Sex-dependent differences are frequently observed in the biological and behavioural effects of substances of abuse, including cannabis. We recently demonstrated a modulating effect of sex and oestrous cycle on cannabinoid-taking and seeking behaviours. Here, we investigated the influence of sex and oestrogen in the regulation of cannabinoid CB1 receptor density and function, measured by [(3)H]CP55940 and CP55940-stimulated [(35)S]GTPγS binding autoradiography, respectively, in the prefrontal cortex (Cg1 and Cg3), caudate- putamen, nucleus accumbens, amygdala and hippocampus of male and cycling female rats, as well as ovariectomised (OVX) rats and OVX rats primed with oestradiol (10 µg/rat) (OVX+E). CB1 receptor density was significantly lower in the prefrontal cortex and amygdala of cycling females than in males and in OVX females, a difference that appeared to be oestradiol-dependent, because it was no more evident in the OVX+E group. CP55940-stimulated [(35)S]GTPγS binding was significantly higher in the Cg3 of OVX rats relative to cycling and OVX+E rats. No difference was observed in CB1 receptor density or function in any of the other brain areas analysed. Finally, sex and oestradiol were also found to affect motor activity, social behaviour and sensorimotor gating in rats tested in locomotor activity boxes, social interaction and prepulse inhibition tasks, respectively. Our findings provide biochemical evidence for sex- and hormone- dependent differences in the density and function of CB1 receptors in selected brain regions, and in behaviours associated with greater vulnerability to drug addiction, revealing a more vulnerable behavioural phenotype in female than in male rats.

Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25–6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality.

Summary The enzyme 5α-reductase (5αR) catalyzes the conversion of testosterone and other Δ4-3-ketosteroids into their 5α-reduced metabolites. Of the five members of the 5αR family the type 2 enzyme (5αR2) plays a key role in androgen metabolism, and is abundantly distributed in the urogenital system. Although 5αR2 has been reported to be highly expressed in the brain during early developmental stages, little is currently known on its anatomical and cellular distribution in the adult brain. Thus, the present study was designed to determine the detailed localization of 5αR2 in the adult rat brain, using a highly specific polyclonal antibody against this isoform. Parasagittal and coronal sections revealed 5αR2 immunoreactivity throughout most brain regions, with strong immunolabeling in the layers III and VI of the prefrontal and somatosensory cortex, olfactory bulb, thalamic nuclei, CA3 field of hippocampus, basolateral amygdala and Purkinje cell layer of cerebellum. Lower 5αR2 levels were detected in the hypothalamus and midbrain. Moreover, double labeling fluorescence with confocal laser scanning microscopy (CLSM) revealed that 5αR2 is localized in neurons, but not in glial cells. Specifically, the enzyme was documented in the pyramidal neurons of the cortex by CLSM analysis of simultaneous Golgi-Cox and immunofluorescent staining. Finally, low levels of 5αR2 expression were identified in GABAergic cells across the cortex, hippocampus and striatum. These findings show that, in the adult brain, 5αR2 is distributed in critical regions for behavioral regulation, suggesting that the functional role of this isoform is present throughout the entire lifespan of the individual.

Two recently reported hit compounds, COR627 and COR628, underpinned the development of a series of 2-(acylamino)thiophene derivatives. Some of these compounds displayed significant activity in vitro as positive allosteric modulators of the GABAB receptor by potentiating GTPγS stimulation induced by GABA at 2.5 and 25 μM while failing to exhibit intrinsic agonist activity. Compounds were also found to be effective in vivo, potentiating baclofen-induced sedation/hypnosis in DBA mice when administered either intraperitoneally or intragastrically. Although displaying a lower potency in vitro than the reference compound GS39783, the new compounds 6, 10, and 11 exhibited a higher efficacy in vivo: combination of these compounds with a per se nonsedative dose of baclofen resulted in shorter onset and longer duration of the loss of righting reflex in mice. Test compounds showed cytotoxic effects at concentrations comparable to or higher than those of GS39783 or BHF177.

