Angelo Di Clemente

Bifeprunox: a partial agonist at dopamine D2 and serotonin1A receptors, influences nicotine‐seeking behaviour in response to drug‐associated stimuli in rats

Environmental stimuli repeatedly associated with the self‐administered drugs may acquire motivational importance. Because dopamine (DA) D2/D3 partial agonists and D3 antagonists interfere with the ability of drug‐associated cues to induce drug‐seeking behaviour, the present study investigated whether bifeprunox, 7‐[4‐([1,1′biphenyl]‐3‐ylmethyl)‐1‐piperazinyl]‐2(3H)‐benzoxazolone mesylate), a high‐affinity partial agonist of the D2 subfamily of DA receptors and of serotonin1A receptors, influences reinstatement of drug‐associated cue‐induced nicotine‐seeking behaviour. The study also explored whether bifeprunox reduced motivated behaviour by evaluating its effects on reinstatement induced by stimuli conditioned to sucrose. To verify whether bifeprunox interferes with the primary reinforcing properties of either drug or sucrose, we compared its effects on nicotine self‐administration and on sucrose‐reinforced behaviour. Different groups of experimentally naïve, food‐restricted Wistar rats were trained to associate a discriminative stimulus with response‐contingent availability of nicotine or sucrose and tested for reinstatement after extinction of nicotine or sucrose‐reinforced behaviour. Bifeprunox (4–16 µg/kg, s.c.) dose‐dependently attenuated the response‐reinstating effects of nicotine‐associated cues. Higher doses (64–250 µg/kg, s.c.) reduced spontaneous locomotor activity and suppressed operant responding induced by sucrose‐associated cues and by the primary reinforcing properties of nicotine or sucrose. Provided they can be extrapolated to abstinent human addicts, these results suggest the potential therapeutic use of partial DA D2 receptor agonist to prevent cue‐controlled nicotine‐seeking and relapse. The profile of action of high doses of bifeprunox remains to be examined for potential sedation or anhedonia effects.

Short-term abstinence from cocaine self-administration, but not passive cocaine infusion, elevates αCaMKII autophosphorylation in the rat nucleus accumbens and medial prefrontal cortex

Increases in alpha calcium/calmodulin-dependent protein kinase type II (αCaMKII) activity in the nucleus accumbens shell has been proposed as a core component in the motivation to self-administer cocaine and in priming-induced drug-seeking. Since cocaine withdrawal promotes drug-seeking, we hypothesized that abstinence from cocaine self-administration should enhance αCaMKII as well. We found that short-term abstinence from contingent, but not non-contingent, cocaine i.v. self-administration (2 h/d for 14 d; 0.25 mg/0.1 ml, 6 s infusion) elevates αCaMKII autophosphorylation, but not the kinase expression, in a dynamic, time- and brain region-dependent manner. Increased αCaMKII autophosphorylation in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC), but not dorsolateral striatum (dlS), was found 24 h, but not immediately, after the last cocaine self-administration session. Notably, in the mPFC, but not NAc and dlS, αCaMKII autophosphorylation was still enhanced 7 d later. The persistent enhancement in the mPFC of abstinent rats may represent a previously unappreciated contribution to initial incubation of cocaine-seeking.

Gamma-hydroxybutyrate does not maintain self-administration but induces conditioned place preference when injected in the ventral tegmental area

Gamma-hydroxybutyric acid (GHB) is an endogenous brain substance that has diverse neuropharmacological actions, including rewarding properties in different animal species and in humans. As other drugs of abuse, GHB affects the firing of ventral tegmental neurons (VTA) in anaesthetized animals and hyperpolarizes dopaminergic neurons in VTA slices. However, no direct behavioural data on the effects of GHB applied in the VTA or in the target regions of its dopaminergic neurons, e.g. the nucleus accumbens (NAc), are available. Here, we investigated the effects of various doses of intravenous GHB in maintaining self-administration (from 0.001 to 10 mg/kg per infusion), and its ability to induce conditioned place preference (CPP) in rats when given orally (175-350 mg/kg) or injected directly either in the VTA or NAc (from 10 to 300 microg/0.5 microl per side). Our results indicate that while only 0.01 mg/kg per infusion GHB maintained self-administration, although not on every test day, 350 mg/kg GHB given orally induced CPP. CPP was also observed when GHB was injected in the VTA (30-100 microg/0.5 microl per side) but not in the NAc. Together with recent in-vitro findings, these results suggest that the rewarding properties of GHB mainly occur via disinhibition of VTA dopaminergic neurons.

