1995). Second, several studies which is in line with a direct stimulation of paraventricular oxy- show that penile erection induced by the stimulation of par- tocinergic neurons by dopamine, has been suggested recently aventricular D2 receptors, is mediated by oxytocin released in by the discovery of a G protein-coupled dopamine D4 recep- these areas. Accordingly, apomorphine given at doses that induce tor, a member of the D2 receptor family (D2, D3 and D4), penile erection, was found able to increase oxytocin concen- the stimulation of which increases Ca2+ influx in cell prepa- tration, not only in plasma of rats and monkeys (Melis et al., rations containing a cloned version of this receptor subtype 1989a; Cameron et al., 1992), but also in extra-hypothalamic (Moreland et al., 2004). More importantly, a selective D4 receptor brain areas, such as the hippocampus (Melis et al., 1990). In agonist (e.g., ABT 724) (N-methyl-4-(2-cyanophenyl)piperazynil- line with these results, apomorphine injected into the paraven- 3methylbenzamide maleate) was found capable of inducing penile tricular nucleus at a dose that induces penile erection was erection in male rats when given systemically (Brioni et al., recently shown to be able to increase also extra-cellular dopamine 2004). This effect was not found with the selective D2 receptor concentration in the nucleus accumbens, an effect reduced by subtype agonist PNU-95666E (R-5,6-dihydro-N,N-dimethyl-4H- 2 8 the oxytocin receptor antagonist d(CH2)5Tyr(Me) -Orn -vasotocin imidazo[4,5,1-i]quinolin-5-amine) (Hsieh et al., 2004), which was injected into the ventral tegmental area (Succu et al., 2007; also unable to increase Ca2+ influx in the cell preparations contain- Melis et al., 2009a) (see also Section 4). Third, bilateral elec- ing the cloned version of the D4 receptor subtype (Brioni et al., trolytic lesions of the paraventricular nucleus, which almost 2004; Moreland et al., 2004). In line with the above hypothesis and completely eliminate oxytocin from extra-hypothalamic brain findings, PD 168,077 (N-methyl-4-(2-cyanophenyl)piperazynil- areas (Hawthorn et al., 1985), abolish apomorphine-induced penile 3methylbenzamide maleate), PIP-3EA (2-[4-(2-methoxyphenyl) erection (Argiolas et al., 1987a), and selective oxytocin recep- piperazin-1-ylmethyl]imidazo[1,2-a]pyridine) and other selective tor antagonists given into the lateral ventricles, but not into the D4 receptor agonists (Heier et al., 1997; Melis et al., 2006b; Löber paraventricular nucleus, reduce dose-dependently apomorphine- et al., 2009), were found able to induce penile erection when induced penile erection with a potency parallel to that of these injected systemically, i.c.v. and into the paraventricular nucleus, compounds in blocking oxytocin receptors (Melis et al., 1989b). although less effectively than apomorphine. The pro-erectile effect Oxytocin receptor antagonists are also extremely potent in reduc- of these D4 receptor agonists was prevented by L-745,870 (3-(4- ing the facilitation of male sexual behaviour induced not only [chlorophenyl] piperazin-1-yl)-methyl-1H-pyrrolo[2,3-B]pyridine by oxytocin, but also by apomorphine (Argiolas et al., 1988, trihydrochloride), a selective D4 receptor antagonist (Patel et al., 1989). 1997; Melis et al., 2005, 2006b; Löber et al., 2009). Finally, the As to the mechanism by which D2 receptors activated by pro-erectile effect of the above D4 receptor agonists was also dopamine or by dopamine receptor agonists, increase the activity reduced by nitric oxide-synthase inhibitors, given into the par- 2 8 of oxytocinergic neurons, thereby releasing oxytocin in extra- aventricular nucleus, and by d(CH2)5Tyr(Me) -Orn -vasotocin, a hypothalamic brain areas and in the spinal cord, numerous selective oxytocin receptor antagonist given i.c.v. but not in the experimental data support the hypothesis that the stimulation paraventricular nucleus. These results are in line with the hypoth- of D2 receptors increases the concentration of intracellular Ca2+ esis that D4 receptor agonists also stimulate oxytocinergic neurons ions inside the cell bodies of oxytocinergic neurons, leading to the by activating nitric oxide-synthase, and releasing oxytocin in extra- activation of nitric oxide-synthase, a Ca2+-calmodulin-dependent hypothalamic brain areas, which in turn facilitates penile erection, enzyme, which is present in these cell bodies (Vincent and Kimura, as shown for apomorphine and classical D2 agonists (Melis et al., 1992; Torres et al., 1993; Sanchez et al., 1994; Sato-Suzuki et al., 2005, 2006b; Löber et al., 2009). The above findings also support the 1998). The increased nitric oxide production causes in turn the hypothesis that dopamine induces penile erection by acting on D4 activation of oxytocinergic neurons. Accordingly, (1) apomorphine- receptors located on the cell bodies of paraventricular oxytocin- induced penile erection was prevented by organic calcium channel ergic neurons, and which cause an increased Ca2+ influx into the blockers and by ␻-conotoxin GVIA, a potent and selective blocker of cell bodies of oxytocinergic neurons, leading to an increased nitric voltage-dependent Ca2+ channels of the N-type (McCleskey et al., oxide production. Nitric oxide in turn activates oxytocinergic neu- 1987), given into the paraventricular nucleus (see Argiolas et al., rons to release oxytocin in extra-hypothalamic brain areas and in 1990, and references therein); (2) apomorphine-induced penile the spinal cord, as already discussed. In this regard, it is noteworthy erection was prevented by nitric oxide-synthase inhibitors given that dopamine receptors have been identified in the cell bodies of into the paraventricular nucleus (Melis et al., 1994c); and (3) oxytocinergic neurons in the paraventricular nucleus only recently apomorphine and other D2 receptor agonists given at doses that by double immuno-fluorescence studies with high selective D2, D3 induce penile erection increased nitric oxide production in the par- and D4 receptor antibodies and with oxytocin antibodies. These aventricular dialysate obtained by intra-cerebral microdialysis, an studies have shown the expression of all three D2 receptor subtypes increase that was reduced by inhibitors of paraventricular nitric (D2, D3 and D4), which co-localized separately in the cell bodies of oxide-synthase given at doses that reduce D2 receptor agonist- oxytocinergic neurons in the paraventricular nucleus (and also in induced penile erection (Melis et al., 1996). The mechanism by the supraoptic nucleus and the medial preoptic area) (Baskerville means of which nitric oxide activates paraventricular oxytociner- and Douglas, 2008; Baskerville et al., 2009). This provides strong gic neurons, is still unknown, although available