Stefania Angeletti

Compulsive Drug‐Seeking Behavior and Relapse

Abstract: The development of addiction and vulnerability to relapse following withdrawal is proposed to be the result of neuroadaptive processes within the central nervous system that oppose the acute reinforcing actions of drugs of abuse. These changes lead to impairment in the mechanisms that mediate positive reinforcement and the emergence of affective changes such as anxiety, dysphoria, and depression during withdrawal. Considerable evidence exists implicating perturbations in DA and 5‐HT transmission in the nucleus accumbens—neurochemical systems that are activated by cocaine and ethanol self‐administration and deficient during withdrawal—as potential substrates for these affective changes. In addition, growing evidence suggests that enhanced CRF release in the central nucleus of the amygdala represents a mechanism underlying the anxiogenic and stress‐like consequences of withdrawal that are common to all drugs of abuse. A growing body of evidence also implicates dysregulation of the non‐neuroendocrine CRF stress system within the central nucleus of the amygdala as a common factor in the anxiogenic and aversive consequences of withdrawal from drugs of abuse. Moreover, a possible link may exist between long‐lasting abnormalities in CRF function in the CeA and vulnerability to relapse during protracted abstinence. Another presumably critical element contributing to the chronic relapsing nature of drug addiction is the learned responses to drug‐related stimuli. The long‐lasting efficacy of drug‐ and alcohol‐associated contextual stimuli in eliciting drug‐seeking behavior in animal models of relapse resembles the endurance of conditioned cue reactivity and cue‐induced cocaine craving in humans and confirms a significant role of learning factors in the long‐lasting addictive potential of cocaine. With cocaine, D1‐dependent neural mechanisms within the medial prefrontal cortex and basolateral amygdala may be important substrates for the motivating effects of drug‐related environmental stimuli. With ethanol, available data suggest a role for opioid receptors in the mediation of conditioned drug‐seeking behavior. Finally, conditioning factors (i.e., exposure to drug‐associated stimuli) and stress can interact to augment vulnerability to relapse. This finding emphasizes that it will be important to consider the simultaneous effects of multiple environmental triggers for relapse in the development of treatment and medication strategies.

Effect of nociceptin/orphanin FQ on the rewarding properties of morphine.

The present study investigated the effect of nociceptin/orphanin FQ, the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, on the rewarding properties of morphine in the place conditioning paradigm. Intracerebroventricular (i.c.v.) injections of nociceptin/orphanin FQ, 500 or 1000 (but not 250) ng/rat, abolished conditioned place preference induced by subcutaneous (s.c.) injections of morphine (3 mg/kg). These doses of nociceptin/orphanin FQ induced neither place aversion nor preference per se. The same doses did not modify the rat performance in the Morris water test, suggesting that they do not disrupt spatial learning and memory. Moreover, these doses of nociceptin/orphanin FQ did not modify the development of morphine-induced locomotor sensitization, suggesting that they do not interfere with sensitization processes to morphine. The present results confirm and extend previous reports that nociceptin/orphanin FQ is able to abolish morphine-induced conditioned place preference, and raise interest for the possible role of nociceptin/orphanin FQ and ORL1 receptors in the control of opiate abuse.

Nociceptin/orphanin FQ and drugs of abuse.

Nociceptin/orphanin FQ (NC) binds with high affinity to the opioid receptor-like1 (ORL1) receptor. NC has been reported to block opioid-induced supraspinal analgesia, and it has been proposed that it may represent a functional antiopioid peptide in the control of brain nociceptive processes. The wide distribution of NC and of its receptors in the central nervous system suggests, however, that it may be involved in the control of a variety of biologic functions. Increasing evidence indicates that it may influence the rewarding and reinforcing properties of drugs of abuse. NC has been shown to abolish the rewarding properties of ethanol and morphine in the place conditioning paradigm, to reduce ethanol consumption in alcohol-preferring rats and to inhibit stress-induced alcohol-seeking behavior. These findings suggest that drugs directed at central NC receptors may represent an interesting approach to the treatment of ethanol and opiate abuse.

Effects of Hypericum perforatum extract on ethanol intake, and on behavioral despair: a search for the neurochemical systems involved.

The present study investigated the possible involvement of sigma receptors and of serotonergic mechanisms in the effects of Hypericum perforatum extract (HPE) on immobility time in the forced swimming test (FST) and on ethanol intake in Marchigian Sardinian alcohol-preferring rats. The HPE employed was a dry extract containing 0.3% hypericin and 3.8% hyperforin. Intraperitoneal pretreatment with 20 mg/kg of the sigma receptor antagonist rimcazole (RIM), 30 min prior to HPE, completely suppressed the antiimmobility effect of HPE (3 intragastric injections of 250 mg/kg). Intracerebroventricular pretreatment with 5, 7-dihydroxytryptamine (5,7-DHT), which produced a marked depletion of brain serotonin, reduced the antiimmobility effect, although this reduction was not as pronounced as that of RIM. On the other hand, the inhibitory effect of HPE on 10% ethanol intake was modified neither by 5,7-DHT nor by RIM pretreatment. These results suggest that the antidepressant-like effect of HPE in the FST may be mediated by interaction with sigma receptors and to some extent by increased serotonergic neurotransmission. On the other hand, these mechanisms appear to be unimportant for the effect of HPE on ethanol intake.

