Santiago M. Coria

Sex-specific disturbances of the glutamate/GABA balance in the hippocampus of adult rats subjected to adolescent cannabinoid exposure

Adolescence is a period of active synaptic remodelling and plasticity and as such, a developmental phase of particular vulnerability to the effects of environmental insults. The endogenous cannabinoid system regulates central nervous system development and cannabinoid exposure during adolescence has been linked to several alterations to hippocampal-dependent processes such as cognition and emotion, which rely on intact glutamatergic and GABAergic systems. Here we show that K(+)-induced γ-amino butyric acid (GABA) release increases in the CA1 hippocampal field of Wistar rats of both sexes that were treated chronically with the cannabinoid agonist CP 55,940 (CP55940) during adolescence. GABA(B) receptors levels also increased in cannabinoid-exposed rats. In addition, CP55940-treated females exhibit reduced GABA transporter gene expression (GAT-1), increased GABA(A) receptor expression, as well as decreased K(+)-induced glutamate release and NMDA receptor levels. CP55940 administration did not affect the glial (EAAT2) or neuronal (EAAT3) glutamate transporter gene expression in either males or females, and nor were any changes in the mGlu5 receptor protein levels observed. Taken together, these results show that while the exacerbated GABA release induced by early cannabinoid exposure may be compensated by an increment in GABA(B) receptors, which normally function as inhibitory autoreceptors, adolescent cannabinoid exposure in the females disturbs the normal balance between glutamate and GABA transmission. These observations may provide important insight into the neuronal basis of the well-documented alterations in cognitive and emotional processes induced by adolescent cannabinoid exposure.

Cocaine self-administration differentially modulates the expression of endogenous cannabinoid system-related proteins in the hippocampus of Lewis vs. Fischer 344 rats.

The endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) are modulators of glutamate and γ-aminobutyric acid (GABA), two transmitters involved in cocaine addiction. However, little is known on the effects of cocaine on the enzymes that produce and degrade endocannabinoids. The present work addresses the effects of cocaine self-administration on the immunohistochemical expression of endocannabinoid signalling (ECS)-related proteins in the hippocampus. The study has been performed on two different strains of rats, Lewis (Lew) and Fischer 344 (F344), which are characterized for displaying a differential sensitivity to cocaine, thus making them suitable in the study of vulnerability to drug addiction. Both strains showed differences in the expression of ECS-related proteins in the hippocampus, i.e. Lew rats exhibited lower CB1 expression but higher CB2 expression than F344 rats. After setting similar cocaine self-administration, both strains showed clear differences in the expression of ECS-related proteins, which were differentially restricted to either the 2-AG or anandamide signalling pathways in a self-administration training/drug-dependent manner. The decreases observed in CB1 expression and N-acyl phosphatidylethanolamine phospholipase D:fatty acid amino hydrolase ratio after saline self-administration were enhanced only in cocaine self-administered Lew rats. CB2 expression increase and diacylglycerol lipase α:monoacylglycerol lipase ratio decrease detected after saline self-administration were blocked only in cocaine self-administered F344 rats. These findings indicate that cocaine may regulate hippocampal GABA/glutamate synapses by directly modulating endocannabinoid production/degradation enzymes and that these actions are strain-dependent. This differential response suggests that the endogenous cannabinoid system might contribute to genotype/strain differences on the sensitivity to self-administration training and cocaine addiction.

The Effects of Cocaine Self-Administration on Dendritic Spine Density in the Rat Hippocampus Are Dependent on Genetic Background

Chronic exposure to cocaine induces modifications to neurons in the brain regions involved in addiction. Hence, we evaluated cocaine-induced changes in the hippocampal CA1 field in Fischer 344 (F344) and Lewis (LEW) rats, 2 strains that have been widely used to study genetic predisposition to drug addiction, by combining intracellular Lucifer yellow injection with confocal microscopy reconstruction of labeled neurons. Specifically, we examined the effects of cocaine self-administration on the structure, size, and branching complexity of the apical dendrites of CA1 pyramidal neurons. In addition, we quantified spine density in the collaterals of the apical dendritic arbors of these neurons. We found differences between these strains in several morphological parameters. For example, CA1 apical dendrites were more branched and complex in LEW than in F344 rats, while the spine density in the collateral dendrites of the apical dendritic arbors was greater in F344 rats. Interestingly, cocaine self-administration in LEW rats augmented the spine density, an effect that was not observed in the F344 strain. These results reveal significant structural differences in CA1 pyramidal cells between these strains and indicate that cocaine self-administration has a distinct effect on neuron morphology in the hippocampus of rats with different genetic backgrounds.