The termination of serotonin (5-hydroxytryptamine, 5-HT) neurotransmission is regulated by its uptake by the 5-HT transporter (5-HTT), as well as its degradation by monoamine oxidase (MAO)-A. MAO-A deficiency results in a wide set of behavioral alterations, including perseverative behaviors and social deficits. These anomalies are likely related to 5-HTergic homeostatic imbalances; however, the role of 5-HTT in these abnormalities remains unclear. To ascertain the role of 5-HTT in the behavioral anomalies associated to MAO-A deficiency, we tested the behavioral effects of its blocker fluoxetine on perseverative, social and aggressive behaviors in transgenic animals with hypomorphic or null-allele MAO-A mutations. Acute treatment with 5-HTT blocker fluoxetine (10 mg/kg, i.p.) reduced aggressive behavior in MAO-A knockout (KO) mice and social deficits in hypomorphic MAO-ANeo mice. Furthermore, this treatment also reduced perseverative responses (including marble burying and water mist-induced grooming) in both MAO-A mutant genotypes. Both MAO-A mutant lines displayed significant reductions in 5-HTT expression across the prefrontal cortex, amygdala and striatum, as quantified by immunohistochemical detection; however, the down-regulation of 5-HTT in MAO-ANeo mice was more pervasive and widespread than in their KO counterparts, possibly indicating a greater ability of the hypomorphic line to enact compensatory mechanisms with respect to 5-HT homeostasis. Collectively, these findings suggest that the behavioral deficits associated with low MAO-A activity may reflect developmental alterations of 5-HTT within 5-HTergic neurons. Furthermore, the translational implications of our results highlight 5-HT reuptake inhibition as an interesting approach for the control of aggressive outbursts in MAO-A deficient individuals.

The potential efficacy of GABA B receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA B receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA B receptor may constitute a novel approach in the pharmacological manipulation of the GABA B receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA B PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA-and baclofen-stimulated guanosine 5Ј-O-(3-[ 35 S]thio)-triphosphate binding to native GABA B receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA B receptor antago-, both COR627 and COR628 increased the affinity of high-and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA B receptor.

Methamphetamine (METH) is a potent psychostimulant with neurotoxic properties. Heavy use increases the activation of neuronal nitric oxide synthase (nNOS), production of peroxynitrites, microglia stimulation, and induces hyperthermia and anorectic effects. Most METH recreational users also consume cannabis. Preclinical studies have shown that natural (Δ9-tetrahydrocannabinol, Δ9-THC) and synthetic cannabinoid CB1 and CB2 receptor agonists exert neuroprotective effects on different models of cerebral damage. Here, we investigated the neuroprotective effect of Δ9-THC on METH-induced neurotoxicity by examining its ability to reduce astrocyte activation and nNOS overexpression in selected brain areas. Rats exposed to a METH neurotoxic regimen (4×10 mg/kg, 2 hours apart) were pre- or post-treated with Δ9-THC (1 or 3 mg/kg) and sacrificed 3 days after the last METH administration. Semi-quantitative immunohistochemistry was performed using antibodies against nNOS and Glial Fibrillary Acidic Protein (GFAP). Results showed that, as compared to corresponding controls (i) METH-induced nNOS overexpression in the caudate-putamen (CPu) was significantly attenuated by pre- and post-treatment with both doses of Δ9-THC (−19% and −28% for 1 mg/kg pre- and post-treated animals; −25% and −21% for 3 mg/kg pre- and post-treated animals); (ii) METH-induced GFAP-immunoreactivity (IR) was significantly reduced in the CPu by post-treatment with 1 mg/kg Δ9-THC1 (−50%) and by pre-treatment with 3 mg/kg Δ9-THC (−53%); (iii) METH-induced GFAP-IR was significantly decreased in the prefrontal cortex (PFC) by pre- and post-treatment with both doses of Δ9-THC (−34% and −47% for 1 mg/kg pre- and post-treated animals; −37% and −29% for 3 mg/kg pre- and post-treated animals). The cannabinoid CB1 receptor antagonist SR141716A attenuated METH-induced nNOS overexpression in the CPu, but failed to counteract the Δ9-THC-mediated reduction of METH-induced GFAP-IR both in the PFC and CPu. Our results indicate that Δ9-THC reduces METH-induced brain damage via inhibition of nNOS expression and astrocyte activation through CB1-dependent and independent mechanisms, respectively.