Changes in D1 but not D2 dopamine or mu-opioid receptor expression in limbic and motor structures after lateral hypothalamus electrical self-stimulation: A quantitative autoradiographic study.

Intracranial self-stimulation (ICSS) of the lateral hypothalamus (LH) is involved in the activation of neuroanatomical systems that are also associated with the processing of natural and other artificial rewarding stimuli. Specific components of this behavior (hedonic impact, learning, and motor behavior) may involve changes in different neurotransmitters, such as dopamine and opioids. In this study, quantitative autoradiography was used to examine changes in mu-opioid and D1/D2-dopamine receptor expression in various anatomical regions related to the motor and mesolimbic reward systems after intracranial self-stimulation of the LH. Results of the behavioral procedure and subsequent radiochemical assays show selective changes in D1 but not D2 or mu receptors in Accumbens-Shell, Ventral Pallidum, Caudate-Putamen, and Medial Globus Pallidus. These findings are discussed in relation to the different psychobiological components of the appetitive motivational system, identifying some dissociation among them, particularly with respect to the involvement of the D1-dopamine subsystem (but not D2 or mu receptors) in goal-directed behaviors.

Differential gene expression in the nucleus accumbens and frontal cortex of lewis and Fischer 344 rats relevant to drug addiction.

Drug addiction results from the interplay between social and biological factors. Among these, genetic variables play a major role. The use of genetically related inbred rat strains that differ in their preference for drugs of abuse is one approach of great importance to explore genetic determinants. Lewis and Fischer 344 rats have been extensively studied and it has been shown that the Lewis strain is especially vulnerable to the addictive properties of several drugs when compared with the Fischer 344 strain. Here, we have used microarrays to analyze gene expression profiles in the frontal cortex and nucleus accumbens of Lewis and Fischer 344 rats. Our results show that only a very limited group of genes were differentially expressed in Lewis rats when compared with the Fischer 344 strain. The genes that were induced in the Lewis strain were related to oxygen transport, neurotransmitter processing and fatty acid metabolism. On the contrary genes that were repressed in Lewis rats were involved in physiological functions such as drug and proton transport, oligodendrocyte survival and lipid catabolism.These data might be useful for the identification of genes which could be potential markers of the vulnerability to the addictive properties of drugs of abuse.

Chronic periadolescent cannabinoid treatment enhances adult hippocampal PSA-NCAM expression in male Wistar rats but only has marginal effects on anxiety, learning and memory.

Pubertal and adolescent exposure to cannabinoids is associated with enduring alterations in anxiety and memory. However, periadolescence virtually remains unexplored. Here, we measured anxiety in the Elevated Plus Maze (EPM) in adult Wistar rats treated at periadolescence (P28-P38) with the cannabinoid agonist CP 55,940 (CP) (0.4 mg/kg; 2 ml/kg i.p., 1 daily injection), and we also defined their recognition memory in the novel object paradigm and spatial learning and memory in the water maze. Additionally, we measured the expression of hippocampal PSA-NCAM (Polysialic Acid-Neural Cell Adhesion Molecule) and long-term potentiation (LTP) as well as, given their role in mnemonic processing, the levels of plasma corticosterone and estradiol. We found that CP had no robust effects on anxiety or in recognition memory. In the water maze, only a slight decreased percentage of failed trials in the reference memory task and an improvement in an indirect index of attention were observed. However, we detected an up-regulation of hippocampal PSA-NCAM expression, only in CP-males, although this effect was not related to changes in LTP. No hormonal alterations were evident. Based on our data, minimal long-term effects on anxiety, learning and memory appear to result from cannabinoid exposure during the periadolescent period.

Toluene and TCE decrease binding to mu-opioid receptors, but not to benzodiazepine and NMDA receptors in mouse brain.

