La somministrazione di ossitocina nel nucleo del letto della stria terminale del ratto induce erezione peniena e sbadiglio: studi comportamentali, neurofarmacologici e neurochimici sul meccanismo d’azione.

The bed nucleus of the stria terminalis (BNST) is a complex forebrain structure that plays an key role in autonomic, neuroendocrine and behavioral responses such as anxiety, fear, stress and sexual behavior. The control of these functions by the BNST is mediated by neurotransmitters as GABA, glutamic acid, dopamine, serotonin, nitric oxide (NO), and neuropeptides as corticotrophin releasing factor (CRF) and oxytocin. Interestingly, i) oxytocin and oxytocin receptors are present in the BNST, ii) all oxytocin neurons present in the central nervous system originate from the paraventricular nucleus of the hypothalamus, and iii) some of these neurons control penile erection, sexual behavior and yawning. This raises the possibility that oxytocin may also play a role in the control of these responses at the level of the BNST. To test this possibility, the effect of oxytocin injected into the BNST on these responses was studied. Oxytocin (5-100ng), but not Arg8-vasopressin (100ng), injected unilaterally into the BNST induced penile erection and yawning in a dose-dependent manner in male rats. The minimal effective dose was 20ng for penile erection and 5ng for yawning. Oxytocin responses were abolished by the oxytocin receptor antagonist d(CH2)5Tyr(Me)2-Orn8-vasotocin (1μg), (+) MK-801 (1μg), an excitatory amino acid receptor antagonist of the N-methyl-d-aspartic acid (NMDA) subtype, SCH 23390 (1μg), a D1 receptor antagonist, but not haloperidol (1μg), a D2 receptor antagonist, and SMTC (40μg), an inhibitor of neuronal nitric oxide synthase, injected into the BNST 15min before oxytocin. Oxytocin-induced penile erection, but not yawning, was also abolished by CNQX (1μg), an excitatory amino acid receptor antagonist of the AMPA subtype. In contrast, oxytocin responses were not reduced by bicuculline (20ng), a GABAA receptor antagonist, phaclofen (5μg), a GABAB receptor antagonist, CP 376395, a CRF receptor-1 antagonist (5μg), or astressin 2B, a CRF receptor-2 antagonist (150ng). Since NMDA (100 ng) also induced penile erection and yawning when injected into the BNST, but NMDA responses were not antagonized by pretreatment with d(CH2)5Tyr(Me)2-Orn8-vasotocin, these results suggest that oxytocin induces penile erection and yawning by activating glutamatergic neurotransmission in the BNST. This in turn leads to the activation of neural pathways projecting back to the paraventricular nucleus, medial preoptic area, ventral tegmental area, and/or ventral subiculum/amygdala, thereby inducing penile erection and yawning. In line with this mechanism of action, intracerebral microdialysis experiments showed that i) oxytocin (100 ng) injected into the BNST not only induces penile erection and yawning, but also increases the concentrations of extracellular glutamic acid and NO2- ions (a measure of NO production) in the dialysate obtained from the BNST, and ii) both effects are abolished by pre-treatment with d(CH2)5Tyr(Me)2-Orn8-vasotocin (1μg) given into the BNST before oxytocin

New insights into methoxetamine mechanisms of action: Focus on serotonergic 5-HT 2 receptors in pharmacological and behavioral effects in the rat

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action

Dopamine, Noradrenaline and Differences in Sexual Behavior between Roman High and Low Avoidance Male Rats: A Microdialysis Study in the Medial Prefrontal Cortex