Genetic differences in the modulation of accumbal glutamate and γ‐amino butyric acid levels after cocaine‐induced reinstatement

The Lewis (LEW) and Fischer 344 (F344) inbred rat strains are frequently used to study the role of genetic factors in vulnerability to drug addiction and relapse. Glutamate and γ‐amino butyric acid (GABA) transmission are significantly altered after cocaine‐induced reinstatement, although whether LEW and F344 rats differ in their accumbal glutamate and GABA responsiveness to cocaine‐induced reinstatement remains unknown. To investigate this, we measured by in vivo microdialysis extracellular glutamate and GABA levels in the core division of the nucleus accumbens after extinction of cocaine self‐administration and during cocaine‐induced reinstatement (7.5 mg/kg, i.p.) in these two strains of rats. No strain differences were evident in cocaine self‐administration or extinction behavior, although cocaine priming did induce a higher rate of lever pressing in LEW compared with F344 rats. After extinction, F344 rats that self‐administered cocaine had less GABA than the saline controls, while the glutamate levels remained constant in both strains. There was more accumbal glutamate after cocaine priming in LEW rats that self‐administered cocaine, while GABA levels were unaffected. By contrast, GABA increased transiently in F344 rats that self‐administered cocaine, while glutamate levels were unaltered. In F344 saline controls, cocaine priming provoked contrasting effects in glutamate and GABA levels, inducing a delayed increase in glutamate and a delayed decrease in GABA levels. These amino acids were unaffected by cocaine priming in LEW saline rats. Together, these results suggest that genetic differences in cocaine‐induced reinstatement reflect different responses of the accumbal GABA and glutamate systems to cocaine priming.

DEPOTENTIATION OF HIPPOCAMPAL LONG-TERM POTENTIATION DEPENDS ON GENETIC BACKGROUND AND IS MODULATED BY COCAINE SELF-ADMINISTRATION

Lewis (LEW) and Fischer 344 (F344) rats differ in their response to drugs and are frequently used as an experimental model to study vulnerability to drug addiction. We have previously reported that significant differences in hippocampal synaptic plasticity exist between LEW and F344 rats after non-contingent chronic cocaine administration. However, given the several biochemical differences between contingent and non-contingent administration of drugs, we have studied here the possible genetic differences in synaptic plasticity after contingent cocaine self-administration. LEW and F344 animals self-administered cocaine (1 mg/kg i.v.) or saline under a fixed ratio 1 schedule of reinforcement for 20 days. After self-administration, electrophysiological experiments were carried out in which hippocampal slices were tetanized with three high frequency pulses in order to induce long-term potentiation (LTP). After a 20 min period of LTP stabilization, a train of low frequency stimulation (LFS; 900 pulses, 1 Hz) was applied to induce depotentiation of LTP. Data showed no differences between cocaine self-administered LEW or F344 rats in the induction of saturated-LTP compared to saline animals. LEW saline self-administered rats showed normal LTP depotentiation whereas cocaine self-administration impaired depotentiation in this rat strain. In the F344 strain, depotentiation of saturated-LTP was impaired both in saline and cocaine self-administered rats. The present results corroborate previous findings showing differences in basal hippocampal synaptic plasticity between LEW and F344 rats. These differences seem to modulate cocaine effects in a manner independent of contingency of drug administration.

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.

Strain differences in the expression of endocannabinoid genes and in cannabinoid receptor binding in the brain of Lewis and Fischer 344 rats.

