Michela Rosas

Effects of L-cysteine on reinstatement of ethanol-seeking behavior and on reinstatement-elicited extracellular signal-regulated kinase phosphorylation in the rat nucleus accumbens shell.

BACKGROUND Alcoholism is a neuroadaptive disorder, and the understanding of the mechanisms of the high rates of relapse, which characterize it, represents one of the most demanding challenges in alcoholism and addiction research. The extracellular signal-regulated kinase (ERK) is an intracellular kinase, critical for neuroplasticity in the adult brain that is suggested to play a fundamental role in the molecular mechanisms underlying drug addiction and relapse. We previously observed that a nonessential amino acid, L-cysteine, significantly decreases oral ethanol (EtOH) self-administration, reinstatement of EtOH-drinking behavior, and EtOH self-administration break point. METHODS Here, we tested whether L-cysteine can affect the ability of EtOH priming to induce reinstatement of EtOH-seeking behavior. In addition, we determined the ability of EtOH priming to induce ERK phosphorylation as well as the ability of L-cysteine to affect reinstatement-elicited ERK activation. To these purposes, Wistar rats were trained to nose-poke for a 10% v/v EtOH solution. After stable drug-taking behavior was obtained, nose-poking for EtOH was extinguished, and reinstatement of drug seeking, as well as reinstatement-elicited pERK, was determined after an oral, noncontingent, priming of EtOH (0.08 g/kg). Rats were pretreated with either saline or L-cysteine (80 to 120 mg/kg) 30 minutes before testing for reinstatement. RESULTS The findings of this study confirm that the noncontingent delivery of a nonpharmacologically active dose of EtOH to rats, whose previous self-administration behavior had been extinguished, results in significant reinstatement into EtOH-seeking behavior. In addition, the results indicate that reinstatement selectively activates ERK phosphorylation in the shell of the nucleus accumbens (Acb) and that pretreatment with L-cysteine reduces either reinstatement of EtOH seeking and reinstatement-elicited pERK in the AcbSh. CONCLUSIONS Altogether, these results indicate that L-cysteine could be an effective pharmacological agent for the prevention of behavioral and molecular correlates of EtOH-primed reinstatement of EtOH seeking and that the shell of the Acb represents a critical neural substrate for priming-elicited reinstatement mechanisms involving ERK phosphorylation.

Acquisition and expression of conditioned taste aversion differentially affects extracellular signal regulated kinase and glutamate receptor phosphorylation in rat prefrontal cortex and nucleus accumbens

Conditioned taste aversion (CTA) can be applied to study associative learning and its relevant underpinning molecular mechanisms in discrete brain regions. The present study examined, by immunohistochemistry and immunocytochemistry, the effects of acquisition and expression of lithium-induced CTA on activated Extracellular signal Regulated Kinase (p-ERK) in the prefrontal cortex (PFCx) and nucleus accumbens (Acb) of male Sprague-Dawley rats. The study also examined, by immunoblotting, whether acquisition and expression of lithium-induced CTA resulted in modified levels of phosphorylation of glutamate receptor subunits (NR1 and GluR1) and Thr34- and Thr75-Dopamine-and-cAMP-Regulated PhosphoProtein (DARPP-32). CTA acquisition was associated with an increase of p-ERK-positive neurons and phosphorylated NR1 receptor subunit (p-NR1) in the PFCx, whereas p-GluR1, p-Thr34- and p-Thr75-DARPP-32 levels were not changed in this brain region. CTA expression increased the number of p-ERK-positive neurons in the shell (AcbSh) and core (AcbC) but left unmodified p-NR1, p-GluR1, p-Thr34- and p-Thr75-DARPP-32 levels. Furthermore, post-embedding immunogold quantitative analysis in AcbSh revealed that CTA expression significantly increased nuclear p-ERK immunostaining as well as p-ERK-labeled axo-spinous contacts. Overall, these results indicate that ERK and NR1, but not GluR1 and DARPP-32, are differentially phosphorylated as a consequence of acquisition and expression of aversive associative learning. Moreover, these results confirm that CTA represents an useful approach to study the molecular basis of associative learning in rats and suggest the involvement of ERK cascade in learning-associated synaptic plasticity.

From Ethanol to Salsolinol: Role of Ethanol Metabolites in the Effects of Ethanol

In spite of the global reputation of ethanol as the psychopharmacologically active ingredient of alcoholic drinks, the neurobiological basis of the central effects of ethanol still presents some dark sides due to a number of unanswered questions related to both its precise mechanism of action and its metabolism. Accordingly, ethanol represents the interesting example of a compound whose actions cannot be explained as simply due to the involvement of a single receptor/neurotransmitter, a scenario further complicated by the robust evidence that two main metabolites, acetaldehyde and salsolinol, exert many effects similar to those of their parent compound. The present review recapitulates, in a perspective manner, the major and most recent advances that in the last decades boosted a significant growth in the understanding on the role of ethanol metabolism, in particular, in the neurobiological basis of its central effects.

