Effects of heroin self-administration and forced withdrawal on the expression of genes related to the mTOR network in the basolateral complex of the amygdala of male Lewis rats

Rationale The development of substance use disorders involves long-lasting adaptations in specific brain areas that result in an elevated risk of relapse. Some of these adaptations are regulated by the mTOR network, a signalling system that integrates extracellular and intracellular stimuli and modulates several processes related to plasticity. While the role of the mTOR network in cocaine- and alcohol-related disorders is well established, little is known about its participation in opiate use disorders. Objectives To use a heroin self-administration and a withdrawal protocol that induce incubation of heroin-seeking in male rats and study the associated effects on the expression of several genes related to the mTOR system and, in the specific case of Rictor, its respective translated protein and phosphorylation. Results We found that heroin self-administration elicited an increase in the expression of the genes Igf1r, Igf2r, Akt2 and Gsk3a in the basolateral complex of the amygdala, which was not as evident at 30 days of withdrawal. We also found an increase in the expression of Rictor (a protein of the mTOR complex 2) after heroin self-administration compared to the saline group, which was occluded at the 30-day withdrawal period. The activation levels of Rictor, measured by the phosphorylation rate, were also reduced after heroin self-administration, an effect that seemed more apparent in the protracted withdrawal group. Conclusions These results suggest that heroin self-administration under extended access conditions modifies the expression profile of activators and components of the mTOR complexes and show a putative irresponsive mTOR complex 2 after withdrawal from heroin use

Impulsive Action and Impulsive Choice Are Differentially Associated With Gene Expression Variations of the GABAA Receptor Alfa 1 Subunit and the CB1 Receptor in the Lateral and Medial Orbitofrontal Cortices

The orbitofrontal cortex (OFC) is a key brain region for decision-making, action control and impulsivity. Quite notably, previous research has identified a double dissociation regarding the role of this cortical territory in impulsive choice. While medial orbitofrontal lesions increase preference for a large but delayed reward, lateral orbitofrontal lesions have the opposite effect. However, there are no data regarding this anatomical dissociation in impulsive action. The neurochemical basis of impulsivity is still being elucidated, however, in recent years a role for the endocannabinoids and the related glutamatergic and GABAergic neurotransmitter systems has been suggested. Here, we submitted male Wistar rats to a delay-discounting task (DDT) or a two-choice serial reaction time task (2-CSRTT) and classified them as high impulsive or low impulsive in either task using cluster analysis. We then examined the gene expression of several elements of the endocannabinoid system or different subunits of certain glutamatergic or GABAergic ionotropic receptors (AMPA, NMDA, or GABAA) in the lateral or medial divisions of their orbitofrontal cortices. Our results confirm, at the gene expression level, the dissociation in the participation of the medial, and lateral divisions of the orbitofrontal cortex in impulsivity. While in the 2-CSRTT (inhibitory control) we found that high impulsive animals exhibited lower gene expression levels of the α1 GABAA receptor subunit in the lateral OFC, no such differences were evident in the medial OFC. When we analyzed DDT performance, we found that high impulsive animals displayed lower levels of CB1 gene expression in the medial but not in the lateral OFC. We propose that GABAergic dynamics in the lateral OFC might contribute to the inhibitory control mechanisms that are altered in impulsive behavior while endocannabinoid receptor gene transcription in the medial OFC may subserve the delay-discounting processes that participate in certain types of impulsiveness

Chronic cannabinoid administration to periadolescent rats modulates the metabolic response to acute cocaine in the adult brain