data suggest that neuroanatomical support to the possibility that dopamine and nitric oxide acts as an intracellular messenger and that guanylate dopamine receptor agonists of the D2 type induce penile erection cyclase is not involved. Indeed, the active phosphodiesterase- by activating directly oxytocinergic neurons projecting to the extra- resistant analogue of cyclic GMP, 8-bromo-cyclic GMP, was found hypothalamic brain areas recalled above, e.g., the spinal cord, the unable to induce penile erection when given into the paraventric- ventral tegmental area, the hippocampus and the amygdala. How- M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 947 ever, these findings do not provide any help for the identification receptor knockout animals) have usually added further confusion of the D2 receptor subtype/s, whose stimulation causes the erectile and complications in the confirmation of the putative sexual role response. Unfortunately, no help can be obtained even from studies of neurotransmitters and/or neuropeptides and their receptors. aimed at identifying oxytocinergic neurons activated by dopamine Namely, oxytocin gene ablation produces oxytocin knock out mice receptor agonists in the paraventricular nucleus. Indeed, in spite of that mate and copulate normally, as if oxytocin was unnecessary the different activity on the various dopamine receptor subtypes, for mating and copulation. Also the homozygous female oxytocin either mixed dopamine receptor agonists (e.g., apomorphine), or knockout mice show normal mating and parturition, although with selective D2 receptor agonists (e.g., quinpirole, which acts on all a marked impairment of milk letdown (Nishimori et al., 1996; D2 receptor subtypes) or selective D4 receptor agonists cause the Young et al., 1996). The ablation of the gene encoding neuronal activation of oxytocinergic neurons, as measured by the increase of nitric oxide synthase, also produces nitric oxide synthase knock FOS protein in parvocellular oxytocinergic neurons of the paraven- out mice that mate and copulate normally (Huang et al., 1993). tricular nucleus (Bitner et al., 2006). However, this finding has been This occurs in spite of the fact that nitric oxide synthase produces recently questioned, as the FOS protein increase in paraventricular nitric oxide, which is one of the main physiological mediators of oxytocinergic neurons was found only when penile erection was penile erection at the penile level (Burnett et al., 1992; Rajfer et al., induced by quinerolane, which acts mainly on D2 and D3 recep- 1992) and at the central level, in the paraventricular nucleus of tor subtypes, but not by PD 168077, a D4 receptor agonist, in spite the hypothalamus (Argiolas, 1994; Benelli et al., 1995; Melis et al., of the ability of both compounds to induce the sexual response 1998). However, these findings probably indicate an important (Baskerville et al., 2009). Further experiments with selective ago- feature of reproductive physiology, i.e., the redundancy of the sys- nists of the other D2 receptor subtypes (mainly D2 and D3) are then tems involved in its control at central and peripheral level. Such necessary to identify the exact role of each dopamine receptor sub- redundancy has certainly an evolutionary origin, since it guaran- type in the control of erectile function at the paraventricular level. tees the passage of genes to the next generation for the survival In this regard, as already recalled before, apomorphine, which acts of the species. Therefore, the fact that ablation of the D4 receptor potently on all dopamine receptor subtypes (see Brioni et al., 2004, gene does not alter the pro-erectile effect of apomorphine, sug- and references therein), is much more effective than D4 receptors gests that D4 receptors, like oxytocin and nitric oxide, are only agonists in inducing penile erection when injected into the par- a few of the mediators working in the systems controlling erec- aventricular nucleus. This might be explained by a higher affinity tile function, rather than suggesting that there is no role for these of apomorphine on D4 receptors when compared to that of the receptors in the control of penile erection and sexual behaviour. tested D4 receptor agonists, or alternatively, D4 receptor agonists The failure of D4 agonists to induce penile erection when given may act as D4 receptor partial agonists, or the concomitant acti- systemically to male rats of different strains has been recently vation of different dopamine receptor subtypes by apomorphine reported by another study (Depoortère et al., 2009). However, in may produce a higher activation of oxytocinergic neurons mediat- contrast to the work of Collins et al. (2009), and to make the pic- ing penile erection, than the activation by D2 receptor agonists of ture on the role of the different D2 receptor subtypes in the control the D4 receptor subtype only. Interactions between dopamine D1 of penile erection even more puzzling, this study also shows that and D2 receptors have been already described in the control of sex- putative selective D3 receptor antagonists given systemically were ual behaviour at the level of the medial preoptic area (see Hull et al., unable to reduce apomorphine-induced penile erection in male rats 1989). In the case that the inability of selective D2 receptor ago- of the strain more sensitive to the pro-erectile effect of apomor- nists to induce penile erection (Hsieh et al., 2004) will be confirmed phine, while the sexual response (and yawning) was antagonized (but see Depoortère et al., 2009), for instance even also after injec- by the selective D2 antagonist L-741,626 (3-[[4-(4-chlorophenyl)- tion of these compounds into the paraventricular nucleus, a major 4-hydroxypiperidin-1-yl]methyl-1H-indole), leading the authors role for D3 receptors alone or together with that of D4 receptors in to suggest that D2 receptors, rather than D3 and D4 receptors, are the activation of oxytocinergic neurons mediating penile erection those playing a major role in D2 agonist-induced penile erection should be analyzed in detail (see Baskerville et al., 2009). Unfortu- (Depoortère et al., 2009). Finally, the possibility that the excita- nately, selective D2 and D3 receptor agonists (e.g., which differ in tory effect of dopamine receptor agonists on oxytocinergic neurons their affinity for these two receptor subtypes for at least four/five mediating penile erection, at least in part, is indirect rather than orders of magnitude in vitro) are not available at the moment. direct, e.g., mediated or influenced by changes in the activity of For this reason, the recent suggestion that D3 receptors mediate other neurotransmitters/neuropeptides able to modulate the activ- penile erection induced by classical D2 receptor