Mechanism of action for reduction of ethanol intake in rats by the tachykinin NK-3 receptor agonist aminosenktide.

Intracerebroventricular (i.c.v.) injection of tachykinin (TK) NK-3 receptor agonists inhibits alcohol intake in genetically selected alcohol-preferring rats. The present study investigated the mechanism of action by which the selective TK NK-3 receptor agonist aminosenktide (NH2-SENK) attenuates ethanol intake in Marchigian Sardinian alcohol-preferring (msP) rats. The effect of NH2-SENK was studied by i.c.v. injection in the conditioned taste aversion (CTA) and in the conditioned place preference (CPP) paradigms; moreover, the effect of NH2-SENK on blood alcohol levels (BAL) following intragastric ethanol administration was investigated. The i.c.v. dose of 125 ng/rat of NH2-SENK, that markedly reduces ethanol intake, did not modify BAL, nor did it increase the CTA induced by intraperitoneal injection of ethanol, 1 g/kg body weight. These findings suggest that the effect of NH2-SENK on alcohol consumption is not related to modification of the pharmacokinetics of ethanol, nor to increase of the aversive properties of ethanol. On the other hand, the same i.c.v. dose of NH2-SENK evoked a pronounced and statistically significant CPP. This finding indicates that the TK NK-3 receptor agonist NH2-SENK possesses rewarding properties in msP rats and suggests that its inhibitory effect on ethanol consumption may be due to substitution of the rewarding properties of ethanol, thus making its consumption redundant.

Autoradiographic analysis of 5-HT2A binding sites in the brain of Sardinian alcohol-preferring and nonpreferring rats

The density of 5-HT2A binding sites in the brain of Sardinian alcohol-preferring (sP) and nonpreferring (sNP) rats was evaluated, using [3H]ketanserin for quantitative autoradiography. The highest [3H]ketanserin binding levels were found in the anterior olfactory nucleus, prefrontal cortex, medial prefrontal cortex, post-genual anterior cingulate cortex, insular cortex and claustrum. Statistically significant differences between sP and sNP rats were found in prefrontal cortex, medial prefrontal cortex and post-genual anterior cingulate cortex, where sP rats showed about 20% lower [3H]ketanserin binding levels. No significant difference was found in other areas, although some of them showed slightly lower [3H]ketanserin binding density in sP rats. The 5-HT2A receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane hydrochloride (DOI), microinjected into the medial prefrontal cortex, induced a lower number of wet dog shakes in sP than in sNP rats. These results indicate a different density of 5-HT2A binding sites, and a different functional regulation of 5-HT2A receptor mechanisms in discrete brain areas of sP, in comparison to sNP rats. These findings, and those showing lower levels of 5-HT in the frontal cortex of sP rats, suggest that altered 5-HT function in fronto-cortical areas could be linked to the genetic predisposition to high voluntary ethanol intake in these rats.

Conditioned Taste Aversion Induced by Ethanol in Alcohol-Preferring Rats: Influence of the Method of Ethanol Administration

A recent study of our group has shown that ethanol evokes conditioned place preference (CPP) in Marchigian Sardinian alcohol-preferring (msP) rats following intragastric (IG) administration by means of an indwelling IG catheter, but not following administration by gavage or by intraperitoneal (IP) injection. The present study evaluated in ethanol-naive msP rats the influence of the method of administration (IG injection by indwelling catheter vs. IP injection) on ethanol-induced conditioned taste aversion (CTA). The dose of 0.35 g/kg of ethanol did not evoke aversion either by IG or by IP administration. Following IG injection, 0.7 g/kg of ethanol, the amount that msP rats voluntarily ingest in a short (2-5 min) drinking episode, did not evoke CTA, and 1.5 g/kg induced a modest CTA. On the other hand, IP injection of 0.7 g/kg of ethanol evoked CTA, and 1.5 g/kg induced a very pronounced CTA. These findings show that the aversive properties of ethanol in msP rats are influenced by the method of administration, and suggest that the IG injection by catheter may reveal more faithfully than the IP injection the motivational properties of amounts of ethanol that alcohol-preferring rats voluntarily ingest.

