Paola Devoto

Cannabis is the most common illicit drug abused worldwide, and its consumption has substantially increased among pregnant women. We previously demonstrated that male preadolescent offspring prenatally exposed to Δ ⁹ -tetrahydrocannabinol (THC), a model of prenatal cannabinoid exposure (PCE), exhibit a mesolimbic dopamine (DA) neuron dysfunction contributing to at-risk psychotic-like (endo)phenotypes that are unmasked by acute THC exposure at preadolescence. Dysregulation of mesocortical DA signaling along with prefrontal cortex (PFC) function is also a central feature of psychotic disorders. Furthermore, studies investigating the impact of PCE on PFC in the offspring at preadolescence, a window of heightened plasticity and vulnerability, are limited. To fill this gap, we applied a multiscale analysis of mesocortical DA transmission and PFC function in PCE preadolescent offspring by integrating behavioral, neurochemical, electrophysiological, and molecular approaches. PCE enhanced spontaneous repetitive behaviors in a male-specific manner. PCE also abolished sex differences in the intrinsic excitability of PFC pyramidal neurons and Netrin-1 expression. In addition, PCE altered the expression of genes associated with endocannabinoid signaling without changing basal and THC-induced extracellular levels of DA in the PFC. Collectively, these findings demonstrate that prenatal THC exposure disrupts both proper maturation and sexual differentiation of PFC circuitry, thus extending the impact of PCE from previously described mesolimbic abnormalities to mesocortical pathway. Finally, our data identify early cortical molecular and cellular alterations that may contribute to neuropsychiatric vulnerability later in life. Highlights: • Preadolescent male rats exposed in utero to THC display repetitive behavior • Prenatal cannabinoid exposure (PCE) does not alter dopamine transmission in the PFC • PCE potentiates AMPA-mediated transmission in male pyramidal cells • PCE abolishes sex differences in Netrin-1 expression levels in the PFC

Experimental and clinical evidence indicates a deficit of release and function of dopamine in schizophrenia and suggests that α₂-adrenoceptor antagonists rescue dopamine deficit and improve the antipsychotic efficacy of D₂-receptor antagonists. In anesthetized male rats, we investigated how the blockade of α₂- and D₂-receptors by atipamezole and raclopride, respectively, modified the firing of noradrenergic neurons in the locus coeruleus (LC) and dopaminergic neurons in the ventral tegmental area (VTA). In freely moving rats, we studied how atipamezole and raclopride modified extracellular noradrenaline, dopamine, and DOPAC levels in the medial prefrontal cortex (mPFC) through microdialysis. When administered alone, atipamezole activated LC noradrenaline but not VTA dopamine cell firing. Combined with raclopride, atipamezole activated dopamine cell firing above the level produced by raclopride. Atipamezole increased extracellular dopamine to the same level, whether administered alone or combined with raclopride. In the presence of the noradrenaline transporter (NET) inhibitor, atipamezole combined with raclopride increased extracellular dopamine beyond the level produced by either compound administered alone. The results suggest that a) the D₂-autoreceptor blockade is required for LC noradrenaline to activate VTA cell firing; b) the level of dopamine released from dopaminergic terminals is determined by NET; c) the elevation of extracellular dopamine levels in the mPFC is the resultant of dopamine uptake and release from noradrenergic terminals, independent of dopaminergic cell firing and release; and d) LC noradrenergic neurons are an important target for treatments to improve the prefrontal deficit of dopamine in neuropsychiatric pathologies.

Cannabis use among pregnant women is increasing worldwide along with permissive sociocultural attitudes toward it. Prenatal cannabis exposure (PCE), however, is associated with adverse outcome among offspring, ranging from reduced birth weight to child psychopathology. We have previously shown that male rat offspring prenatally exposed to Δ9-tetrahydrocannabinol (THC), a rat model of PCE, exhibit extensive molecular, cellular, and synaptic changes in dopamine neurons of the ventral tegmental area (VTA), resulting in a susceptible mesolimbic dopamine system associated with a psychotic-like endophenotype. This phenotype only reveals itself upon a single exposure to THC in males but not females. Here, we characterized the impact of PCE on female behaviors and mesolimbic dopamine system function by combining in vivo single-unit extracellular recordings in anesthetized animals and ex vivo patch clamp recordings, along with neurochemical and behavioral analyses. We find that PCE female offspring do not show any spontaneous or THC-induced behavioral disease-relevant phenotypes. The THC-induced increase in dopamine levels in nucleus accumbens was reduced in PCE female offspring, even when VTA dopamine activity in vivo and ex vivo did not differ compared to control. These findings indicate that PCE impacts mesolimbic dopamine function and its related behavioral domains in a sex-dependent manner and warrant further investigations to decipher the mechanisms determining this sex-related protective effect from intrauterine THC exposure.