agonists, which ity of oxytocinergic neurons in the paraventricular nucleus, cannot is based mainly on the ability of putative D3 receptor antagonists be completely ruled out. characterized in in vitro experiments, to reduce penile erection induced by classical D2 agonists, such as apomorphine, quinpirole 3.2. Glutamic acid–oxytocin interaction in the paraventricular and pramipexole, which potently activate all dopamine D2 recep- nucleus tor subtypes (Collins et al., 2009), needs certainly to be validated with other experiments. This validation is necessary also because The paraventricular nucleus of the hypothalamus is very rich no effect of D4 receptor agonists on penile erection was found in this in synapses containing an excitatory amino acid as a neuro- study, in striking contrast with the results of the studies cited above, transmitter (e.g., glutamic acid and aspartic acid) (Van Den which demonstrate a pro-erectile effect of D4 receptor agonists. Pol, 1991). Excitatory amino acids in this nucleus are involved Indeed, even the ability of apomorphine to induce penile erec- in numerous functions, including penile erection and sexual tion (and yawning) in D4 receptor knockout mice with a potency behaviour (Roeling et al., 1991; Melis et al., 1994b, 2000, identical to that seen in wild type D4 receptor knockout mice and 2004b). Accordingly, N-methyl-d-aspartic acid (NMDA), a selective the ability of D3 receptor antagonists to abolish the apomorphine agonist of the NMDA receptor subtype, but not (±)-␣-amino-3- response in these animals (Collins et al., 2009) cannot be consid- hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), a selective ered as a definitive evidence for a selective role of the D3 receptor agonist of the AMPA receptor subtype or (±)-trans(1)-amino-1,3- subtype in D2 receptor agonist-induced penile erection. Species dif- cyclopentane dicarboxilic acid (ACPD), a selective agonist of the ferences apart, studies with neurotransmitter/neuropeptide and/or metabotropic receptor subtype, was found capable of inducing neurotransmitter/neuropeptide receptor gene ablation (neuro- penile erection when injected into the paraventricular nucleus of transmitter/neuropeptide and/or neurotransmitter/neuropeptide freely moving rats (Melis et al., 1994b). The pro-erectile effect of 948 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955

NMDA was prevented by (+)MK-801, a non-competitive NMDA 141761A, given into the lateral ventricles or directly into the par- receptor antagonist (Woodruff et al., 1987), injected into the par- aventricular nucleus at doses that induce penile erection (see aventricular nucleus (Melis et al., 1994b). In line with these results, Succu et al., 2006; Castelli et al., 2007). Such increase led to the in telemetry studies aimed at monitoring intracavernous pressure, activation of nitric oxide-synthase in the cell bodies of oxytocin- NMDA was found much more active than agonists of the other ergic neurons, increasing nitric oxide production. Nitric oxide in excitatory amino acid receptor subtypes when injected into the turn activates oxytocinergic neurons mediating penile erection paraventricular nucleus in increasing intracavernosal pressure in as described above. In line with such mechanism, SR 141761A- awake or anaesthetized male rats (Zahran et al., 2000; Chen and induced penile erection was reduced by the blockade of NMDA Chang, 2003). receptors and by nitric oxide-synthase inhibitors, but not by the As suggested above for oxytocin and dopamine, it is likely blockade of dopamine or oxytocin receptors in the paraventricular that NMDA receptors mediating penile erection are located in nucleus, while it was prevented by the blockade of central oxytocin the cell bodies of oxytocinergic neurons, since excitatory amino receptors by oxytocin receptor antagonists given i.c.v. (Melis et al., acid nerve endings impinge on oxytocinergic cell bodies in the 2004a, 2006a). paraventricular nucleus (Van Den Pol, 1991). In analogy to what found with dopamine receptor agonists, the pro-erectile effect of 3.3. Oxytocin–dopamine interaction in the ventral tegmental area NMDA is apparently mediated by the activation of oxytociner- gic neurotransmission, being abolished by the oxytocin antagonist Oxytocin induces penile erection when injected into the caudal 2 8 d(CH2)5Tyr(Me) -Orn -vasotocin given i.c.v., but not into the par- part of the ventral tegmental area in a dose-dependent manner aventricular nucleus (see Argiolas and Melis, 1995, 2004, 2005 and (Melis et al., 2007). The lowest active dose injected unilater- references therein). Likewise, NMDA-induced activation of oxy- ally was 20 ng, while the highest dose tested was 100 ng. The tocinergic neurotransmission is also secondary to the activation oxytocin effect is mediated by the activation of oxytocinergic of nitric oxide-synthase, since NMDA-induced penile erection is receptors, as the sexual response is abolished by the prior injec- 2 8 prevented by nitric oxide-synthase inhibitors (N-Nitro-N-methyl- tion of the oxytocin antagonist d(CH2)5Tyr(Me) -Orn -vasotocin l-arginine methyl ester and N-methyl-thio-l-citrulline) given into in the caudal ventral tegmental area. These receptors are local- the paraventricular nucleus, and NMDA injected into the paraven- ized in the cell bodies of dopaminergic neurons, which project tricular nucleus at doses that induce penile erection, increases nitric mainly to the shell of the nucleus accumbens. Accordingly, (1) dou- oxide production in the hypothalamic nucleus (see Argiolas and ble immuno-fluorescence studies show that in the caudal ventral Melis, 1995, 2004, 2005 and references therein). As for dopamine tegmental area oxytocinergic fibres are in close contact with the receptor agonists, the NMDA induced activation of nitric oxide- cell bodies of dopaminergic neurons, the majority of which were synthase may be also secondary to an increased Ca2+ influx in positively labeled for tyrosine-hydroxylase and containing the ret- oxytocinergic cell bodies through the Ca2+ channel-coupled NMDA rograde tracer Fluorogold previously injected into the shell of the receptors, as shown in several neural preparations (for a review nucleus accumbens (Melis et al., 2007), and (2) ventral tegmen- see Snyder, 1992; Southam and Garthwaite, 1993; Schuman and tal area oxytocin-induced penile erection occurs concomitantly Madison, 1994 and references therein). Nitric oxide in turn acti- with an increase in the concentration of extra-cellular dopamine vates oxytocinergic neurotransmission (see above). The origin of in the dialysate obtained from the shell of the nucleus accum- glutamatergic projections that activate paraventricular oxytociner- bens (Melis et al., 2007). Oxytocin-induced penile erection also