In vitro and in vivo studies have shown that abused solvents affect different neurotransmitter systems, including the GABAergic, glutamatergic, and opioidergic. The first purpose of this study was to determine in mice whether an acute exposure to 4,000 ppm toluene or 12,000 ppm 1,1,1-trichloroethane (TCE) modifies receptor binding levels to: (a) DAMGO, a mu-opioid receptor selective agonist; (b) MK-801, a noncompetitive selective NMDA-receptor antagonist; and (c) flunitrazepam, a benzodiazepine binding site selective agonist. In addition, in separate groups of animals, nociceptive effects of toluene alone or co-administered with morphine were evaluated in the hot-plate test. Mice were exposed to toluene or TCE in static exposure chambers for 30 min, and their brains were removed 24 h later for autoradiography. Acute toluene inhalation produced a significant decrease in mu-opioid receptor binding levels in cingulate and piriform cortices, caudate putamen, thalamus, amygdala, and periaqueductal gray, whereas TCE significantly decreased mu-opioid receptor levels, but only in thalamus and periaqueductal gray. Both toluene and TCE decreased benzodiazepine receptor binding levels in discrete brain areas, but had no effect on NMDA receptor levels. In the hot-plate test, a single toluene exposure counteracted morphine antinociceptive response when the solvent exposure time was immediately followed by morphine treatment, but not when morphine was administered 24, 48, 72, and 96 h later. However, co-administration of morphine and toluene 24, 48, 72, and 96 h after the single solvent exposure resulted in morphine-induced analgesia blockade. Present results suggest that mu-opioid receptors are an important molecular target for organic solvents, and that the inhalation of these compounds may affect the analgesic properties of opioids.

Glutamate and aspartate levels in the nucleus accumbens during cocaine self-administration and extinction: a time course microdialysis study.

RATIONALE: Accumbal excitatory amino acid (EAA) transmission has been implicated in cocaine addiction. However, the time course effects of extinction of cocaine self-administration on EAAs are unknown. OBJECTIVES: The objective of this study was to define the time course of cocaine self-administration and extinction effects on glutamate and aspartate levels in the nucleus accumbens. MATERIALS AND METHODS: Rats were trained to self-administer cocaine for 20 days, and the levels of extracellular glutamate and aspartate were measured by in vivo microdialysis both during cocaine self-administration and after a priming cocaine injection at different time points after extinction (1, 5, or 10 days). A food-reinforced control group was also included in this study. Furthermore, the effect of acute i.v. cocaine administration (0, 1, 2, or 4 mg/kg) on glutamate and aspartate levels was also evaluated. RESULTS: At any of the dose tested, acute i.v. cocaine did not affect the levels of glutamate or aspartate in the Nacc. In contrast, glutamate levels were reduced in animals trained to self-administer cocaine, although they augmented substantially during a subsequent session of cocaine self-administration, and similar changes were not observed in food-reinforced controls. After 1 or 5, but not after 10 days of extinction, the glutamate levels were also reduced, and the ability of i.v. cocaine priming injections to increase glutamate levels followed a similar time course. These effects were specific, as aspartate levels were not affected by any administration protocol. CONCLUSIONS: These data suggest that glutamatergic transmission could be involved in the maintenance of cocaine self-administration and in the early phases of abstinence.

Cocaine self-administration improves performance in a highly demanding water maze task.