Roman High- (RHA) and Low-Avoidance (RLA) outbred rats, which differ for a respectively rapid vs. poor acquisition of the active avoidance response in the shuttle-box, display differences in sexual activity when put in the presence of a sexually receptive female rat. Indeed RHA rats show higher levels of sexual motivation and copulatory performance than RLA rats, which persist also after repeated sexual activity. These differences have been correlated to a higher tone of the mesolimbic dopaminergic system of RHA rats vs. RLA rats, revealed by the higher increase of dopamine found in the dialysate obtained from the nucleus accumbens of RHA than RLA rats during sexual activity. This work shows that extracellular dopamine and noradrenaline (NA) also, increase in the dialysate from the medial prefrontal cortex (mPFC) of male RHA and RLA rats put in the presence of an inaccessible female rat and more markedly during direct sexual interaction. Such increases in dopamine (and its main metabolite 3,4-dihydroxyphenylacetic acid, DOPAC) and NA were found in both sexually naïve and experienced animals, but they were higher: (i) in RHA than in RLA rats; and (ii) in sexually experienced RHA and RLA rats than in their naïve counterparts. Finally, the differences in dopamine and NA in the mPFC occurred concomitantly to those in sexual activity, as RHA rats displayed higher levels of sexual motivation and copulatory performance than RLA rats in both the sexually naïve and experienced conditions. These results suggest that a higher dopaminergic tone also occurs in the mPFC, together with an increased noradrenergic tone, which may be involved in the different copulatory patterns found in RHA and RLA rats, as suggested for the mesolimbic dopaminergic system

Neuroplastic changes in c-Fos, ΔFosB, BDNF, trkB, and Arc expression in the hippocampus of male Roman rats: differential effects of sexual activity

Sexual activity causes differential changes in the expression of markers of neural activation (c-Fos and Delta FosB) and neural plasticity (Arc and BDNF/trkB), as determined either by Western Blot (BDNF, trkB, Arc, and Delta FosB) or immunohistochemistry (BDNF, trkB, Arc, and c-Fos), in the hippocam pus of male Roman high (RHA) and low avoidance (RLA) rats, two psychogenetically selected rat lines that display marked differences in sexual behavior (RHA rats exhibit higher sexual motivation and better copulatory performance than RLA rats). Both methods showed (with some differences) that sexual activity modifies the expression levels of these markers in the hippocampus of Roman rats depending on: (i) the level of sexual experience, that is, changes were usually more evident in sexually naive than in experienced rats; (ii) the hippocampal partition, that is, BDNF and Arc increased in the dorsal but tended to decrease in the ventral hippocampus; (iii) the marker considered, that is, in sexually experienced animals BDNF, c-Fos, and Arc levels were similar to those of controls, while Delta FosB levels increased; and (iv) the rat line, that is, changes were usually larger in RHA than RLA rats. These findings resemble those of early studies in RHA and RLA rats showing that sexual activity influences the expression of these markers in the nucleus accumbens, medial prefrontal cortex, and ventral tegmental area, and show for the first time that also in the hippocampus sexual activity induces neural activation and plasticity, events that occur mainly during the first phase of the acquisition of sexual experience and depend on the genotypic/phenotypic characteristics of the animals

Oxytocin induces penile erection and yawning when injected into the bed nucleus of the stria terminalis: involvement of glutamic acid, dopamine and nitric oxide

