Exposure to glyphosate during pregnancy induces neurobehavioral alterations and downregulation of Wnt5a-CaMKII pathway.

Glyphosate-based formulations are the most popular herbicide used around the world. These herbicides are widely applied in agriculture to control weeds on genetically modified crops. Although there is much evidence showing that glyphosate-based herbicides induce toxic effect on reproductive and hepatic systems, and also cause oxidative damage on cells, studies from recent years revealed that the nervous system may represent a key target for their toxicity. In the present work, we evaluated the effect of glyphosate (without adjuvants) in neonate rats after gestational exposure. Particularly, we examined whether glyphosate during gestation affected the nervous system function at early development. Pregnant Wistar rats were treated with 24 or 35 mg/kg of pure glyphosate every 48 h and neurobehavioral studies were performed. Our results indicated that gestational exposure to glyphosate induced changes in reflexes development, motor activity and cognitive function, in a dose-dependent manner. To go further, we evaluated whether prenatal exposure to glyphosate affected the Ca+2-mediated Wnt non-canonical signaling pathway. Results indicated that embryos exposed to glyphosate showed an inhibition of Wnt5a-CaMKII signaling pathway, an essential cascade controlling the formation and integration of neural circuits. Taken together, these findings suggest that gestational exposure to glyphosate leads to a downregulation of Wnt/Ca+2 pathway that could induce a developmental neurotoxicity evidenced by deficits at behavioral and cognitive levels in rat pups.

Social Isolation in Male Rats During Adolescence Inhibits the Wnt/ß-Catenin Pathway in the Prefrontal Cortex and Enhances Anxiety and Cocaine-Induced Plasticity in Adulthood.

In adult animals, it is well established that stress has a proactive effect on psychostimulant responses. However, whether only a short period of stress during adolescence can also affect cocaine responses later in life and what mechanisms are involved are unknown. Here, we showed that 5 days of social isolation during rat adolescence had a long-term impact on anxiety-like behaviors, cocaine-induced conditioned place preference, and the expression of sensitization during adulthood. At the molecular level, social isolation decreased the activity of the Wnt/ß-catenin pathway in the prefrontal cortex (PFC). Furthermore, after the expression of cocaine sensitization, isolated rats showed an increase in this pathway in the nucleus accumbens. Together, these findings suggest that, adolescent social isolation by altering the Wnt/ß-catenin pathway in the developing PFC might increase the cocaine responses during adulthood, introducing this pathway as a novel neuroadaptation in the cortical-accumbens connection that may mediate a stress-induced increase in vulnerability to drugs.

Wnt/ß-catenin pathway in the prefrontal cortex is required for cocaine-induced neuroadaptations.

Behavioral sensitization is a progressive and enduring enhancement of the motor stimulant effects elicited by repeated administration of drugs of abuse. It can be divided into two distinct temporal and anatomical domains, termed initiation and expression, which are characterized by specific molecular and neurochemical changes. This study examines the role of the Wnt canonical pathway mediating the induction of cocaine sensitization. We found that ß-catenin levels in the prefrontal cortex (PFC), amygdala (Amyg) and dorsal striatum (CPu) are decreased in animals that show sensitization. Accordingly, GSK3ß activity levels are increased in the same areas. Moreover, ß-catenin levels in nuclear fraction, mRNA expression of Axin2 and Wnt7b are decreased in the PFC of sensitized animals. Then, in order to demonstrate that changes in the PFC are crucial for initiation of sensitization, we either rescue ß-catenin levels with a systemic treatment of a GSK3ß inhibitor (Lithium Chloride) or inhibit Wnt/ß-catenin pathway with an intracerebral infusion of Sulindac before each cocaine injection. As expected, rescuing ß-catenin levels in the PFC as well as CPu and Amyg blocks cocaine-induced sensitization, while decreasing ß-catenin levels exclusively in the PFC exacerbates it. Therefore, our results demonstrate a new role for the Wnt/ß-catenin pathway as a required neuroadaptation in inducing behavioral sensitization.

Role of Wnt/ß-catenin pathway in the nucleus accumbens in long-term cocaine-induced neuroplasticity: a possible novel target for addiction treatment.