Previous results indicate that dopamine (DA) release in the medial prefrontal cortex (mPFC) is modified by α₂ adrenoceptor- but not D2 DA receptor- agonists and antagonists, suggesting that DA measured by microdialysis in the mPFC originates from noradrenergic terminals. Accordingly, noradrenergic denervation was found to prevent α₂-receptor-mediated rise and fall of extracellular DA induced by atipamezole and clonidine, respectively, in the mPFC. The present study was aimed to determine whether DA released by dopaminergic terminals in the mPFC is not detected by in vivo microdialysis because is readily taken up by norepinephrine transporter (NET). Accordingly, the D2-antagonist raclopride increased the electrical activity of DA neurons in the ventral tegmental area (VTA) and enhanced extracellular DOPAC but failed to modify DA in the mPFC. However, in rats whose NET was either inactivated by nisoxetine or eliminated by noradrenergic denervation, raclopride still elevated extracellular DOPAC and activated dopaminergic activity, but also increased DA. Conversely, the D2-receptor agonist quinpirole reduced DOPAC but failed to modify DA in the mPFC in control rats. However, in rats whose NET was eliminated by noradrenergic denervation or inhibited by locally perfused nisoxetine, quinpirole maintained its ability to reduce DOPAC but acquired that of reducing DA. Moreover, raclopride and quinpirole, when locally perfused into the mPFC of rats subjected to noradrenergic denervation, were able to increase and decrease, respectively, extracellular DA levels, while being ineffective in control rats. Transient inactivation of noradrenergic neurons by clonidine infusion into the locus coeruleus, a condition where NET is preserved, was found to reduce extracellular NE and DA in the mPFC, whereas noradrenergic denervation, a condition where NET is eliminated, almost totally depleted extracellular NE but increased DA. Both transient inactivation and denervation of noradrenergic neurons were found to reduce the number of spontaneously active DA neurons and their bursting activity in the VTA. The results indicate that DA released in the mPFC by dopaminergic terminals is not detected by microdialysis unless DA clearance from extracellular space is inactivated. They support the hypothesis that noradrenergic terminals are the main source of DA measured by microdialysis in the mPFC during physiologically relevant activities.

Dopamine agonists have been proposed as therapeutic tools for cocaine addiction. We have recently demonstrated that indirect dopamine agonists, including levodopa (L‐DOPA), markedly increase cocaine‐induced dopamine release in the medial prefrontal cortex (mPFC) of rats leading to the suppression of cocaine‐seeking behavior. This study was aimed to understand the behavioral and neurochemical effects of L‐DOPA on cocaine‐taking and cocaine‐seeking in rats. After reaching a stable pattern of intravenous cocaine self‐administration under a continuous fixed ratio (FR‐1) schedule of reinforcement, male rats were treated with L‐DOPA at different steps of the self‐administration protocol. We found that L‐DOPA reduced cocaine self‐administration under FR‐1 schedule of reinforcement and decreased the breaking points and the amount of cocaine self‐administered under the progressive ratio schedule of reinforcement. Levodopa also decreased cocaine‐seeking behavior both in a saline substitution test and in the cue priming‐induced reinstatement test, without affecting general motor activity. Importantly, L‐DOPA greatly potentiated cocaine‐induced dopamine release in the mPFC of self‐administering rats while reducing their cocaine intake. In the same brain area, L‐DOPA also increased dopamine levels during cue priming‐induced reinstatement of cocaine‐seeking behavior. The potentiating effect was also evident in the mPFC but not nucleus accumbens core of drug‐naïve rats passively administered with cocaine. Altogether, these findings demonstrate that L‐DOPA efficaciously reduces the reinforcing and motivational effects of cocaine likely potentiating dopamine transmission in the mPFC. Its ability to prevent cue priming‐induced reinstatement of cocaine‐seeking suggests that it might be effective in reducing the risk to relapse to cocaine in abstinent patients.