OXYTOCIN INDUCES PENILE ERECTION AND YAWNING WHEN INJECTED INTO THE BED NUCLEUS OF THE STRIA TERMINALIS: INVOLVEMENT OF GLUTAMIC ACID, DOPAMINE AND NITRIC OXIDE. The bed nucleus of the stria terminalis (BNST) is a complex forebrain structure divided in different subregions, which plays an important role in autonomic, neuroendocrine and behavioral responses such as anxiety, fear, stress and sexual behavior. The control of physiological and behavioral activities exerted by the BNST is mediated through the action of various neurotransmitters including GABA, glutamic acid, dopamine, serotonin, nitric oxide (NO), and neuropeptides as corticotrophin releasing factor (CRF) and oxytocin [1]. The presence of oxytocin and oxytocin receptors in the BNST together with the fact that all oxytocin neurons present in the central nervous system originate from the paraventricular nucleus of the hypothalamus and that some of these neurons exert a facilitatory role on penile erection and sexual behavior and on yawning as well [2,3], raise the possibility that oxytocin may play a role in the control of penile erection, sexual behavior and yawning at the level of this brain area. In order to verify such possibility, we studied the effect of the injection of oxytocin into the BNST on penile erection and yawning and its possible interaction with other BNST neurotransmitter systems in inducing these effects. Briefly, male Sprague Dawley rats were stereotaxically implanted unilaterally with a stainless-steel guide cannula (22 gauge) aimed at the BNST under isoflurane anaesthesia. After surgery, rats were given 7 days for recovery. Microinjections of oxytocin and other compounds usually dissolved in saline or appropriate vehicles were performed through an internal cannula (28 gauge) extending 6 mm below tip of the guide cannula (Paxinos & Watson, The stereotaxic atlas of the rat brain, 2004) in a volume of 0.3 µL in 2 min. Rats injected with saline or vehicle alone were used as controls. Oxytocin (5-100 ng) but not vasopressin (100 ng) injected into the BNST, dose-dependently induced penile erection and yawning. The minimal effective dose of oxytocin was 20 ng for penile erection and 5 ng for yawning, while the maximum effect was found for both responses with the dose of 100 ng. Oxytocin responses were completely abolished by the oxytocin receptor antagonist d(CH2)5Tyr(Me)2-Orn8-vasotocin (1 µg) injected into the BNST 15 min before oxytocin. Penile erection and yawning were also abolished by the prior injection into the BNST of (+) MK-801 (1 µg) and CNQX (1 µg), excitatory amino acid receptor antagonists, the first of the NMDA and the second of the AMPA receptor subtype, respectively, by SCH 23390 (1 µg), a D1 receptor antagonist and by SMTC (40 µg), an inhibitor of neuronal nitric oxide synthase. In contrast, no effect on oxytocin-induced penile erection and yawning was found when haloperidol, a D2 receptor antagonist, (1 µg), bicuculline (20 ng) and phaclophen (5 µg), a GABAA and GABAB receptor antagonist, respectively, and CP 376395 (5 µg) and astressin 2B (150 ng), CRF receptor antagonists, the first of the CRF-1 and the second of the CRF-2 receptor subtype, were given into the BNST 15 min prior oxytocin. Finally, the excitatory amino acid N-methy-D-aspartic acid (NMDA) (100 ng) per sé was able to induce penile erection and yawning when injected into the BNST, and these effects were antagonized by the prior injection of (+) MK-801 (1 µg) into the BNST. Taken together, the results of this study show that oxytocin injected into the BNST induces penile erection and yawning in a dose dependent manner and that this effect can be attributed to the interaction of BNST oxytocin neurons with BNST dopaminergic, glutamatergic and nitric oxide pathways and not through CRF and/or GABAergic pathways. The involvement of glutamic acid and NMDA receptors in the pro-erectile and proyawning effects of oxytocin at the level of the BNST is also supported by the ability of NMDA given into the BNST to induce both penile erection and yawning

Oxytocin induces penile erection and yawning when injected into the bed nucleus of the stria terminalis: A microdialysis and immunohistochemical study

Oxytocin (5, 20 and 100 ng) injected unilaterally into the bed nucleus of the stria terminalis (BNST) of male rats stereotaxically implanted with a microinjection cannula coupled to a microdialysis probe, induces penile erection and yawning that occur concomitantly with a dose-dependent increase in the extracellular concentration of glutamic acid, dopamine and its main metabolite 3,4-dihydroxyphenilacetic acid (DOPAC), and nitrites (NO2 −) in the dialysate obtained from the BNST by intracerebral microdialysis. The responses induced by oxytocin (100 ng) were all abolished by the oxytocin receptor antagonist d(CH2)5Tyr(Me)2-Orn8-vasotocin (1 μg), and reduced by CNQX (1 μg), a competitive antagonist of the AMPA receptors, both given into the BNST 25 min before oxytocin. In contrast, (+) MK-801 (1 μg), a non-competitive antagonist of NMDA receptors, and SCH 23390 (1 μg), a selective dopamine D1 receptor antagonist, reduced penile erection and yawning, but not glutamic acid and dopamine increases in the BNST dialysate induced by oxytocin. Immunohistochemistry revealed oxytocin-labelled neuronal structures in close proximity to tyrosine hydroxylase-labelled neurons or nitric oxide synthase-labelled cell bodies surrounded by intense vesicular glutamate transporter1-stained synapses in BNST sections where oxytocin injections induce the above responses. Together, these findings show that oxytocin injected into the BNST induces penile erection and yawning by activating not only the glutamatergic (and nitrergic) but also the dopaminergic neurotransmission, leading in turn to the activation of neural pathways mediating penile erection and yawning

Oxytocin-conjugated saporin injected into the substantia nigra of male rats alters the activity of the nigrostriatal dopaminergic system: A behavioral and neurochemical study