gic neurons mediating penile erection is unknown, although some occurs concomitantly with an increase in nitric oxide production neuroanatomical and electrophysiological evidence suggest that in the ventral tegmental area, being both responses antagonized 2 8 they may originate, at least in part, in the hippocampus (Saphier not only by d(CH2)5Tyr(Me) -Orn -vasotocin and by the nitric and Feldman, 1987; Chen et al., 1992). Although further work oxide synthase inhibitor S-methyl-thio-l-citrulline, but also by ␻- is necessary to characterize better the origin of the glutamater- conotoxin, a voltage-dependent Ca2+ channels blocker, and by ODQ gic projections to the paraventricular nucleus, the involvement (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one), a potent inhibitor of glutamic acid in the paraventricular nucleus in the control of of guanylate cyclase, all given in the caudal ventral tegmen- penile erection and sexual behaviour is clearly supported by micro- tal area before oxytocin (Succu et al., 2008). As many of the dialysis studies. Accordingly, the extra-cellular concentrations of Fluorogold labeled dopaminergic cell bodies contacted by oxy- glutamic acid and aspartic acid were increased in the dialysate tocinergic fibres, found to be positive for tyrosine hydroxylase in obtained from the paraventricular nucleus of male rats showing the caudal ventral tegmental area, were also positively labeled for noncontact erections when put in the presence of inaccessible nitric oxide-synthase and guanylate cyclase (Succu et al., 2008), estrogen + progesterone-primed receptive female rats (Melis et al., oxytocin-induced penile erection may be mediated by the fol- 2004b), penile erections that are also mediated by the activation lowing mechanism. The activation of oxytocinergic receptors in of central oxytocinergic transmission (Melis et al., 1999a,b). Such dopaminergic cell bodies by the peptide increases Ca2+ influx increases were found even higher when copulation with the recep- inside the cell bodies of dopaminergic neurons. This activates nitric tive female was allowed (Melis et al., 2004a). In line with the oxide-synthase, a Ca2+-calmodulin-dependent enzyme, thereby hypothesis that an increased activity of excitatory amino acids increasing nitric oxide production. Nitric oxide in turn activates occurs in the paraventricular nucleus during penile erection and guanylate cyclase, leading to an increased concentration of cyclic copulation, both noncontact erections and copulatory behaviour GMP. Cyclic GMP activates dopaminergic neurons projecting to (during which in copula penile erections occur) are reduced by the nucleus accumbens. The role of cyclic GMP in penile erection blockade of NMDA receptors in the paraventricular nucleus, and induced by oxytocin injected into the caudal ventral tegmen- this reduction is followed by a decrease in the increase of nitric tal area is also supported by the ability of 8-bromo-cyclic GMP, oxide production that occurs in this hypothalamic nucleus in these an active phosphodiesterase-resistant analogue of cyclic GMP, physiological contexts (Melis et al., 2000). An increase in extra- to induce penile erection when injected into the caudal ventral cellular glutamic acid concentration secondary to a decreased tegmental area, and to increase extra-cellular dopamine in the GABA release from GABAergic nerve endings impinging on exci- dialysate from the nucleus accumbens (Succu et al., 2008; Melis tatory amino acidergic synapses juxtaposed to oxytocinergic cell et al., 2009a). This is at variance with the mechanism by which bodies, was also found in the paraventricular nucleus after the nitric oxide activates oxytocinergic neurons in the paraventricu- blockade of cannabinoid CB1 receptors by the CB1 antagonist SR lar nucleus, being 8-bromo-cyclic GMP unable to induce penile M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 949 erection when injected in this nucleus (Melis and Argiolas, 1995b) penile erection, possibly through neural efferent projections from (Fig. 2). The role of dopamine released in the nucleus accumbens in the ventral subiculum to extra-hippocampal brain areas. In line penile erection induced by oxytocin injected into the caudal ventral with this hypothesis, NMDA injected into the ventral subiculum tegmental area is supported by the ability of haloperidol, a potent induces penile erection in a dose-dependent manner, and this effect dopamine receptor antagonist injected into the nucleus accum- is antagonized completely by the prior injection into the same site bens to reduce the oxytocin response (Melis et al., 2007). As to the of (+)MK-801, but not by S-methyl-thio-l-citrulline, haemoglobin 2 8 neural pathways activated by dopamine in the nucleus accumbens or d(CH2)5Tyr(Me) -Orn -vasotocin (Melis et al., 2010). As to the leading to penile erection, these are still unknown. However, the neural efferent pathways projecting to extra-hippocampal brain available data suggest that these pathways activate dopamine neu- areas activated by excitatory amino acids (i.e., glutamic acid) in the rotransmission in the paraventricular nucleus of the hypothalamus. ventral subiculum, it is likely that these are glutamatergic, as are Accordingly, oxytocin-induced penile erection occurs concomi- the majority of hippocampal efferent projections. At the moment, it tantly to an increase in extra-cellular dopamine not only in the might be only suggested that these projections modulate the activ- nucleus accumbens, but also in the paraventricular nucleus, and ity of mesolimbic dopaminergic neurons, which in turn modulate is antagonized by the dopamine receptor antagonist haloperidol the activity of incerto-hypothalamic dopaminergic neurons in the injected into the paraventricular nucleus (Melis et al., 2007). All paraventricular nucleus, leading to the activation of oxytocinergic together, these results support the idea that oxytocinergic neu- neurons mediating penile erection as already discussed (see above). rons originating in the paraventricular nucleus and projecting to Accordingly penile erection induced by ventral subiculum oxy- the caudal ventral tegmental area, when activated release oxy- tocin occurs concomitantly with an increase in the concentration of tocin in this area, thereby activating a NO-cyclic GMP signaling extra-cellular dopamine in the shell of the nucleus accumbens, and 2 system, which in turn activates mesolimbic dopaminergic neurons this increase, like penile erection, is abolished by d(CH2)5Tyr(Me) - (Melis et al., 2007, 2009a; Succu et al., 2008). Dopamine released in Orn8-vasotocin given into the ventral subiculum before oxytocin the nucleus accumbens in turn activates neural pathways leading (Melis et al., 2009b). Moreover, since ventral