José Antonio López-Moreno

Genetic polymorphisms among C57BL/6 mouse inbred strains

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina® Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyrc−2j/J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.

The pharmacology of the endocannabinoid system: functional and structural interactions with other neurotransmitter systems and their repercussions in behavioral addiction

Addiction is a chronic, recurring and complex disorder. It is characterized by anomalous behaviors that are linked to permanent or long‐lasting neurobiological alterations. Furthermore, the endocannabinoid system has a crucial role in mediating neurotransmitter release as one of the main neuromodulators of the mammalian central nervous system. The purpose of the present review is to instruct readers about the functional and structural interactions between the endocannabinoid system and the main neurotransmitter systems of the central nervous system in the context of drug addiction. With this aim, we have systematically reviewed the main findings of most of the existing literature that explores cross‐talk in the five brain areas that are most traditionally implicated in addiction: amygdala, prefrontal cortex, nucleus accumbens, hippocampus and ventral tegmental area (VTA). The neurotransmission systems influenced by the pharmacology of the endocannabinoid system in these brain areas, which are reviewed here, are gamma‐aminobutyric acid (GABA), glutamate, the main biogenic amines (dopamine, noradrenaline and serotonin), acetylcholine and opioids. We show that all of these neurotransmitter systems can be modulated differentially in each brain area by the activation or deactivation of cannabinoid CB1 brain receptors. Specifically, most of the studies relate to the hippocampus and nucleus accumbens. Moreover, the neurotransmitter with the fewest number of related studies is acetylcholine (excepting in the hippocampus), whereas there is a large number that evaluates GABA, glutamate and dopamine. Finally, we propose a possible interpretation of the role of the endocannabinoid system in the phenomenon of addiction.

Long-Lasting Increase of Alcohol Relapse by the Cannabinoid Receptor Agonist WIN 55,212-2 during Alcohol Deprivation

Alcoholism is characterized by successive relapses. Recent data have shown a cross-talk between the cannabinoid system and ethanol. In this study, male Wistar rats with a limited (30 min sessions), intermittent, and extended background of alcohol operant self-administration were used. The relapse to alcohol after 1 week of alcohol deprivation was evaluated. Two weeks later, the animals were treated with the cannabinoid agonist WIN 55,212-2 (R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate) (0, 0.4, 2.0, and 10.0 mg/kg, s.c.) during a similar alcohol deprivation period, and alcohol relapse during 2 weeks was assessed. A conditioned place preference (CPP) paradigm was used to study the rewarding properties of the cannabinoid agonist. Locomotor activity was also recorded. All doses of WIN 55,212-2 produced aversion in the CPP paradigm. The doses of 2.0 and 10.0 mg/kg resulted in an important suppression of spontaneous locomotor activity and a progressive weight loss during the next 2 weeks. The single alcohol deprivation was followed by a transient increase in their responding for alcohol from a range of 20-24 lever presses at baseline to a range of 38-48 responses in the first and second days (alcohol deprivation effect). However, the administration of WIN 55,212-2 during ethanol deprivation produced similar increased responses for alcohol but in a long-term way (at least over 2 weeks). These findings suggest that noncontingent chronic exposure to cannabinoids during alcohol deprivation can potentiate the relapse into alcohol use, indicating that functional changes in the cannabinoid brain receptor may play a key role in ethanol relapse.

Common single nucleotide variants underlying drug addiction: more than a decade of research.

Drug‐related phenotypes are common complex and highly heritable traits. In the last few years, candidate gene (CGAS) and genome‐wide association studies (GWAS) have identified a huge number of single nucleotide polymorphisms (SNPs) associated with drug use, abuse or dependence, mainly related to alcohol or nicotine. Nevertheless, few of these associations have been replicated in independent studies. The aim of this study was to provide a review of the SNPs that have been most significantly associated with alcohol‐, nicotine‐, cannabis‐ and cocaine‐related phenotypes in humans between the years of 2000 and 2012. To this end, we selected CGAS, GWAS, family‐based association and case‐only studies published in peer‐reviewed international scientific journals (using the PubMed/MEDLINE and Addiction GWAS Resource databases) in which a significant association was reported. A total of 371 studies fit the search criteria. We then filtered SNPs with at least one replication study and performed meta‐analysis of the significance of the associations. SNPs in the alcohol metabolizing genes, in the cholinergic gene cluster CHRNA5‐CHRNA3‐CHRNB4, and in the DRD2 and ANNK1 genes, are, to date, the most replicated and significant gene variants associated with alcohol‐ and nicotine‐related phenotypes. In the case of cannabis and cocaine, a far fewer number of studies and replications have been reported, indicating either a need for further investigation or that the genetics of cannabis/cocaine addiction are more elusive. This review brings a global state‐of‐the‐art vision of the behavioral genetics of addiction and collaborates on formulation of new hypothesis to guide future work.