Saporin conjugated to oxytocin (OXY-SAP) destroys neurons expressing oxytocinergic receptors. When injected unilaterally in the substantia nigra of male rats, OXY-SAP causes a dose-dependent decrease up to 55 % in nigral Tyrosine Hydroxylase (TH)-immunoreactivity compared to control mock peptide BLANK-SAP- and PBS-treated rats or the contralateral substantia nigra. TH decrease was parallel to a dopamine content decrease in the ipsilateral striatum compared to BLANK-SAP- or PBS-treated rats or the contralateral striatum. OXY-SAP-treated rats showed a small but significant increase of locomotor activity 28 days after intranigral injection in the Open field test compared to BLANK-SAP- or PBS-treated rats, in line with an inhibitory role of nigral oxytocin on locomotor activity. OXY-SAP-, but not BLANK-SAP- or PBS-treated rats, also showed marked dose-dependent rotational turning ipsilateral to the injected substantia nigra when challenged with D-amphetamine, but not with apomorphine. Under isoflurane anesthesia OXY-SAP-treated rats showed levels of extracellular dopamine in the dialysate from the ipsilateral striatum only half those of BLANK-SAP- or PBS-treated rats or the contralateral striatum. When treated with D-amphetamine, OXY-SAP_60/120 rats showed increased extracellular dopamine levels in the dialysate from the ipsilateral striatum two third/one third only of those found in BLANK-SAP- or PBS-treated rats or the contralateral striatum, respectively. These results show that OXY-SAP destroys nigrostriatal dopaminergic neurons expressing oxytocin receptors leading to a reduced striatal dopamine function

Attività del sistema dopaminergico mesocorticale durante il comportamento sessuale: ruolo dell’esperienza e delle differenze individuali

Attività del sistema dopaminergico mesocorticale durante il comportamento sessuale: ruolo dell’esperienza e delle differenze individuali. Il sistema dopaminergico (DA) mesocorticolimbico, costituito da neuroni che dall’area ventrale del tegmento (VTA) proiettano al nucleo accumbens (NAcc) ed alla corteccia prefrontale (PFC), gioca un ruolo chiave nei comportamenti motivati (assunzione di cibo, attività sessuale, abuso di sostanze). Questo studio indaga l’attività del sistema DA mesocorticale durante il comportamento sessuale e le possibili differenze dovute all’esperienza ed alle caratteristiche individuali nelle linee di ratti Roman (RHA e RLA): i primi sono impulsivi e proni all’abuso di sostanze, mentre i secondi sono iper-emotivi e proni a sviluppare sintomi depressivi. Metodi. Sono stati misurati i livelli di DA nel dializzato ottenuto con la microdialisi intracerebrale dalla mPFC di ratti RHA e RLA naïve (mai esposti prima a stimoli sessuali) o esperienti, esposti ad una femmina recettiva inaccessibile e/o durante la copula, insieme a diversi indici motivazionali e di performance sessuale. I risultati sono stati analizzati con ANOVA per disegni misti seguita da test post hoc (P < 0.05). Risultati. L’esposizione alla femmina inaccessibile ed ancora di più l’accoppiamento causano un aumento della liberazione di DA nella mPFC. Quest’aumento è maggiore nei ratti esperienti rispetto ai naïve di entrambe le linee, sia nella fase appetitiva che durante la copula. Inoltre l’aumento che si osserva nei ratti RHA è maggiore di quello dei ratti RLA. Tali differenze sono in linea con quelle osservate negli indici comportamentali dei ratti RHA e RLA. Conclusioni. Questo studio è il primo a mostrare che durante l’attività sessuale c’è un aumento della liberazione di DA nella mPFC, come nel NAcc. L’entità dell’aumento dipende sia dal livello di esperienza sessuale che dalle caratteristiche individuali degli animali. Altri studi sono necessari per chiarire l’esatto ruolo del sistema DA mesocorticale nei diversi aspetti del comportamento sessuale e nelle differenze esistenti tra i ratti RHA e RLA

Involvement of dopamine in the differences in sexual behaviour between roman high and low avoidance rats: behavioral, pharmacological and neurochemical findings