subiculum oxytocin- to the activation of incerto-hypothalamic dopaminergic neurons, induced penile erection is also reduced by (+)MK-801 injected which stimulate paraventricular oxytocinergic neurons project- into the ventral tegmental area, but not into the nucleus accum- ing to the spinal cord mediating penile erection. At the same bens (Melis et al., 2009b) and occurs concomitantly to an increase time, dopamine released in the paraventricular nucleus might also of extra-cellular glutamic acid in the ventral tegmental area, but activate oxytocinergic neurons projecting to extra-hypothalamic not in the nucleus accumbens, being both responses abolished by 8 brain areas such as the ventral tegmental area, the hippocampus, d(CH2)5Tyr(Me)-Orn -vasotocin, injected into the ventral subicu- the amygdala and perhaps other brain areas. As recalled above, lum before oxytocin (see Fig. 3), it is likely that the activation of in line with this hypothesis, apomorphine injected into the par- mesolimbic dopaminergic neurons is secondary to an increased aventricular nucleus at a dose that induces penile erection also glutamatergic neurotransmission in the ventral tegmental area. increases extra-cellular dopamine concentration in the nucleus This suggests that a glutamic acid–dopamine interaction control- accumbens, an effect reduced by the oxytocin receptor antagonist ling penile erection exists in the ventral tegmental area. Further 2 8 d(CH2)5Tyr(Me) -Orn -vasotocin injected into the ventral tegmen- studies are necessary to ascertain whether the pro-erectile effer- tal area (Succu et al., 2007; Melis et al., 2009a). Together, the above ent glutametergic pathways from the subiculum to the ventral neural pathways might constitute a hypothetical neural circuitry tegmental area are direct or indirect, i.e., through the prefrontal involving dopamine, oxytocin and other neurotransmitters (e.g., cortex or other brain areas (see Melis et al., 2009b and refer- glutamic acid, see below) influencing not only sexual performance, ences therein). Since the paraventricular nucleus also receives but also sexual motivation and sexual rewarding (see Section 4). glutamatergic projections from the hippocampus (see above and Saphier and Feldman, 1987), and glutamic acid activates paraven- 3.4. Oxytocin–glutamic acid interaction in the ventral subiculum tricular oxytocinergic neurons including those projecting to the of the hippocampus ventral tegmental area (see Argiolas and Melis, 2005 and references therein), and oxytocin in the ventral tegmental area induces penile Oxytocin induces penile erection when injected into the ven- erection and increases the activity of mesolimbic dopaminergic tral, but not in the dorsal subiculum, in a dose dependent manner neurons (see above), it is tempting to speculate that paraventric- (Melis et al., 2009b). The sexual response is mediated by the stimu- ular oxytocinergic neurons may be also involved, at least in part, lation of oxytocin receptors, being abolished by the prior injection in the activation of mesolimbic dopaminergic neurons by oxytocin 2 8 of d(CH2)5Tyr(Me) -Orn -vasotocin given into the same site of oxy- injected into the ventral subiculum (see Section 4). tocin, as found in other brain areas (see above). As to the localization of these receptors, the available data suggest that they are local- ized in the cell bodies of neurons rich in nitric oxide-synthase. 4. Concluding remarks Accordingly, microdialysis studies show that oxytocin-induced penile erection occurs concomitantly with an increase in nitric The studies reviewed above confirm and extend early findings oxide production in the ventral subiculum, and this increase is showing that in male rats oxytocin plays a key role in the cen- abolished not only by the prior injection of the nitric oxide- tral control of penile erection at the level of the paraventricular synthase inhibitor S-methyl-thio-l-citrulline and by the nitric nucleus of the hypothalamus and of the spinal cord. In particular, 2 8 oxide scavenger haemoglobin, but also by d(CH2)5Tyr(Me) -Orn - the most recent studies show that oxytocin influences penile erec- vasotocin, all given into the same site of oxytocin at doses that tion also by acting in other brain areas, i.e., the ventral tegmental antagonize penile erection (Melis et al., 2010). More important, area, the ventral subiculum, and the posteromedial cortical nucleus oxytocin-induced penile erection occurs also concomitantly with of the amygdala. At the paraventricular level, the most impor- an increase in the concentration of extra-cellular glutamic acid in tant new finding is perhaps the discovery of the expression of all the ventral subiculum, which is only partially antagonized by the dopamine receptors of the D2 family (D2, D3 and D4) in the cell bod- non-competitive NMDA receptor antagonist (+)MK-801 given into ies of oxytocinergic neurons in the paraventricular nucleus (and in the ventral subiculum (Melis et al., 2010). Together, these results the supraoptic nucleus and the medial preoptic area) (Baskerville suggest that newly formed nitric oxide, by acting as an intercellular and Douglas, 2008; Baskerville et al., 2009). This provides strong messenger, activates glutamic acid neurotransmission leading to neuroanatomical support to the hypothesis that dopamine and 950 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955

Fig. 3. Effect of oxytocin injected into the ventral subiculum on penile erection and on extra-cellular glutamic acid concentration in the dialysate obtained from the ventral tegmental area (A) or the nucleus accumbens (B). Oxytocin (0.1 ␮g) dissolved in saline or saline alone was injected into the ventral subiculum of male rats implanted with intra-cerebral microdialysis cannulas aimed at the caudal ventral tegmental area or at the shell of the nucleus accumbens and chronic guide cannulas for microinjections into 2 8 the ventral subiculum. d(CH2)5Tyr(Me) -Orn -vasotocin (OA) dissolved in saline or saline alone was given 15 min before oxytocin. Intra-cerebral microdialysis conditions and other experimental conditions were identical to those reported in Melis et al. (2004b, 2007, 2009a). Values are means ± SEM of 6 rats per group. *P < 0.001 with respect to saline-treated rats values (Mann–Whitney U test). dopamine receptor agonists may activate directly oxytocinergic rat or manipulation of the genitalia, when injected into the paraven- neurons involved in erectile function and projecting not only to tricular nucleus and the CA1 field of the hippocampus, but not in the spinal cord, but also to extra-hypothalamic brain areas. In this the dorsal subiculum, the lateral septum, the caudate nucleus, the regard, it is also important the discovery that dopamine receptor medial preoptic