Purpose: To analyze brain metabolic response to acute cocaine in male and female Wistar rats with or without a history of cannabinoid exposure during periadolescence. Procedures: The synthetic cannabinoid agonist CP 55,940 (CP) or its vehicle (VH), were administered to male and female rats during periadolescence. When these animals reached adulthood, saline and cocaine-induced changes in 2-deoxy-2-[18F]fluoro D glucose (FDG) uptake were studied by positron emission tomography. Results: The baseline (post-saline) metabolism in the septal nuclei was higher in CP-females than in VH-females, although septal metabolism was lower in CP-females after cocaine, reaching similar values to those of VH-females at baseline. Cocaine did not affect metabolism in VH-females. Periadolescent cannabinoid treatment did not influence baseline metabolism in males although cocaine reduced the FDG uptake in the dorsal striatum of males that received the VH but not CP. Conclusions: These results suggest that cannabinoids during periadolescence modify baseline and cocaine-evoked brain metabolism in a sex-dependent manner. In the case of CP-females, the involvement of septal metabolic alterations in their susceptibility to the rewarding effects of cocaine should be further investigated.This work was supported by grants from the Ministerio de Educación y Ciencia (Grants nº SAF2004-08148 and SAF2007-064890); Ministerio de Sanidad y Consumo (Grants RD06/ 00170029 of Instituto de Salud Carlos III, PNSD 2004 2007 and 2008 2010); Dirección General de Investigación de la Comunidad de Madrid (Grant S-SAL/0261/2006, I+D CANNAB-CM Consortium); and UNED (Plan de Promoción de la Investigación) to EA, and grants from the “Ministerio de Ciencia y Tecnología” (TEC2004-07052-C02-01/TCM), “Ministerio de Sanidad y Consumo” (CIBER CB06/01/0079, PNSD 2007 2010, FIS CP08/00017), “Ministerio de Industria” (CENIT program) and “Fundación de Investigación Médica Mutua Madrileña” (2007 2010 and 2008 2011) to MD.Publicad

Parafascicular thalamic nucleus deep brain stimulation decreases NMDA receptor GluN1 subunit gene expression in the prefrontal cortex

The rodent parafascicular nucleus (PFn) or the centromedian-parafascicular complex of primates is a posterior intralaminar nucleus of the thalamus related to cortical activation and maintenance of states of consciousness underlying attention, learning and memory. Deep brain stimulation (DBS) of the PFn has been proved to restore arousal and consciousness in humans and to enhance performance in learning and memory tasks in rats. The primary expected effect of PFn DBS is to induce plastic changes in target neurons of brain areas associated with cognitive function. In this study, Wistar rats were stimulated for 20mins in the PFn following a DBS protocol that had previously facilitated memory in rats. NMDA and GABAB receptor binding, and gene expression of the GluN1subunit of the NMDA receptor (NMDAR) were assessed in regions related to cognitive functions, such as the prefrontal cortex and hippocampus. The results showed that PFn DBS induced a decrease in NMDAR GluN1 subunit gene expression in the cingulate and prelimbic cortices, but no significant statistical differences were found in the density of NMDA or GABAB receptors in any of the analyzed regions. Taken together, our findings suggest a possible role for the NMDAR GluN1 subunit in the prefrontal cortex in the procognitive actions of the PFn DBS

Interaction between maternal immune activation and peripubertal stress in rats: impact on cocaine addiction-like behaviour, morphofunctional brain parameters and striatal transcriptome.