mGluR2/3 mediates short-term control of nicotine-seeking by acute systemic N-acetylcysteine

Chronic self‐administration of nicotine induces maladaptive changes in the cortico‐accumbal glutamate (Glu) network. Consequently, re‐exposure to nicotine‐associated cues raises extracellular Glu in the nucleus accumbens reinstating drug‐seeking. Restoring basal concentrations of extracellular Glu, thereby increasing tonic activation of the presynaptic group II metabotropic Glu receptors (mGluR2/3) with N‐acetylcysteine (N‐AC), might offer a valid therapeutic approach for maintaining smoking abstinence. Although N‐AC modulates nicotine‐seeking behavior by drug‐associated stimuli in abstinent rats, it is still unclear whether it occurs through activation of mGluR2/3. Male Wistar rats were trained to associate discriminative stimuli (SDs) with the availability of intravenous nicotine (0.03 mg/kg/65 µl/2‐second/infusion) or oral saccharin (100 µl of 50 mg/l) self‐administration versus non‐reward. Reinforced response was followed by a cue signaling 20‐second time‐out (CSs). Once the training criterion was met, rats underwent lever press extinction, without reinforcers, SDs and CSs. Re‐exposure to nicotine or saccharin SD+/CS+, but not non‐reward SD−/CS−, revived responding on the previously reinforced lever. Acute N‐AC, 100 but not 60 or 30 mg/kg i.p., reduced cue‐induced nicotine‐seeking. N‐AC 100 mg/kg did not modify cue‐induced saccharin‐seeking behavior or influenced locomotor activity. Blocking mGluR2/3 with the selective antagonist LY341495, 1 mg/kg i.p., completely prevented the antirelapse activity of N‐AC. The finding that N‐AC prevents cue‐induced nicotine‐seeking by stimulating mGluR2/3 might indicate a therapeutic opportunity for acute cue‐controlled nicotine‐seeking. Future studies could evaluate the persistent effects of chronic N‐AC in promoting enduring suppression of nicotine‐cue conditioned responding.

The effect of exposure to low frequency electromagnetic fields (EMF) as an integral part of the housing system on anxiety-related behaviour, cognition and welfare in two strains of laboratory mouse

Electromagnetic field (EMF) technology has the potential to improve scientific data capture and welfare assessment by allowing automated data collection from individual cages. However, it is important to determine any impact that a new technology itself may have on animal welfare, and previous studies have found contrasting results of EMF on laboratory rodent anxiety-like behaviour and cognition. We therefore investigated whether there was an effect of low frequency EMF experienced continuously over a six-week period, as an integral part of the animal housing system, on measures of mouse anxiety-related behaviour, cognition and welfare. We housed mice (N = 80) of two strains (BALB/cAnNCrl and C57BL/6NCrl) separately in Individually Ventilated Cages (IVCs) in groups of four, either with the EMF plate turned ‘on’ or ‘off’ (n = 5). Some measures, e.g. food and water utilisation, were collected at regular intervals, whereas measures of anxiety-like behaviour (e.g. open field test) and cognitive performance (novel-object recognition test) were collected only at the end of the study. We found expected strong strain differences in most measures, e.g. latency to leave the starting square in an open field test, with C57BL/6NCrl mice moving away sooner, and interactions between strain and time for those measures recorded at more than one time point, e.g. significant weight gain over time for both strains, but with BALB/cAnNCrl mice weighing more. However, we found no significant effects of treatment (EMF ‘on’/‘off’) for any of the measures collected. These results indicate that, for the measures recorded here, there was no measurable impact on the behaviour and welfare of low frequency EMF exposure experienced continuously over a six-week period. Housing systems that include EMF monitoring technology may therefore be suitable for use without influencing either animal welfare or scientific outcomes.