area, the ventromedial nucleus and the supraop- agonist-induced increase in intracavernous pressure is reduced by tic nucleus (Melis et al., 1986). In all these studies penile erection the blockade of oxytocinergic receptors in the lumbo-sacral spinal was usually counted when the penis emerged from the penile cord (Baskerville et al., 2009). Indeed, although such evidence has sheath by an observer who was unaware of the given treatments been obtained in anaesthetized male rats, the finding confirms directly during the experiment or later by observing the experi- the activation of a paraventriculo-spinal oxytocinergic descend- ment recorded on a videotape with a video camera apparatus. Each ing pathway involved in dopamine receptor agonist-induced penile penile erection episode lasts for 0.5–1 min and is usually accom- erection. However, it has still to be ascertained whether penile erec- panied by penile grooming and/or hip flexions. No experiment is tion induced by the stimulation of dopamine receptors present in usually done in these rats to ascertain the effect of sexual experi- oxytocinergic cell bodies is secondary to the activation of a spe- ence, age or if these rats can be divided in low or high responders to cific dopamine receptor subtype of the D2 family (D2, D3 or D4) the pro-erectile effect of oxytocin injected into the different brain or if these receptor subtypes co-operate in modulating the erectile areas. This applies also to the majority of studies on the pro-erectile response, possibly in different ways depending on the context in effect of other neuropeptides and drugs that increase spontaneous which penile erection occurs (see Moreland et al., 2004; Enguehard- penile erection episodes, including dopamine agonists, excitatory Gueiffier et al., 2006; Melis et al., 2006a,b; Löber et al., 2009; Collins amino acids, ACTH-MSH, hexarelin and VGF peptides. However, et al., 2009; Depoortère et al., 2009; Baskerville et al., 2009). the pro-erectile effect of these compounds has been repeatedly Another important new finding is that oxytocin induces penile confirmed by telemetry methods, which determine the occurrence erection when injected not only into the paraventricular nucleus of penile erection by the increase in intracavernous pressure that or the CA1 field of the hippocampus, but also into the ventral occurs spontaneously or after administration of these compounds tegmental area, the ventral subiculum, and the posteromedial cor- by different routes, i.e., systemically, intracerebroventricularly or tical nucleus of the amygdala. These brain areas were not tested directly into specific brain nuclei, after the implant of a pres- in the earlier studies showing that oxytocin increased spontaneous sure microtransducer directly into the cavernous corpora (see penile erection episodes in male rats, although they receive like Bernabè et al., 1999). In the ventral tegmental area, the ventral the lumbo-sacral spinal cord oxytocinergic projections from the subiculum and the posteromedial nucleus of the amygdala also oxy- paraventricular nucleus. Oxytocin was indeed found capable of tocin induces penile erection by acting on oxytocinergic receptors. increasing spontaneous penile erection episodes, which occur in This leads to the activation of mesolimbic dopaminergic neurons adult male rats in the absence of any sexual stimuli, such as those originating in the ventral tegmental area and projecting to the which originate from the presence of an accessible or inaccessible shell of the nucleus accumbens, as measured by the increases in receptive (estrogen–progesterone primed) ovariectomized female extra-cellular dopamine concentration in the dialysate obtained M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 951 from the shell of nucleus accumbens and by the reduction in oxytocin-induced penile erection seems secondary to the acti- the erectile response induced by the peptide injected into these vation of oxytocinergic receptors located in the cell bodies of extra-hypothalamic areas, found after the blockade of dopamin- nitric oxide-synthase-containing neurons. This causes an increase ergic receptors in the nucleus accumbens (see below). As to the in the production of nitric oxide, which activates glutamatergic mechanisms activated by the stimulation of oxytocinergic recep- neurotransmission by acting as an intercellular messenger with tors in these brain areas, which lead to the activation of mesolimbic a mechanism similar to that described for long term potentia- dopaminergic neurons and to penile erection, the best clarified tion (see Snyder, 1992; Southam and Garthwaite, 1993; Schuman are those occurring in the caudal ventral tegmental area. Indeed, and Madison, 2004). In line with this hypothesis oxytocin-induced here pharmacological and immuno-fluorescence results show that penile erection occurs concomitantly with an increase in extra- oxytocin nerve endings impinge on the cell bodies of dopamin- cellular glutamic acid in the dialysate from the ventral subiculum, ergic neurons projecting to the shell of the nucleus accumbens and stimulation of excitatory amino acid receptors in the ventral (Melis et al., 2007, 2009a; Succu et al., 2008). Many of these neu- subiculum by NMDA, induces penile erection. Glutamic acid in turn rons are rich in nitric oxide synthase and in guanylate cyclase. The activates neural efferent projections, which lead to the activation of stimulation of oxytocinergic receptors in the cell bodies of these mesolimbic dopaminergic neurons in the ventral tegmental area, dopaminergic neurons causes the activation of nitric oxide syn- as above reported. If these mechanisms are operative also in the thase leading to an increased nitric oxide production. Nitric oxide posteromedial nucleus of the amygdala is unknown at present. in turn activates guanylate cyclase, thereby increasing the concen- Moreover, further studies are necessary to prove that (1) oxy- tration of cyclic GMP, which leads to the activation of mesolimbic tocinergic nerve endings and receptors in the ventral subiculum dopaminergic neurons and to the release of dopamine in the and in the posteromedial nucleus of the amygdala are localized nucleus accumbens, as measured by the increase in extra-cellular in cell bodies of neurons containing nitric oxide-synthase, (2) if dopamine in the dialysate from the nucleus accumbens obtained these neurons are eventually under the control of excitatory amino by intra-cerebral microdialysis (Succu et al., 2008). Dopamine acid (glutamatergic) synapses and (3) to identify the signaling sys- released in the nucleus accumbens in turn activates neural path- tem activated by nitric oxide. In this regard, it is noteworthy that ways leading to penile erection. This is supported by the