INVOLVEMENT OF DOPAMINE IN THE DIFFERENCES IN SEXUAL BEHAVIOUR BETWEEN ROMAN HIGH AND LOW AVOIDANCE RATS: BEHAVIORAL, PHARMACOLOGICAL AND NEUROCHEMICAL FINDINGS. The Roman lines of rats (High – RHA, and Low Avoidance - RLA) display opposite behavioral traits: RHA rats are active copers, impulsive and prone to abuse drugs while RLA rats are reactive copers, hyperemotional and prone to develop depressive-like symptoms. These differences are linked to differences in brain monoamine function and neuroendocrine responses to stress. RHA and RLA rats differ also in sexual behavior, with RHA rats displaying higher motivation and better copulatory performances than RLA rats [1]. Recently, we found that the differences observed in sexual behavior between the two Roman lines occur concomitantly to differences in the activation of the mesolimbic dopaminergic system, which plays a key role in motivated behaviors (i.e. goal-directed behaviors), as assessed by the differences in dopamine concentrations in the dialysates obtained from the nucleus accumbens shell by means of intracerebral microdialysis before and during sexual activity [2]. Together with the well known role of dopamine in sexual behavior [3], these findings suggest that the sexual differences between RHA and RLA rats may be due to differences in dopamine neurotransmission. Another brain area involved in motivated behaviors is the medial prefrontal cortex (mPFC), which receives, as the nucleus accumbens, dopaminergic projections from the ventral tegmental area (VTA). The mPFC is involved in several functions, from decision making to coping to stress, from behavioral inhibition to incentive evaluation. This raises the possibility that the mPFC might be another brain area in which the Roman lines display differences in dopaminergic (monoaminergic) activity related to the observed differences in sexual behavior. In order to test this hypothesis, naïve (never exposed to a receptive female) and sexually experienced (which underwent five copulation tests) RHA and RLA rats implanted with a microdialysis probe aimed at the mPFC, were used in a classical appetitive/consummatory test of sexual behavior, during which copulatory parameters were recorded and dialysate aliquots collected from the mPFC for the determination of dopamine and noradrenaline by HPLC-ECD. The results show that the higher sexual motivation and better performance of RHA vs. RLA rats occurred concomitantly with a higher dopamine and noradrenaline release in the mPFC, as shown by the higher concentrations found in the mPFC dialysates of RHA vs. RLA rats. These differences between the two lines were greater in naïve animals and persisted, although attenuated, in experienced animals. Furthermore, sexually experienced rats of both lines displayed higher dopamine, and to a lesser extent noradrenaline, release in the mPFC compared to their sexually naïve counterparts. These findings confirm that a different mesocorticolimbic dopaminergic tone exists in RHA and RLA rats, which may be responsible, together with differences in noradrenaline release, at least in part, for their different copulatory patterns, and could provide further insights into the differences among individuals in the neural basis of motivated behaviors and their relationship with vulnerability to abuse natural and/or drug rewards or to develop depressive disorders

The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: a neurochemical, immunohistochemical and behavioral study

Experimental evidence shows that the phenylpyrazole pesticide fipronil exerts neurotoxic effects at central level in rodents, and in particular on nigrostriatal dopaminergic neurons, whose degeneration is well known to cause motor and non-motor deficits in animals and in humans. In order to characterize better the central neurotoxic effect of fipronil, we injected fipronil (15 and 25 μg) dissolved in dimethyl sulfoxide (DMSO) unilaterally into the substantia nigra of male rats. Male rats injected with DMSO unilaterally into the substantia nigra were used as controls. Control and fipronil-treated rats were then tested in different motor (i.e., open field arena, rotarod, tail flick) and non motor tests (novel object recognition, social interaction) 15 days after injection. A systemic challenge dose of the dopamine-agonist apomorphine was also used to study the presence of a rotational behavior. Sixteen days after fipronil or DMSO injection into the substantia nigra, rats were sacrificed, and either striatal dopamine content or substantia nigra tyrosine hydroxylase (TH) immunoreactivity were measured. The results confirm that the unilateral injection of fipronil into the substantia nigra caused the degeneration of nigrostriatal dopaminergic neurons, which leads to a decrease around 50 % in striatal dopamine content and substantia nigra TH imunoreactivity. This occurred together with changes in motor activity and coordination, and in nociception but not in recognition memory and in social interaction, as revealed by the results of the behavioral experiments performed in fipronil-treated rats compared to vehicle-treated rats 15 days after treatment, as found with other compounds that destroy nigrostriatal dopaminergic neurons