Sensitivity of Brain Sites to the Inhibitory Effect on Alcohol Intake of the Tachykinin Aminosenktide

The present study evaluated the sensitivity of several brain sites to the inhibitory effect of the tachykinin (TK) NK-3 receptor agonist aminosenktide (NH2-SENK) on 10% ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring rats. Attention was focused on limbic structures involved in alcohol-seeking behavior and endowed with TK NK-3 receptors. NH2-SENK was bilaterally injected into the shell of the nucleus accumbens (NACC), the medial amygdala (AMY), the dorsal hippocampus (HIPP), the ventral tegmental area (VTA), the bed nucleus of the stria terminalis (BNST), the lateral hypothalamus (LH), and the nucleus basalis magnocellularis (NBM). NH2-SENK (injected up to 25-75 ng/site) into the NACC, AMY, HIPP, and VTA did not significantly modify ethanol intake. Injection of NH2-SENK into the BNST reduced ethanol intake at doses of 25 ng/site or higher, but the same doses also reduced water intake in water-deprived rats and food intake in food-deprived rats. Injection of NH2-SENK into the LH or the NBM at doses of 0.5, 5, or 25 ng/site inhibited 10% ethanol intake even at the lowest dose tested without affecting either food or water consumption in deprived animals. Present results indicate that the LH and the NBM are highly sensitive to the inhibitory effect of the TK NK-3 receptor agonist NH2-SENK on ethanol intake. TK peptides have been shown to evoke conditioned place preference following injection in the LH or the NBM, suggesting that in these brain sites the effect of TK agonists on ethanol intake might be due to interference with reward processes.

Preprotachykinin-A gene expression in the forebrain of Sardinian alcohol-preferring and -nonpreferring rats.

Increasing evidence suggests that TKergic mechanisms might play a role in ethanol intake control. Preprotachykinin-A (PPT-A) mRNA brain levels were measured in Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats. PPT-A mRNAs were about 50% lower in sP than in sNP rats in the bed nucleus of the stria terminalis (BNST), whereas levels in the olfactory tubercle (Tu) were about 30% higher in sP than in sNP rats. Our findings suggest that altered PPT-A gene expression might contribute to the different ethanol preference and intake of sP opposite to sNP rats.

Regulation of preprotachykinin-A mRNA in genetic hypertensive and normotensive rats

It is well-known that central administration of tachykinins (Tks) inhibit salt intake in rats. Recent studies have shown that conditions that arouse salt appetite, such as adrenalectomy and sodium depletion, induce a decrease in preprotachykinin-A (PPT-A) mRNA in discrete regions of the rat brain, suggesting that reduced levels of PPT-A mRNA in the brain may have a permissive role on the expression of salt appetite. It has also been shown that spontaneously hypertensive rats (SHR) show higher avidity for salty solutions than their normotensive control Wistar-Kyoto (WKY) rats. In this regard, the present study tested whether SHR and WKY rats differ in expression of the gene coding for PPT-A, the precursor for Tks peptides. Using semi-quantitative in situ hybridization histochemistry, we examined the level of PPT-A mRNA in discrete rat brain regions of SHR and WKY rats under no treatment, after 1 or 3 days of Na+ depletion. Levels of PPT-A mRNA were analysed in the olfactory tubercle (Tu), in the lateral olfactory tubercle (LOT), in the dorsal and ventral caudate putamen (d/v CPu), in the medial preoptic area (mPOA), in the bed nucleus of the stria terminalis (BNST), in the habenula (Hb) and in the postero-dorsal part of the amygdala (MePD). Semi-quantitative analysis of silver grains revealed a 27.5% lower expression of the PPT-A mRNA levels in SHR opposite to WKY rats under no treatment in v-CPu, mPOA, BNST and Hb. 1 day of Na+ depletion reduced PPT-A mRNA levels when opposite to Na+-repleted animals in Tu and mPOA in both SHR and WKY rats. On the other hand, when comparing SHR and WKY rats after 1 day of Na+ depletion, a 26% lower level of PPT-A mRNA was detected in Tu and d-CPu of SHR opposite to WKY rats whereas a 14% and an 18% lower level was detected in v-CPu and Hb, respectively. A lower expression of PPT-A mRNA in SHR compared to WKY rats was also found in BNST and MePD, although no statistical significance was detected in these two brain areas. In the last experiment, 3 days of Na+ depletion reduced PPT-A mRNA levels in mPOA while negligibly increased mRNA levels in d-CPu and v-CPu, in BNST, Hb and MePD, both in SHR and WKY rats. Conversely, when making comparisons between the two strains, a 35% lower level of PPT-A mRNA in SHR with respect to WKY rats was found after 3 days of Na+ depletion in d-CPu, v-CPu and mPOA. A lower gene expression, even though not statistically significant, was found in Tu, LOT, MePD. These findings show a consistent difference of PPT-A mRNA levels in discrete regions of the SHR brain opposite to WKY rats and confirm that 1 day of Na+ depletion reduces PPT-A mRNA in discrete brain regions. Since SHR are notoriously more salt-avid than WKY rats and Tks are potent inhibitors of sodium intake, the down-regulation of PPT-A mRNA may contribute to the higher natriophilia and, therefore, to the etiology of the hypertensive disease.