ability of the ability of oxytocin to increase nitric oxide-synthase activity dopamine receptor antagonists haloperidol and/or cis-flupentixol in the cell bodies of dopaminergic neurons in the caudal ventral injected into the nucleus accumbens to reduce ventral tegmental tegmental area and of still unknown neurons in the ventral subicu- area oxytocin-induced penile erection (Succu et al., 2008). One of lum and the posteromedial nucleus of the amygdala, recalls the the pro-erectile pathways seems to activate incerto-hypothalamic ability of oxytocin to activate nitric oxide-synthase in the cell bod- dopaminergic neurons, in particular those that project to the cell ies of oxytocinergic neurons in the paraventricular nucleus (Melis bodies of paraventricular oxytocinergic neurons. Indeed oxytocin et al., 1997). However, while in the ventral tegmental area the injected into the caudal ventral tegmental area increases extra- increased production of nitric oxide in the cell bodies of dopamin- cellular dopamine not only in the nucleus accumbens but also in the ergic neurons leads to the activation of these neurons by activating paraventricular nucleus, and the blockade of dopamine receptors in guanylate cyclase and increasing cyclic GMP, this does not occur the paraventricular nucleus reduces significantly ventral tegmen- in the paraventricular nucleus. Accordingly, 8-bromo-cyclic GMP tal area oxytocin-induced penile erection (Succu et al., 2007, injected into the paraventricular nucleus does not induce penile 2008; Melis et al., 2007, 2009a). The existence of these nucleus erection, while it does so when injected into the ventral tegmental accumbens dopamine–paraventricular dopamine–paraventricular area. Another signaling pathway different from the nitric oxide- oxytocin–ventral tegmental area oxytocin–dopamine links is also cyclic GMP system is then involved at the paraventricular level in suggested by the ability of a pro-erectile dose of apomorphine and the activation of oxytocinergic neurons mediating penile erection of the D4 receptor agonist PD 168077 injected into the paraven- by endogenous and/or exogenous nitric oxide (Melis and Argiolas, tricular nucleus to increase extra-cellular dopamine in the shell of 1995b; Melis et al., 1997)(Fig. 1). On the other hand, cyclic GMP in the nucleus accumbens (Succu et al., 2007), response that is abol- the ventral tegmental area seems to play also a key role in the acti- 8 ished by d(CH2)5Tyr(Me)-Orn -vasotocin given into the ventral vation of mesolimbic dopaminergic neurons and in the increase tegmental area (Melis et al., 2009a, see also below). However, fur- in extra-cellular dopamine occurring in the dialysate obtained ther work is necessary to identify the neural pathways that connect from the shell of the nucleus accumbens of male rats selected the nucleus accumbens to the incerto-hypothalamic dopaminergic for showing or not showing noncontact penile erections when system. put in the presence of an inaccessible ovariectomized receptive The mechanism by which oxytocin induces penile erection and (oestrogen + progesterone treated) female rat. In these experimen- activates mesolimbic dopaminergic neurons when injected into the tal conditions, in male rats showing noncontact penile erections, ventral subiculum or into the posteromedial nucleus of the amyg- an increase in extra-cellular dopamine concentration is found as dala is only partially understood at the moment. In these areas also, expected, and this increase is further increased, although only mod- oxytocin activates its own receptors which lead to the activation estly, by phosphodiesterase inhibitors given into the caudal ventral of nitric oxide-synthase, thereby increasing nitric oxide produc- tegmental area (Sanna et al., 2009). In contrast, in male rats not tion. Nitric oxide in turn activates unknown efferent projections, showing noncontact erections, in which no appreciable increase in which apparently increase glutamatergic neurotransmission in the extra-cellular dopamine in the nucleus accumbens is found, extra- ventral tegmental area. Glutamic acid then stimulates mesolimbic cellular dopamine concentration is found markedly increased by dopaminergic neurons leading to penile erection. This hypothe- phosphodiesterase inhibitors given into the caudal ventral tegmen- sis is supported mainly by the ability of oxytocin injected into tal area and noncontact erections are observed (Sanna et al., 2009). the ventral subiculum to increase extra-cellular glutamic acid in The ability of oxytocin injected into the ventral tegmental the ventral tegmental area (Fig. 3), and of the non-competitive area, in the ventral subiculum and in the posteromedial cortical NMDA receptor antagonist (+)MK-801 injected into the ventral nucleus of the amygdala, together with that of dopamine agonists tegmental area, but not in the nucleus accumbens, to reduce injected into the paraventricular nucleus, to induce penile erec- penile erection induced by oxytocin injected either into the ven- tion and to activate mesolimbic dopaminergic neurons deserves tral subiculum or into the posteromedial nucleus of the amygdala some comment. First, mechanisms similar to those recalled above (Melis et al., 2009b). At the moment, more details are available may be operative when penile erection occurs in physiological for the ventral subiculum oxytocin-induced penile erection. Here contexts, such as during copulation (when in copula penile erec- 952 M.R. Melis, A. Argiolas / Neuroscience and Biobehavioral Reviews 35 (2011) 939–955 tions occur) or during noncontact penile erections. These erections are pheromone-mediated penile erections indistinguishable from those induced by drugs or oxytocin, which occur when sexually potent male rats are put into the presence of an inaccessible recep- tive (ovariectomized estrogen + progesterone primed) female rat and are considered as an index of sexual arousal (Sachs, 1997, 2007). Indeed, although these results do not demonstrate that oxy- tocin in these areas plays a role in penile erection occurring in physiological contexts or after drug administration, they add fur- ther strength to early findings suggesting that these brain areas belong to those where oxytocin given centrally acts to increase not only spontaneous penile erection episodes seen after pro-erectile drugs, but also to improve male (and female) sexual behaviour (see Argiolas and Melis, 2004 and references therein). Accord- ingly, oxytocin concentration increases in the hippocampus of male rats treated with a pro-erectile dose of apomorphine, a classical 8 dopamine agonist (Melis et al., 1990) and d(CH2)5Tyr(Me)-Orn - vasotocin, which blocks oxytocin receptors, is extremely effective Fig. 4. Schematic representation of a hypothetical neural