Substance use disorders are more prevalent in schizophrenia, but the causal links between both conditions remain unclear. Maternal immune activation (MIA) is associated with schizophrenia which may be triggered by stressful experiences during adolescence. Therefore, we used a double-hit rat model, combining MIA and peripubertal stress (PUS), to study cocaine addiction and the underlying neurobehavioural alterations. We injected lipopolysaccharide or saline on gestational days 15 and 16 to Sprague-Dawley dams. Their male offspring underwent five episodes of unpredictable stress every other day from postnatal day 28 to 38. When animals reached adulthood, we studied cocaine addiction-like behaviour, impulsivity, Pavlovian and instrumental conditioning, and several aspects of brain structure and function by MRI, PET and RNAseq. MIA facilitated the acquisition of cocaine self-administration and increased the motivation for the drug; however, PUS reduced cocaine intake, an effect that was reversed in MIA + PUS rats. We found concomitant brain alterations: MIA + PUS altered the structure and function of the dorsal striatum, increasing its volume and interfering with glutamatergic dynamics (PUS decreased the levels of NAA + NAAG but only in LPS animals) and modulated specific genes that could account for the restoration of cocaine intake such as the pentraxin family. On its own, PUS reduced hippocampal volume and hyperactivated the dorsal subiculum, also having a profound effect on the dorsal striatal transcriptome. However, these effects were obliterated when PUS occurred in animals with MIA experience. Our results describe an unprecedented interplay between MIA and stress on neurodevelopment and the susceptibility to cocaine addiction.This work has been funded by the Spanish Ministry of Economy and Competitiveness (Project no.: PSI2016-80541-P to EA and AH-M); Ministry of Science (PID2019- 104523RB-I00 to A-HM and PID2019-111594RB-100 to EA), Spanish Ministry of Health, Social Services and Equality (Network of Addictive Disorders - Project no.: RTA-RD16/ 020/0022 of the Institute of Health Carlos III and National Plan on Drugs, Project no.: 2016I073 to EA and 2017I042 to A H-M); The BBVA Foundation (Leonardo Grants) to AH-M; The European Union (Project no.: JUST- 2017- AG- DRUG-806996-JUSTSO) to EA; and the UNED (Plan for the Promotion of Research) to EA and AH-M. MLS-M was supported by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (project PI17/01766), co-financed by the European Regional Development Fund (ERDF), ‘A way to make Europe’; project PID2021-128862OB-I00 funded by MCIN/AEI/ 10.13039/501100011033/FEDER, UE, CIBER de Salud Mental - Instituto de Salud Carlos III (project number CB07/09/0031); Delegación del Gobierno para el Plan Nacional sobre Drogas (project number 2017/085, 2022/008917); and Fundación Alicia Koplowitz. Fundación Tatiana Pérez de Guzmán el Bueno supported MC-V. MD’s work was supported by Ministerio de Ciencia e Innovación (MCIN) and Instituto de Salud Carlos III (PT20/00044). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Morphine self-administration alters the expression of translational machinery genes in the amygdala of male Lewis rats

Background: Addiction is a chronic disorder with a high risk of relapse. The neural mechanisms mediating addictions require protein synthesis, which could be relevant for the development of more effective treatments. The mTOR signaling pathway regulates protein synthesis processes that have recently been linked to the development of drug addiction. Aims: To assess the effects of morphine self-administration and its subsequent extinction on the expression of several genes that act in this pathway, and on the levels of specific phosphoproteins (Akt, Gsk3α/β, mTOR, PDK1 and p70 S6 kinase) in the amygdala, nucleus accumbens, and the prefrontal cortex. Methods: Male Lewis rats underwent morphine self-administration (1 mg/kg) for 19 days. They subsequently were submitted to extinction training for 15 days. Rats were killed either after self-administration or extinction, their brains extracted, and gene expression or phosphoprotein levels were assessed. Results: We found an increase in Raptor and Eif4ebp2 expression in the amygdala of rats that self-administered morphine, even after extinction. The expression of Insr in the amygdala of control animals decreased over time while the opposite effect was seen in the rats that self-administered morphine

Brain metabolism in lewis and fischer 344 rats after morphine self-administration and extinction: a PET imaging study

Proceeding of: The 72nd Annual Scientific Meeting of the College on Problems of Drug Dependence, Scottsdale, Arizona, June 12-17, 2010.MICINN (SAF2007-064890); Ministerio de Sanidad y Consumo (RD06/001/0029 y CP08/00017 del ISC III; Plan Nacional sobre Drogas 2004-2007 y 2008-2010); Fundación Mutua Madrileña; and CAM (S-SAL/0261/2006)

Augmented acquisition of cocaine self-administration and altered brain glucose metabolism in adult female but not male rats exposed to a cannabinoid agonist during adolescence

Marijuana consumption during adolescence has been proposed to be a stepping stone for adult cocaine addiction. However, experimental evidence for this hypothesis is missing. In this work we chronically injected male and female Wistar rats with either the cannabinoid agonist CP 55,940 (CP; 0.4 mg/kg) or its corresponding vehicle. Adult acquisition (seven 30 min daily sessions) and maintenance (fourteen 2 h daily sessions) of cocaine self administration (1 mg/kg), food reinforced operant learning under conditions of normal (ad libitum access to food), and high motivation (food restriction schedule) were measured. Additionally, brain metabolic activity was analyzed by means of [18F] fluorodeoxyglucose positron emission tomography. During the acquisition phase, female CP treated rats showed a higher rate of cocaine self administration as compared to vehicle treated females and males; no differences were found between both male groups. This effect disappeared in the maintenance phase. Moreover, no differences among groups were evident in the food reinforced operant task, pointing to the cocaine specific nature of the effect seen in self administration rather than a general change in reward processing. Basal brain metabolic activity also changed in CP treated females when compared to their vehicle treated counterparts with no differences being found in the males; more specifically we observed a hyper activation of the frontal cortex and a hypo activation of the amygdalo entorhinal cortex. Our results suggest that a chronic exposure to cannabinoids during adolescence alters the susceptibility to acquire cocaine self administration, in a sex specific fashion. This increased susceptibility could be related to thechanges in brain metabolic activity induced by cannabinoids during adolescenceThis work was supported by Grants FIS G03/05 (Red de Trastornos Adictivos), BSO2001-1099, FIS 01-05-01, Plan Nacional sobre Drogas (PNSD) 2001–2003, PNSD 2004–2007, GR-SAL/0260/2004 to EA and Grants INT/2012/ 2002, CB06/01/0079, and CENIT (2006–2009) to MDPublicad