Brain disposition, metabolism and behavioral effects of the synthetic opioid AH-7921 in rats

ABSTRACT 3,4‐Dichloro‐N‐benzamide (AH‐7921) is a cyclohexyl‐methylbenzamide derivative with analgesic activity, whose abuse was associated with several fatal intoxications, included in Schedule I of UN Single Convention on Narcotic Drugs. We validated an HPLC‐MS/MS method to investigate its brain disposition and metabolism after single and repeated injections; in parallel, we evaluated its central behavioral effects. After an intraperitoneal injection of 10 mg/kg, the analgesic effect appeared after 5 min and persisted up to 4 h; brain absorption was rapid (tmax 30 min) and large (brain‐to‐plasma ratio 16), with active concentration >700 ng/g. By high‐resolution MS we identified several metabolites in plasma and brain, the most important being N‐demethylated and N,N‐didemethylated metabolites; they showed high brain permeability, although they probably do not contribute to the analgesic effect of the parent compound (brain tmax>2 h). Starting 2 h after treatment, the two metabolites showed higher plasma and brain concentrations than the parent molecule, which persisted much longer, and could be used to evaluate drug intake in human consumers. Tolerance was observed after seven daily doses, when the compounds analgesic effect was 14% lower than after the first dose; since brain concentrations did not decrease in parallel, the development of pharmacodynamic tolerance can be suggested. However, pharmacokinetic tolerance is also likely, as brought to light by the data after a dose challenge, given after a 48 h washout period from the 7th dose, showing a lower brain‐to‐plasma ratio. We also describe the rewarding effect of AH‐7921 (conditioned place preference), suggesting a high risk of addiction in humans. HighlightsMethod to measure the narcotic drug AH‐7921 and its metabolites in plasma and brain.The analgesic effect depends on brain levels of AH‐7921 but not of metabolites.Main metabolites are slowly cleared and can be used to assess drug intake in humans.Repeated treatment results in both PD and PK tolerance to the analgesic affect.AH‐7921 has positive motivational properties.

Brain disposition of cis - para -methyl-4-methylaminorex ( cis -4,4'-DMAR) and its potential metabolites after acute and chronic treatment in rats: correlation with central behavioral effects

para-Methyl-4-methylaminorex (4,4′-DMAR) is a phenethylamine derivative with psychostimulant activity whose abuse has been associated with several deaths and a wide range of adverse effects. We recently validated a high-performance liquid chromatography—tandem mass spectrometry method to measure the compound’s concentrations in plasma, and we applied it to describe the pharmacokinetic properties of 4,4′-DMAR after a single dose in rats. In this study, we investigated the brain disposition and metabolism of cis-4,4′-DMAR after intraperitoneal injection as well as its central behavioral effects. Locomotor activity increased after a single injection of 10 mg/kg, peaking at 2 hours and disappearing at 5 hours; in these conditions, brain absorption was very rapid, (tmax = 30–60 minutes) and large (brain-to-plasma ratio = 24); the half-life was approximately 50 minutes. After 14 daily doses, the compound’s effect on locomotor activity was greater (approximately 20% compared with the effect after the first dose), but not for pharmacokinetic reasons. Using high-resolution mass spectrometry, we also identified four metabolites of cis-4,4′-DMAR in the plasma and brain of treated rats. Semiquantitative analysis indicated low brain permeability and very low brain concentrations, suggesting that these metabolites do not contribute to central behavioral effects; however, the metabolite originating from oxidation of the para-methyl group (M2) persisted in the plasma longer and at higher concentrations than the parent molecule and could be used to evaluate drug intake in human consumers. Finally, we describe the rewarding effect of cis-4,4′-DMAR in the conditioning place preference test, suggesting a high risk of addiction in humans.

Abstinence from cocaine-self-administration activates the nELAV/GAP -43 pathway in the hippocampus: A stress-related effect?

We previously demonstrated that nELAV/GAP‐43 pathway is pivotal for learning and its hippocampal expression is up‐regulated by acute stress following repeated cocaine administration. We therefore hypothesized that abstinence‐induced stress may sustain nELAV/GAP‐43 pathway during early abstinence following 2 weeks of cocaine self‐administration. We found that contingent, but not non‐contingent, cocaine exposure selectively increases hippocampal nELAV, but not GAP‐43, expression immediately after the last self‐administration session, an effect that wanes after 24 h and that comes back 7 days later when nELAV activation becomes associated with increased expression of GAP‐43, an effect again observed only in animals self‐administering the psychostimulant. Such effect is specific for nELAV since the ubiquitous ELAV/HuR is unchanged. This nELAV profile suggests that its initial transient alteration is perhaps related to the daily administration of cocaine, while the increase in the nELAV/GAP‐43 pathway following a week of abstinence may reflect the activation of this cascade as a target of stressful conditions associated with drug‐related memories.