RATIONALE: Long-term potentiation (LTP) is considered to be a cellular substrate of learning and memory. Indeed, the involvement of LTP-like mechanisms in spatial learning has consistently been demonstrated in the Morris water maze test. We have previously shown that hippocampal LTP in Lewis rats was modulated by cocaine self-administration, although the performance of cocaine-self-administered rats in the Morris water maze was not altered. OBJECTIVE: Given that the ease of the task previously used could have masked any possible effects of the cocaine-induced LTP enhancement on spatial learning, a new and more difficult water maze task was devised to address this issue. MATERIALS AND METHODS: Animals self-administered cocaine (1 mg/kg) or saline under a fixed ratio 1 schedule of reinforcement for 22 days. Spatial learning was assessed in a difficult water maze task (four sessions, two trials per session with a 90-min intertrial interval), and spatial memory was also evaluated 48 h after training (a 90-s test). Additionally, reversal learning and perseverance were also studied. RESULTS: There was a reduced latency in finding the hidden platform during training, as well as improved memory of the platform location in cocaine-self-administered rats with respect to animals that self-administered saline. No differences were observed in reversal learning or perseverance between groups. CONCLUSIONS: Our data suggest that cocaine self-administration facilitates learning and memory in the water maze test only when animals are submitted to highly demanding tasks, involving working memory or consolidation-like processes during the intertrial interval.

The effects of morphine self-administration on cortical pyramidal cell structure in addiction-prone lewis rats.

The consumption of drugs of abuse provokes sensitization, the development of tolerance, dependency, and eventually addiction. It is thought that these events are partially a consequence of drug-induced alterations in the organization of neuronal circuits in specific areas of the brain. In the present study, we have used intracellular injections of lucifer yellow to examine the alterations that may occur in cortical pyramidal neurons of addiction-prone Lewis rats following 15 days of self-administration of morphine. Specifically, the effects of morphine on the structure, size and branching complexity of the basal dendrites, and spine density were determined in the basal dendritic arbors of layer III pyramidal neurons in both the prelimbic and motor cortex. We found that following morphine self-administration, there was a reduction in the size and branching complexity of the dendritic arbors of pyramidal cells in the motor cortex. In contrast, prelimbic pyramidal neurons from these morphine-treated animals had larger and longer basal dendritic arbors. Furthermore, the spine density on pyramidal neurons was higher in both cortical regions of morphine self-administered rats. These results suggest that at least part of the behavioral changes produced by repeated opiate administration may be attributed to alterations in pyramidal cell structure.

Hippocampal synaptic plasticity and water maze learning in cocaine self-administered rats.

Previously, we have shown that long-term potentiation (LTP) in hippocampus of Lewis rats was significantly modulated by cocaine self-administration. Using a single train of high-frequency stimulation of 100 Hz for 1s (HFS), we found an enhancement of LTP after cocaine self-administration that was maintained even during the extinction of this behavior. However, the effects of cocaine self-administration on a hippocampal-dependent spatial learning task were unknown. Therefore, in the present study our first objective was to analyze if cocaine self-administration might affect the performance in a hippocampus-dependent task, such as the Morris water maze test. Male adult Lewis (LEW) rats self-administered cocaine (1 mg/kg/injection) or saline (0.9% NaCl) for 3 weeks. Three hours after finishing the last self-administration session, animals were submitted to Morris water maze training for 3 consecutives days. A memory test was carried out 24 h after the last training session. No significant differences were found in escape latencies and time spent in the quadrant where the platform was located during training. Given that we did not find any cocaine effect on this spatial learning task, our second objective was to estimate indirectly if brain cocaine levels have failed to modulate LTP in animals that were performing the water maze trials. To this end, we tested if cocaine application to hippocampal slices of naïve subjects was able to evoke LTP. The results indicated that cocaine produced an enhanced LTP in these hippocampal slices. Taking together, the results of the present study suggest that hippocampal LTP-like processes generated after cocaine self-administration are not related to spatial learning hippocampal-dependent tasks, such as the water maze test.

Differential effect of low doses of intracerebroventricular corticotropin-releasing factor in forced swimming test.