Introduction: Disulfiram has been claimed to be useful in cocaine addiction therapy, its efficacy being attributed to dopamine‐beta‐hydroxylase (DBH) inhibition. Our previous results indicate that disulfiram and the selectiveDBHinhibitor nepicastat increase extracellular dopamine (DA) in the rat medial prefrontal cortex (mPFC), and markedly potentiated cocaine‐induced increase. Concomitantly, in rats with cocaine self‐administration history, cocaine‐seeking behavior induced by drug priming was prevented, probably through overstimulation of D1 receptors due to theDAincrease. The present research was aimed at studying the neurochemical mechanisms originating the enhancedDArelease.Methods: Noradrenergic system ablation was attained by intracerebroventricular (i.c.v.) administration of the neurotoxin anti‐DBH‐saporin (aDBH‐sap).DA, noradrenaline (NA), andDOPACwere assessed byHPLCafter ex vivo tissue extraction or in vivo microdialysis. Control and denervated rats were subjected to microdialysis in themPFCand caudate nucleus to evaluate the effect of nepicastat–cocaine combination on extracellularDAlevels and their regulation by α₂‐adrenoceptors.Results: Fifteen days after neurotoxin or its vehicle administration, tissue and extracellularNAwere reduced to less than 2% the control value, while extracellularDAwas increased by approximately 100%. In control rats, nepicastat given alone and in combination with cocaine increased extracellularDAby about 250% and 1100%, respectively. In denervated rats, nepicastat slightly affected extracellularDA, while in combination with cocaine increased extracellularDAby 250%. No differences were found in the caudate nucleus. Clonidine almost totally reversed the extracellularDAelevation produced by nepicastat–cocaine combination, while it was ineffective in denervated rats.Conclusions: This research shows that the increase of extracellularDAproduced by nepicastat alone or in combination with cocaine was prevented by noradrenergic denervation. The results indicate that nepicastat enhancesDArelease from noradrenergic terminals supposedly by removingNAfrom α₂‐autoreceptors. In addition to the inhibition ofDAuptake, the latter mechanism may explain the synergistic effect of cocaine on nepicastat‐inducedDArelease.

Previous investigations indicate that the dopamine‐β‐hydroxylase (DBH) inhibitors disulfiram and nepicastat suppress cocaine‐primed reinstatement of cocaine self‐administration behaviour. Moreover, both inhibitors increase dopamine release in the rat medial prefrontal cortex (mPFC) and markedly potentiate cocaine‐induced dopamine release in this region. This study was aimed to clarify if the suppressant effect of DBH inhibitors on cocaine reinstatement was mediated by the high extracellular dopamine in the rat mPFC leading to a supra‐maximal stimulation of D1 receptors in the dorsal division of mPFC, an area critical for reinstatement of cocaine‐seeking behaviour. In line with previous microdialysis studies in drug‐naïve animals, both DBH inhibitors potentiated cocaine‐induced dopamine release in the mPFC, in the same animals in which they also suppressed reinstatement of cocaine seeking. Similar to the DBH inhibitors, L‐DOPA potentiated cocaine‐induced dopamine release in the mPFC and suppressed cocaine‐induced reinstatement of cocaine‐seeking behaviour. The bilateral microinfusion of the D1 receptor antagonist SCH 23390 into the dorsal mPFC not only prevented cocaine‐induced reinstatement of cocaine seeking but also reverted both disulfiram‐ and L‐DOPA‐induced suppression of reinstatement. Moreover, the bilateral microinfusion of the D1 receptor agonist chloro‐APB (SKF 82958) into the dorsal mPFC markedly attenuated cocaine‐induced reinstatement of cocaine seeking. These results suggest that stimulation of D1 receptors in the dorsal mPFC plays a crucial role in cocaine‐induced reinstatement of cocaine seeking, whereas the suppressant effect of DBH inhibitors and L‐DOPA on drug‐induced reinstatement is mediated by a supra‐maximal stimulation of D1 receptors leading to their inactivation.