The Lewis (LEW) and Fischer 344 (F344) rat strains have been proposed as a model to study certain genetic influences on drug use. These strains differ in terms of the self-administration of several drugs, and in their expression of various components of the dopaminergic, glutamatergic, GABAergic and endogenous opioid neurotransmitter systems. As the endocannabinoid system is linked to these systems, we investigated whether these two strains exhibit differences in cannabinoid receptor binding and in the expression of cannabinoid-related genes. Quantitative autoradiography of [(3)H]-CP 55,940 binding levels and real-time PCR assays were used. F344 rats displayed higher levels of cannabinoid receptor binding in the lateral globus pallidus and weaker CNR1 gene expression in the prefrontal cortex (PFc) than LEW rats. Moreover, the N-acyl phosphatidylethanolamine-specific phospholipase D/fatty acid amide hydrolase ratio was greater in the PFc and NAcc of F344 rats. Our results suggest that the endocannabinoid system may be a mediator of the individual differences that exist in the susceptibility to the rewarding effects of drugs of abuse.

Extended-access methamphetamine self-administration elicits neuroinflammatory response along with blood-brain barrier breakdown.

Methamphetamine (METH) is a highly addictive psychostimulant drug that can lead to neurological and psychiatric abnormalities. Several studies have explored the central impact of METH use, but the mechanism(s) underlying blood-brain barrier (BBB) dysfunction and associated neuroinflammatory processes after chronic METH consumption are still unclear. Important findings in the field are mainly based on in vitro approaches and animal studies using an acute METH paradigm, and not much is known about the neurovascular alterations under a chronic drug use. Thus, the present study aimed to fill this crucial gap by exploring the effect of METH-self administration on BBB function and neuroinflammatory responses. Herein, we observed an increase of BBB permeability characterized by Evans blue and albumin extravasation in the rat hippocampus and striatum triggered by extended-access METH self-administration followed by forced abstinence. Also, there was a clear structural alteration of blood vessels showed by the down-regulation of collagen IV staining, which is an important protein of the endothelial basement membrane, together with a decrease of intercellular junction protein levels, namely claudin-5, occludin and vascular endothelial-cadherin. Additionally, we observed an up-regulation of vascular cell and intercellular adhesion molecule, concomitant with the presence of T cell antigen CD4 and tissue macrophage marker CD169 in the brain parenchyma. Rats trained to self-administer METH also presented a neuroinflammatory profile characterized by microglial activation, astrogliosis and increased pro-inflammatory mediators, namely tumor necrosis factor-alpha, interleukine-1 beta, and matrix metalloproteinase-9. Overall, our data provide new insights into METH abuse consequences, with a special focus on neurovascular dysfunction and neuroinflammatory response, which may help to find novel approaches to prevent or diminish brain dysfunction triggered by this overwhelming illicit drug.

Rat-strain dependent changes of dendritic and spine morphology in the hippocampus after cocaine self-administration.

We previously showed that cocaine self‐administration increases spine density in CA1 hippocampal neurons in Lewis (LEW) but not in Fischer 344 (F344) rats. Dendritic spine morphology is intimately related to its function. Thus, we conducted a 3D morphological analysis of CA1 dendrites and dendritic spines in these two strains of rats. Strain‐specific differences were observed prior to cocaine self‐administration: LEW rats had significantly larger dendritic diameters but lower spine density than the F344 strain. After cocaine self‐administration, proximal dendritic volume, dendritic surface area and spine density were increased in LEW rats, where a higher percentage of larger spines were also observed. In addition, we found a strong positive correlation between dendritic volume and spine morphology, and a moderate correlation between dendritic volume and spine density in cocaine self‐administered LEW rats, an effect that was not evident in any other condition. By contrast, after cocaine self‐administration, F334 rats showed decreased spine head volumes. Our findings suggest that genetic differences could play a key role in the structural plasticity induced by cocaine in CA1 pyramidal neurons. These cocaine‐induced alterations could be related to differences in the memory processing of drug reward cues that could potentially explain differential individual vulnerability to cocaine addiction.

Modulation of the mTOR pathway in male Lewis rats after morphine self-administration and subsequent extinction training

Addiction is a chronic disorder with an elevated risk of relapse, even after long periods of abstinence. Some of 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. Thus, we have assessed 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 some phosphoproteins in three brain areas related to reward learning and extinction: the amygdala, the nucleus accumbens, and the prefrontal cortex. We found an increase in Raptor and Eif4ebp2 gene expression in the amygdala of rats that self-administered morphine, and this persisted beyond the extinction period. 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. We also found a strong correlation between some of the biochemical variables measured and behavioral traits, suggesting a significant role for the genes and phosphoproteins identified, mostly in the amygdala, in the behavioral effects of morphine.