The Basolateral Amygdala to Nucleus Accumbens Core Circuit Mediates the Conditioned Reinforcing Effects of Cocaine-Paired Cues on Cocaine Seeking.

BACKGROUND: Individuals addicted to cocaine spend much of their time foraging for the drug. Pavlovian drug-associated conditioned stimuli exert a major influence on the initiation and maintenance of drug seeking often long into abstinence, especially when presented response-contingently, acting as conditioned reinforcers that bridge delays to drug use. The acquisition of cue-controlled cocaine seeking has been shown to depend on functional interactions between the basolateral amygdala (BLA) and the nucleus accumbens core (NAcC). However, the precise neuronal circuits underlying the acquisition of cue-controlled cocaine-seeking behavior have not been elucidated. METHODS: Here, we used a projection-specific Cre-dependent DREADD (designer receptor exclusively activated by designer drugs)-mediated causal approach to test the hypothesis that the direct projections from the BLA to the NAcC are required for the acquisition of cue-controlled cocaine-seeking behavior. RESULTS: In Sprague Dawley rats with Cre-mediated expression of the inhibitory DREADD hM4D(Gi) in the NAcC-projecting BLA neurons, treatment with clozapine N-oxide, but not vehicle, selectively prevented the impact of cocaine-associated conditioned reinforcers on cocaine seeking under a second-order schedule of reinforcement. This effect was attributable to the chemogenetic inhibition of the NAcC-projecting BLA neurons, as it was reversible, and it was absent in clozapine N-oxide-treated rats expressing an empty control virus. In contrast, chemogenetic inhibition of the anterior insula, which receives collateral projections from NAcC-projecting BLA neurons, was without effect. CONCLUSIONS: These data demonstrate that the acquisition of cue-controlled cocaine seeking that depends on the conditioned reinforcing effects of cocaine cues requires activity in the direct projections from the BLA to the NAcC.Leverhulme Trus

CNR1 gene deletion affects the density of endomorphin-2 binding sites in the mouse brain in a hemisphere-specific manner

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two endogenous tetrapeptides with very high affinities for the mu-opioid receptor. Until recently, the precise neuroanatomical localization of the binding sites for these peptides was unknown. However, the recent synthesis of tritiated forms of these molecules has permitted these binding sites to be analysed with a very high degree of neuroanatomical specificity. Preliminary studies demonstrated a superior binding profile for EM-2, with less non-specific binding than EM-1. As the endogenous cannabinoid and opioid systems interact at several levels, we investigated how deletion of the CNR1 gene, which encodes the cannabinoid receptor 1 (CB1R) protein, affects the brain distribution of EM-2 binding sites. Our results revealed no differences in the average density of EM-2 binding sites in CB1 receptor knockout (CB1R KO) and WT mice. However, when both hemispheres were analysed separately, we detected specific alterations in the distribution of EM-2 binding sites in the right hemisphere of CB1R KO mice. While, the density of EM-2 binding sites in CB1R KO mice was higher in the CA3 hippocampal field and in the pontine tegmental nuclei, it was lower in the superior colliculus and ventral tegmental area than in WT controls. No differences were observed in the left hemisphere for any of the regions analysed. For the first time these findings demonstrate a lateralization effect on cerebral opioid binding sites that may be mediated by the central cannabinoid system. (c) 2012 Elsevier B.V. All rights reserved