Functional Interactions between Endogenous Cannabinoid and Opioid Systems: Focus on Alcohol, Genetics and Drug-Addicted Behaviors

Although the first studies regarding the endogenous opioid system and addiction were published during the 1940s, addiction and cannabinoids were not addressed until the 1970s. Currently, the number of opioid addiction studies indexed in PubMed-Medline is 16 times greater than the number of cannabinoid addiction reports. More recently, functional interactions have been demonstrated between the endogenous cannabinoid and opioid systems. For example, the cannabinoid brain receptor type 1 (CB1) and mu opioid receptor type 1 (MOR1) co-localize in the same presynaptic nerve terminals and signal through a common receptor-mediated G-protein pathway. Here, we review a great variety of behavioral models of drug addiction and alcohol-related behaviors. We also include data providing clear evidence that activation of the cannabinoid and opioid endogenous systems via WIN 55,512-2 (0.4-10 mg/kg) and morphine (1.0-10 mg/kg), respectively, produces similar levels of relapse to alcohol in operant alcohol self-administration tasks. Finally, we discuss genetic studies that reveal significant associations between polymorphisms in MOR1 and CB1 receptors and drug addiction. For example, the SNP A118G, which changes the amino acid aspartate to asparagine in the MOR1 gene, is highly associated with altered opioid system function. The presence of a microsatellite polymorphism of an (AAT)n triplet near the CB1 gene is associated with drug addiction phenotypes. But, studies exploring haplotypes with regard to both systems, however, are lacking.

Nicotine in alcohol deprivation increases alcohol operant self-administration during reinstatement

Tobacco and alcohol are highly co-abused by humans. Most experimental studies have evaluated ethanol consumption in animals exposed concomitantly to nicotine. However, little is known regarding the effects of nicotine administered during periods of alcohol deprivation. In the present study, adult male Wistar rats with an extended background of operant self-administration of ethanol were alcohol-deprived and treated with nicotine (0.1, 0.2, 0.4 and 0.8 mg/kg) or saline during five consecutive days in one chamber of a place conditioning apparatus. Nicotine-induced changes in locomotion were monitored daily, whereas the expression of place conditioning was studied the day after the last nicotine injection. Forty-eight hours after testing for conditioning, the animals resumed operant self-administration of ethanol and their alcohol intake was evaluated during the next 14 days. We observed that alcohol consumption was increased in animals treated with nicotine at doses of 0.2, 0.4 and 0.8 mg/kg but not in animals treated with the dose of 0.1 mg/kg or saline. Additionally, the dose of 0.8 mg/kg of nicotine not only induced persistent changes in alcohol self-administration but also produced conditioned place aversion and depressed locomotor activity. These results indicate that nicotine administration during the ethanol deprivation period can exacerbate the maintenance of alcohol consumption.

The genetic basis of the endocannabinoid system and drug addiction in humans

The cannabinoid receptor (CNR1) and the fatty acid amide hydrolase (FAAH) genes are located on chromosomes 6 and 1 in the 6q15 and 1p33 cytogenetic bands, respectively. CNR1 encodes a seven-transmembrane domain protein of 472 amino acids, whereas FAAH encodes one transmembrane domain of 579 amino acids. Several mutations found in these genes lead to altered mRNA stability and transcription rate or a reduction of the activity of the encoded protein. Increasing evidence shows that these functional mutations are related to dependence upon cocaine, alcohol, marijuana, heroin, nicotine and other drugs. One of the most compelling associations is with the C385A single nucleotide polymorphism (SNP), which is found in the FAAH gene. For all of the genetic polymorphisms reviewed here, it is difficult to form overall conclusions due to the high diversity of population samples being studied, ethnicity, the use of volunteers, heterogeneity of the recruitment criteria and the drug addiction phenotype studied. Care should be taken when generalizing the results from different studies. However, many works have repeatedly associated polymorphisms in the CNR1 and FAAH genes with drug-related behaviours; this suggests that these genes should be examined in further genetic studies focusing on drug addiction and other psychiatric disorders.

Consequences of early life stress on the expression of endocannabinoid-related genes in the rat brain.