Mystic Acetaldehyde: The Never-Ending Story on Alcoholism

After decades of uncertainties and drawbacks, the study on the role and significance of acetaldehyde in the effects of ethanol seemed to have found its main paths. Accordingly, the effects of acetaldehyde, after its systemic or central administration and as obtained following ethanol metabolism, looked as they were extensively characterized. However, almost 5 years after this research appeared at its highest momentum, the investigations on this topic have been revitalized on at least three main directions: (1) the role and the behavioral significance of acetaldehyde in different phases of ethanol self-administration and in voluntary ethanol consumption; (2) the distinction, in the central effects of ethanol, between those arising from its non-metabolized fraction and those attributable to ethanol-derived acetaldehyde; and (3) the role of the acetaldehyde-dopamine condensation product, salsolinol. The present review article aims at presenting and discussing prospectively the most recent data accumulated following these three research pathways on this never-ending story in order to offer the most up-to-date synoptic critical view on such still unresolved and exciting topic.

Differential effects of cocaine on extracellular signal‐regulated kinase phosphorylation in nuclei of the extended amygdala and prefrontal cortex of psychogenetically selected roman high‐ and low‐avoidance rats

Roman high (RHA)‐ and low (RLA)‐avoidance rats are selectively bred for rapid vs. poor acquisition of active avoidance, respectively, and differ markedly in emotional reactivity, coping style, and behavioral and neurochemical responses to morphine and psychostimulants. Accordingly, acute cocaine induces more robust increments in locomotion and dopamine output in the nucleus accumbens shell (AcbSh) of RHA than of RLA rats. Cocaine induces short‐ and long‐term neuronal plasticity via activation of the extracellular signal‐regulated kinase (ERK) pathway. This study compares the effects of acute cocaine on ERK phosphorylation (pERK) in limbic brain areas of Roman rats. In RHA but not RLA rats, cocaine (5 mg/kg) increased pERK in the infralimbic prefrontal cortex and AcbSh, two areas involved in its acute effects, but did not modify pERK in the prelimbic prefrontal cortex and Acb core, which mediate the chronic effects of cocaine. Moreover, cocaine failed to affect pERK immunolabeling in the bed nucleus of stria terminalis pars lateralis and central amygdala of either line but increased it in the basolateral amygdala of RLA rats. These results extend to pERK expression previous findings on the greater sensitivity to acute cocaine of RHA vs. RLA rats and confirm the notion that genetic factors influence the differential responses of the Roman lines to addictive drugs. Moreover, they support the view that the Roman lines are a useful tool to investigate the molecular underpinnings of individual vulnerability to drug addiction.

Differential sensitivity of ethanol-elicited ERK phosphorylation in nucleus accumbens of Sardinian alcohol-preferring and -non preferring rats

Sardinian alcohol-preferring (sP) and -non preferring (sNP) rats have been selectively bred for opposite ethanol preference and consumption; sP rats represent a validated experimental tool to model several aspects of excessive ethanol drinking in humans. Phosphorylated Extracellular signal-Regulated Kinase (pERK) in dopamine-rich terminal areas plays a critical role in several psychopharmacological effects of addictive drugs, including ethanol. This study was aimed at investigating whether ethanol-elicited ERK activation may differ in key brain areas of ethanol-naïve sP and sNP rats. To this end, the effects of ethanol (0, 0.5, 1, and 2 g/kg, administered intra-gastrically [i.g.]) on ERK phosphorylation were assessed by pERK immunohistochemistry in the shell (AcbSh) and core (AcbC) of the nucleus accumbens (Acb) as well as in the prelimbic (PrL) and infralimbic (IL) prefrontal cortex (PFCx), in the bed nucleus of stria terminalis (BSTL) and in the central nucleus of the amygdala (CeA). Ethanol (1 g/kg) significantly increased pERK immunoreactivity in AcbSh and AcbC of sP but not sNP rats. Conversely, ethanol failed to affect pERK expression in PrL and IL PFCx as well as in BSTL and CeA of both sP and sNP rats. These results suggest that selective breeding of these rat lines results in differential effects of acute ethanol on ERK phosphorylation in brain regions critical for the psychopharmacological effects of ethanol.

Role of nucleus accumbens μ opioid receptors in the effects of morphine on ERK1/2 phosphorylation

RationaleDespite the critical role attributed to phosphorylated extracellular signal regulated kinase (pERK1/2) in the nucleus accumbens (Acb) in the actions of addictive drugs, the effects of morphine on ERK1/2 phosphorylation in this area are still controversial.ObjectivesIn order to investigate further this issue, we studied (1) the ability of morphine to affect ERK1/2 phosphorylation in the shell (AcbSh) and core (AcbC) of Sprague-Dawley and Wistar rats and of CD-1 and C57BL/6J mice and (2) the role of dopamine D1 and μ-opioid receptors in Sprague-Dawley rats and CD-1 mice.MethodsThe pERK1/2 expression was assessed by immunohistochemistry.ResultsIn rats, morphine decreased AcbSh and AcbC pERK1/2 expression, whereas in mice, increased it preferentially in the AcbSh compared with the AcbC. Systemic SCH 39166 decreased pERK1/2 expression on its own in the AcbSh and AcbC of Sprague-Dawley rats and CD-1 mice; furthermore, in rats, SCH 39166 disclosed the ability of morphine to stimulate pERK1/2 expression. Systemic (rats and mice) and intra-Acb (rats) naltrexone prevented both decreases, in rats, and increases, in mice.ConclusionsThese findings confirm the differential effects of morphine in rats and mice Acb and that D1 receptors exert a facilitatory role on ERK1/2 phosphorylation; furthermore, they indicate that, in rats, removal of the D1-dependent pERK1/2 expression discloses the stimulatory influence of morphine on ERK1/2 phosphorylation and that the morphine’s ability to decrease pERK1/2 expression is mediated by Acb μ-opioid receptors. Future experiments may disentangle the psychopharmacological significance of the effects of morphine on pERK1/2 in the Acb.