In this work, we studied the effect of low doses of intracerebroventricular corticotropin-releasing factor (CRF) in six sessions of forced swimming test (FST). When CRF (0.01 and 0.1 microg) was administered pre-test, results showed that the 0.1-microg dose significantly increased swimming in SESSION2, SESSION3 and SESSION4, while the 0.01-microg dose proved ineffective. When CRF (0.1 and 0.03 microg) was administered post-test to evaluate retention of swimming response, the dose of 0.1 microg impaired retention, while the dose of 0.03 microg improved it, although these effects only reached significance in SESSION2. In an additional session (SESSION6), testing long-term retention of this swimming response, the 0.1-microg dose significantly impaired retention, whereas the 0.03-microg dose proved ineffective. A high dose of CRF (1 microg) was also included as a control of previous results [García-Lecumberri C, Ambrosio E. Role of corticotropin-releasing factor in forced swimming test. Eur J Pharmacol 1998;343:17-26]. In all the FST sessions, this high dose increased swimming when administered pre-test, while impairing retention when administered post-test. Preliminary data obtained with low doses of CRF suggest that a differential effect on retention of swimming response seems to exist depending on the dose, whereas a high dose of CRF clearly impairs retention. The role of CRF in learning and memory processes in FST is discussed.

The neurobiology of cannabinoid dependence: sex differences and potential interactions between cannabinoid and opioid systems.

Cannabis is the most widely used illicit drug in many western countries. Its psychoactive ingredient, delta9-tetrahydrocannabinol (THC), produces a variety of effects in animals and humans that are probably mediated by specific cannabinoid receptors in the brain and interactions with several neurotransmitter and neuromodulator systems. For instance, recent research has revealed an important mutual functional relationship between cannabinoids and endogenous opioid systems in mediating the pharmacological and behavioral actions produced by these agents, including their reinforcing effects. Perinatal exposure to and interactions between cannabinoids and opioids might also have long-term behavioral consequences lasting into adulthood. In this work, we present preliminary evidence examining the potential effects of maternal exposure to THC on the motivational properties of morphine in male and female adult rats, as measured by an intravenous opiate self-administration paradigm.

Differential basal proenkephalin gene expression in dorsal striatum and nucleus accumbens, and vulnerability to morphine self-administration in Fischer 344 and Lewis rats.

We have previously shown that the acquisition rate of intravenous morphine self-administration under a fixed ratio one (FR1) schedule of reinforcement was greater in Lewis (LEW) than Fischer 344 (F344) rats. The purpose of the present experiment was to examine the relative motivational properties of morphine (1 mg/kg) or food under progressive ratio (PR) schedules of reinforcement in LEW and F344 rats. In addition, by using in situ hybridization histochemistry we have measured in both strains of rats the basal level of proenkephalin (PENK) gene expression in dorsal striatum and nucleus accumbens (NAcc). The results show that LEW rats responded to significantly higher breaking points (BPs) than F344 rats for intravenous morphine self-administration. In contrast, no differences were found in BPs for food pellets. Basal PENK mRNA levels were significantly higher in the dorsal striatum and nucleus accumbens of F344 than in LEW rats. Taken together, these results reveal a strain difference in the reinforcing efficacy of morphine and in the basal PENK gene expression in brain regions involved in the reinforcing actions of opiates. These data also suggest that the strain differences in opiate self-administration behavior found in this and other studies may be related, at least in part, to differences in basal opioid activity between LEW and F344 rats.

Maternal exposure to delta9-tetrahydrocannabinol facilitates morphine self-administration behavior and changes regional binding to central mu opioid receptors in adult offspring female rats.