The endocannabinoid system is involved in several physiological and pathological states including anxiety, depression, addiction and other neuropsychiatric disorders. Evidence from human and rodent studies suggests that exposure to early life stress may increase the risk of psychopathology later in life. Indeed, maternal deprivation (MD) (24 h at postnatal day 9) in rats induces behavioural alterations associated with depressive-like and psychotic-like symptoms, as well as important changes in the endocannabinoid system. As most neuropsychiatric disorders first appear at adolescence, and show remarkable sexual dimorphisms in their prevalence and severity, in the present study, we analysed the gene expression of the main components of the brain cannabinoid system in adolescent (postnatal day 46) Wistar male and female rats reared under standard conditions or exposed to MD. For this, we analysed, by real-time quantitative PCR, the expression of genes encoding for CB1 and CB2 receptors, TRPV1 and GPR55 (Cnr1, Cnr2a, Cnr2b, Trpv1, and Gpr55), for the major enzymes of synthesis, N-acyl phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL) (Nape-pld, Dagla and Daglb), and degradation, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) (Faah, Magl and Cox-2), in specific brain regions, that is, the frontal cortex, ventral and dorsal striatum, dorsal hippocampus and amygdala. In males, MD increased the genetic expression of all the genes studied within the frontal cortex, whereas in females such an increase was observed only in the hippocampus. In conclusion, the endocannabinoid system is sensitive to early life stress at the gene expression level in a sex-dependent and region-dependent manner, and these changes are already evident in the adolescent brain.

Pharmacological activation of CB1 and D2 receptors in rats: predominant role of CB1 in the increase of alcohol relapse.

The classical dopamine D2 receptor has been widely studied in alcoholism. Recently, different studies have explored the role of the CB1 receptor in alcohol‐related behavior. In alcohol addiction, relapse is one of the central features. In light of this, we investigated the functional roles of and interactions between CB1 and D2 receptors in alcohol relapse. We used the learned task of alcohol operant self‐administration in Wistar rats. In order to evaluate alcohol relapse, we set up a protocol essentially based on the alcohol deprivation effect. We found that subchronic activation of CB1 (WIN 55,212–2, 2 mg/kg), but not D2 receptors (quinpirole, 1 mg/kg), during a period of alcohol deprivation increased long‐lasting alcohol relapse. The cannabinoid‐induced potentiation of alcohol relapse was mediated by a motivational and appetitive component, and not merely by alcohol consumption. This potentiation was prevented by the pharmacological inactivation of D2 receptors (raclopride, 0.1–0.3 mg/kg). Together, these results essentially demonstrate that activation of CB1 receptors plays a key role in the increase of alcohol relapse, whereas inactivation of D2 receptors modulates this aberrant behavior. We suggest that there exists a functional and interactive relationship between both receptor systems, which controls alcohol relapse and alcohol‐learned tasks.

Cannabinoid-induced increase in relapse-like drinking is prevented by the blockade of the glycine-binding site of N-methyl-D-aspartate receptors.

The endocannabinoid system is a neuromodulatory system which controls the release of multiple neurotransmitters, including glutamate and both, the endocannabinoid and glutamatergic systems, have been implicated in alcohol relapse. Cannabinoid agonists induce an increase in relapse-like drinking whereas glutamate receptor antagonists could prevent it. Here we hypothesize that cannabinoid-induced increases in relapse-like alcohol drinking could be mediated by glutamatergic N-methyl-d-aspartate (NMDA) receptors. To test this hypothesis, Wistar rats with a background of alcohol operant self-administration were treated with the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl), pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55.212-2, WIN) (2.0 mg/kg) during periods of alcohol deprivation. For five consecutive days, 30 min before the reintroduction of alcohol, rats were injected with the NMDA/glycine receptor antagonist 7-chloro-4-hydroxy-3-(3-phenoxy)phenylquinolin-2-[1H]-one (L-701) (1.25-5.0 mg/kg) and alcohol reinforcement was evaluated. Our results clearly show that L-701 prevented the cannabinoid-induced increase in relapse-like drinking in a dose-dependent manner, whereas L-701 alone, in the absence of WIN treatment, did not significantly alter alcohol intake. The potentiation of relapse-like drinking induced by WIN is not caused by nonspecific anxiogenic effects, since no effect was observed in the elevated-plus maze test. These alcohol-related behaviors are linked to differential changes in CNR1 and NR1 subunit mRNA transcripts. In WIN-treated rats, an increase in CNR1 transcript levels was observed in the hypothalamus and striatum, whereas in the amygdala and anterior cingulate cortex, brain regions involved in emotional processing, a decrease was observed. Interestingly, such changes were blocked after L-701 treatment. Finally, WIN treatment also caused a reduction in NR1 mRNA levels in the amygdala. In conclusion, pharmacological inactivation of the glycine-binding site of NMDA receptors may control cannabinoid-induced relapse-like drinking, which is associated with altered expression of CNR1 and NR1 gene expression as observed after WIN treatment.