circuitry involving not only in impairing copulatory behaviour (Argiolas et al., 1987a) oxytocin influencing sexual motivation, rewarding and sexual performance, as sug- gested by the results of this chapter and previous reports. Oxytocinergic neurons but also the facilitatory effect of apomorphine on male copula- originating in the paraventricular nucleus and projecting to the spinal cord when tory behaviour (Argiolas et al., 1987b) in sexually potent male rats, activated for instance by dopamine and glutamic acid (but also by other neurotrans- during which in copula penile erection occur. d(CH2)5Tyr(Me)- mitters and/or neuropeptides), facilitate penile erection and sexual performance by Orn8-vasotocin is also extremely potent in reducing noncontact activating oxytocinergic neurons projecting to the spinal cord. Dopamine and glu- tamic acid (but also neurotransmitters and neuropeptides) in the paraventricular erections in sexually potent male rats, when given in nanogram nucleus also activates oxytocinergic neurons projecting to the ventral tegmental amounts into the lateral ventricles, but not into the PVN (Melis area, thereby activating mesolimbic dopaminergic neurons projecting to the nucleus et al., 1999a). Second, mesolimbic dopaminergic neurons play a key accumbens, modulating sexual motivation and reward. Dopamine released in the role in the motivational and rewarding properties of natural rein- nucleus accumbens (NAs) activates in turn still unknown neural pathways, which forcing stimuli, such as food, water and sexual activity (Fibiger and increase the activity of incerto-hypothalamic dopaminergic neurons (originating in the A13–A14 groups of Dahlstrom and Fuxe) impinging among others on oxytociner- Phillips, 1988; Wise and Rompre, 1989; Everitt, 1990). In particu- gic neurons, including those that project to the spinal cord, leading to penile erection. lar, dopamine released from these neurons is thought to mediate This circuitry may be also activated by oxytocin injected not only into the caudal the transposition of the motivational aspects of natural stimuli into ventral tegmental area, but also in the ventral subiculum and in the amygdala, which goal directed behaviours, for instance in the case of sexual activity, also receive an oxytocinergic innervation from the paraventricular nucleus, possi- bly through direct or indirect glutamatergic efferent to the ventral tegmental area, the seeking of a sexual partner and of sexual intercourse to reach leading to the modulation of both sexual motivation and penile erection. Finally, reward and satisfaction (Goto and Grace, 2005). Accordingly, extra- the above circuitry may be also activated by sexual stimuli and pheromones, since cellular dopamine concentration increases in dialysate from the extra-cellular dopamine and glutamic acid increase in the paraventricular nucleus nucleus accumbens of sexually potent male rats during exposure (and in the medial preoptic area) during pheromone-mediated noncontact erections to an inaccessible ovariectomized estrogen + progesterone-primed and copulation (for appropriate references see the References list). receptive female rat, and such increase was even higher when the male rat was allowed to copulate with the receptive female (Pfaus and Everitt, 1995). Third, the present results support the hypothe- system providing a neural substrate for explaining the rewarding sis that a neural circuit connects the paraventricular nucleus with properties of sexual activity (Everitt, 1990; Pfaus and Everitt, 1995). the ventral tegmental area directly or indirectly (through the ven- In this regard, it is noteworthy that the mesolimbic dopaminergic tral subiculum and/or the posteromedial cortical nucleus of the system activated by oxytocin injected into the ventral tegmen- amygdala) and the nucleus accumbens, and from here through tal area is the same activated by drugs of abuse such as opiates, unknown pathways back again to the paraventricular nucleus to cannabinoids, amphetamine, cocaine and alcohol (Tanda et al., control the activity of oxytocinergic neurons projecting to the 1997), and that oxytocin was found capable of reducing tolerance spinal cord mediating penile erection and of oxytocinergic neurons and dependence to cocaine, morphine, alcohol and cannabinoids projecting to the ventral tegmental area, the ventral subiculum and (Kovacs et al., 1998; Cui et al., 2001). the posteromedial cortical nucleus of the amygdala, modulating in In conclusion, it seems that oxytocin released not only in the this way the activity of mesolimbic dopaminergic neurons (Fig. 4). ventral tegmental area, but also in the ventral subiculum and This complex neural circuit may play a role in the integration the posteromedial cortical nucleus of the amygdala, can activate of neural activities involved in the control of the consummatory mesolimbic dopaminergic neurons, which may be involved in the (erectile–ejaculatory) and anticipatory (motivational and reward- appetitive and rewarding effects of sexual activity. The activation ing) aspects of male sexual behaviour in physiological contexts. of mesolimbic dopaminergic neurons may be direct, through oxy- Indeed, extra-cellular dopamine increases in the nucleus accum- tocinergic receptors in the cell bodies of mesolimbic dopaminergic bens (Pfaus and Everitt, 1995) and in the paraventricular nucleus neurons, or indirect through the activation of glutamic acid neu- of sexually potent male rats during exposure to an inaccessible rotransmission in the ventral tegmental area. Dopamine released receptive female rat, when noncontact erections occur, and even in the nucleus accumbens shell modulates in turn the activity of more when copulation is allowed, e.g., when in copula penile erec- the incerto-hypothalamic dopaminergic neurons in the paraven- tions occur (Melis et al., 2003). Thus, although further studies tricular nucleus causing either penile erection (via activation of are necessary to clarify the role of endogenous oxytocin in the oxytocinergic neurons projecting to the spinal cord), or sexual ventral tegmental area, the ventral subiculum and the amygdala motivation and reward (via activation of oxytocinergic neurons during noncontact erections and sexual behaviour, it may be then projecting to the ventral tegmental area, the ventral subiculum reasonable to assume that this hypothetical neural circuit, while or the posteromedial cortical nucleus of the amygdala). Since contributing to the consummatory aspects of sexual behaviour, dopamine is also released in the nucleus accumbens shell and in in the same time may also activate the mesolimbic dopaminergic the paraventricular nucleus when penile erection occurs in physio- M.R. Melis, A. 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