Opiates and cannabinoids are among the most widely consumed habit-forming drugs in humans. Several studies have demonstrated the existence of interactions between both kind of drugs in a variety of effects and experimental models. The present study has been focused to determine whether perinatal delta9-tetrahydrocannabinol (Delta9-THC) exposure affects the susceptibility to reinforcing effects of morphine in adulthood and whether these potential changes were accompanied by variations in mu opioid receptor binding in brain regions related to drug reinforcement. Adult female rats born from mothers that were daily treated with delta9-THC during gestation and lactation periods, exhibited a statistically significant increase in the rate of acquisition of intravenous morphine self-administration behavior when compared with females born from vehicle-exposed mothers, an effect that did not exist in delta9-THC-exposed male offspring. This increase was significantly greater on the last day of acquisition period. There were not significant differences when the subjects were lever pressing for food. In parallel, we have also examined the density of mu opioid receptors in the brain of adult male and female offspring that were exposed to Delta9-THC during the perinatal period. Collectively, perinatal exposure to delta9-THC produced changes in mu opioid receptor binding that differed regionally and that were mostly different as a function of sex. Thus, delta9-THC-exposed males exhibited a lower density for these receptors than their respective oil-exposed controls in the caudate-putamen area as well as in the amygdala (posteromedial cortical nucleus). On the contrary, delta9-THC-exposed females exhibited higher density of these receptors than their respective oil-exposed controls in the prefrontal cortex, the hippocampus (CA3 area), the amygdala (posteromedial cortical nucleus), the ventral tegmental area and the periaqueductal grey matter, whereas the binding was lower than control females only in the lateral amygdala. These results support the notion that perinatal delta9-THC exposure alters the susceptibility to morphine reinforcing effects in adult female offspring, in parallel with changes in mu opioid receptor binding in several brain regions.

Role of corticotropin-releasing factor in forced swimming test.

Several aspects of the role of corticotropin-releasing factor (CRF) in the forced swimming test were investigated in this study by using two different administration schedules. I.c.v. microinjection of CRF produced a dose-dependent increase in swimming activity when the administration schedule originally reported for this test to screen antidepressant drugs was followed. The most effective doses were 1 and 3 microg of CRF. A lower dose of CRF (0.5 microg) was also effective when repetitive experimental stress was present. CRF receptor antagonist, alpha-helical CRF-(9-41) (alpha-helical CRF-(9-41)), was able to block CRF-induced increases in swimming in all sessions of the forced swimming test. However, the effects of CRF and CRF receptor antagonist depended on the administration schedule. A decrease in swimming in the forced swimming test was observed when CRF and CRF receptor antagonist were given together, using a different administration schedule. I.c.v. CRF was ineffective and CRF receptor antagonist alone produced an increase in swimming when administered according to this schedule. These behavioural responses were maintained after twelve days without any treatment. The results of the current study suggest that endogenous CRF seems to play a determinant role in behavioural responses in the forced swimming test. The involvement of the level of activation and memory processes in these behavioural responses is discussed.

Effect of yohimbine on the development of morphine dependence in the rat: lack of involvement of cortical beta-adrenoceptor modifications.

The alpha-2 receptor antagonist yohimbine has been previously shown to prevent the development of morphine dependence in a rat behavioral model. This study was directed to clarify the mechanism of this interaction, which is presently unknown. Since upregulation of cortical beta-adrenoceptors has been suggested to be involved in morphine withdrawal, we have tested the possible correlation between receptor density and withdrawal behaviors in the presence of yohimbine. Sprague-Dawley male rats received a s.c. suspension of morphine (300 mg/kg) or the vehicle. Animals received saline or yohimbine (4 mg/kg, IP) 24, 28, 48 and 52 h after morphine and finally naloxone (1 mg/kg i.p) at 72 h; the subsequent signs of withdrawal (mainly wet-dog shakes and escape attempts) were recorded and the cerebral cortex dissected to study [3H]-CGP 12177 binding. Morphine-treated animals displayed a marked withdrawal behavior together with beta-adrenoceptor upregulation; nevertheless, these effects were not correlated. As expected, yohimbine prevented morphine withdrawal behavior but did not reverse the beta-adrenoceptor upregulation induced by the opiate. These results confirm previous evidence against the involvement of beta-adrenoceptor upregulation on morphine withdrawal behaviors and also permit to discard beta-adrenoceptor regulation as the neurochemical basis of the antiwithdrawal effect of yohimbine. The possible contribution of some other neurochemical effects of yohimbine are discussed to explain the inhibition of morphine dependence by that drug.