Effects of Withania somnifera on oral ethanol self-administration in rats.

Recent evidence has shown that Withania somnifera Dunal (Ashwagandha or Indian ginseng), a herbal remedy used in traditional medicine, impairs morphine-elicited place conditioning. Here, we investigated the effect of W. somnifera roots extract (WSE) on motivation for drinking ethanol using operant self-administration paradigms. Wistar rats were trained to self-administer ethanol (10%) by nose-poking. The effects of WSE (25–75 mg/kg) were evaluated on acquisition and maintenance, on ethanol breakpoint under a progressive-ratio schedule of reinforcement and on the deprivation effect and reinstatement of seeking behaviours. Moreover, on the basis of the recent suggestion of an involvement of GABAB receptors in WSE central effects, we studied the interaction between WSE and GABAB ligands. The effect of WSE on saccharin (0.05%) oral self-administration was also tested. The results show that WSE reduced the acquisition, maintenance and breakpoint of ethanol self-administration. WSE also reduced the deprivation effect, reinstatement of ethanol-seeking behaviours and saccharin reinforcement. Furthermore, the GABAB receptor antagonist, phaclofen, counteracted the ability of WSE to impair the maintenance of ethanol self-administration. These findings show that WSE, by an action that may involve GABAB receptors, impairs motivation for drinking ethanol and suggest that further investigations should be performed to determine whether W. somnifera may represent a new approach for the management of alcohol abuse.

Withania somnifera Dunal (Indian ginseng) impairs acquisition and expression of ethanol-elicited conditioned place preference and conditioned place aversion

Withania somnifera Dunal (Indian Ginseng) has recently been shown to impair ethanol self-administration. In order to gain further insights on the ability of the Withania somnifera standardised root extract (WSE) to affect the motivational properties of ethanol, this study investigated whether WSE may also affect ethanol (2 g/kg)-elicited conditioned place preference (CPP) and aversion (CPA). To this end male CD-1 mice were conditioned under two distinct schedules: in backward conditioning experiments ethanol was administered before mice were placed in the conditioning apparatus (CPP) while, in forward conditioning experiments, ethanol was administered immediately after removing mice from the apparatus (CPA). Following these schedules, mice developed significant CPP and CPA, respectively. Administration of WSE significantly impaired both the acquisition (50 and 100 mg/kg) and the expression (50 mg/kg) of CPP and CPA without affecting spatial memory (50 mg/kg), as determined by a two-trial memory recognition task. Overall, the study highlights the ability of WSE to interfere with both positive and negative motivational properties of ethanol and suggests that the effects of WSE may target both ethanol’s motivational properties and underpinning associative learning mechanisms. In conclusion, these results cast new light on Withania somnifera as an agent potentially useful to counteract distinct aspects of ethanol effects.

Differential effects of the MEK inhibitor SL327 on the acquisition and expression of ethanol-elicited conditioned place preference and aversion in mice

The involvement of mitogen-activating extracellular kinase (MEK) in place conditioning may vary depending on the motivational sign (positive or negative) and nature (pharmacological or nociceptive) of the unconditioned stimulus (US) and on the phase (acquisition or expression) of the learning process. This study investigated the role of MEK on the acquisition and expression of ethanol-elicited (given 2 g/kg) backward (preference, CPP) and forward (aversion, CPA) place conditioning. The MEK inhibitor SL327 (50 mg/kg for CPP, and 50 and 100 mg/kg for CPA) was administered to CD-1 mice 60 minutes before an ethanol dose (acquisition) or 60 minutes before the post-conditioning tests (expression). Ethanol significantly elicited CPP and CPA; SL327 (50 mg/kg) significantly blocked the acquisition of ethanol-elicited CPP, but not that of CPA. Moreover, SL327 (50 and 100 mg/kg) significantly reduced the expression of ethanol-elicited CPP, but not that of CPA. Finally, SL327 also prevented ethanol-elicited (given 2 g/kg) increases of phosphorylated extracellular signal regulated kinase (pERK)-positive neurons in the nucleus accumbens and other nuclei of the extended amygdala. Overall, these results confirmed the differential involvement of MEK in the acquisition and expression of drug-elicited place conditioning and suggested its differential involvement in distinct behavioral outcomes, depending on the motivational sign of the (same) US and on the significance of the experimental phase of the learning process.