Cocaine addiction is a chronic relapsing disorder characterized by the loss of control over drug-seeking and taking, and continued drug use regardless of adverse consequences. Despite years of research, effective treatments for psycho-stimulant addiction have not been identified. Persistent vulnerability to relapse arises from a number of long-lasting adaptations in the reward circuitry that mediate the enduring response to the drug. Recently, we reported that the activity of the canonical or Wnt/ß-catenin pathway in the prefrontal cortex (PFC) is very important in the early stages of cocaine-induced neuroadaptations. In the present work, our main goal was to elucidate the relevance of this pathway in cocaine-induced long-term neuroadaptations that may underlie relapse. We found that a cocaine challenge, after a period of abstinence, induced an increase in the activity of the pathway which is revealed as an increase in the total and nuclear levels of ß-catenin (final effector of the pathway) in the nucleus accumbens (NAcc), together with a decrease in the activity of glycogen synthase kinase 3ß (GSK3ß). Moreover, we found that the pharmacological modulation of the activity of the pathway has long-term effects on the cocaine-induced neuroplasticity at behavioral and molecular levels. All the results imply that changes in the Wnt/ß-catenin pathway effectors are long-term neuroadaptations necessary for the behavioral response to cocaine. Even though more research is needed, the present results introduce the Wnt canonical pathway as a possible target to manage cocaine long-term neuroadaptations.

Dopamine and glutamate release in the dorsolateral caudate putamen following withdrawal from cocaine self-administration in rats.

Evidence suggests that cocaine addiction may involve progressive neuroadaptive changes in the dorsolateral caudate putamen (dlCPu). While cocaine seeking following abstinence from chronic self-administration requires intact dlCPu function, in vivo neurotransmitter release in the dlCPu has not been investigated. The current study measured dlCPu dopamine (DA) and glutamate (GLU) release during drug seeking following limited or extended abstinence, as well as in response to a cocaine priming injection alone. Male, Sprague-Dawley rats self-administered cocaine (0.2mg/50µl infusion, i.v.) for 10days (2h/day). In vivo microdialysis occurred in the self-administration chamber after 1 and 14days of abstinence (Experiment 1). A separate set of animals that completed self-administration as well as drug naïve controls received a cocaine priming injection (20mg/kg) during concurrent microdialysis (Experiment 2). DA release increased during drug seeking in the self-administration context at both 1 and 14days post abstinence. In contrast, GLU release only increased after 1day of abstinence. Furthermore, animals with a cocaine self-administration history showed enhanced DA and GLU release following cocaine challenge as compared to drug naïve controls. These results indicate that chronic cocaine self-administration enhances dlCPu DA and GLU under both drug-paired context and drug-primed conditions.

Dorsal striatum mediation of cocaine-seeking after withdrawal from short or long daily access cocaine self-administration in rats.

Accumulating evidence has suggested that prolonged use of cocaine may lead to progressive neuroadaptations proceeding from ventral to more dorsal areas of the corpus striatum. We have previously found that reversible inactivation of the dorsolateral caudate/putamen (dlCPu) significantly attenuated cocaine-seeking in rats following chronic cocaine self-administration and withdrawal. Since the cumulative amount of cocaine intake and the time course of withdrawal emergent patterns have been previously shown to alter subsequent cocaine-seeking, the current study investigated the role of the dlCPu in cocaine-seeking after differing access periods of cocaine self-administration and abstinence time points. Rats were catheterized and implanted with infusion cannulae in the dlCPu, trained on cocaine self-administration (0.2 mg/50 µl/infusion), and then allowed to self-administer cocaine for 1 or 6 h daily sessions. After the final session, animals underwent three separate tests of cocaine-seeking in the self-administration context at days 1, 14, and 60 of abstinence immediately following bilateral infusion of baclofen-muscimol or vehicle into the dlCPu. While inactivation of the dlCPu by baclofen-muscimol resulted in reduced cocaine-seeking in both groups, the degree of inhibited responding varied with access history and withdrawal time point. While these data support a role for dorsal striatal regions in cocaine-seeking, greater previous cocaine intake did not lead to a greater dependence on intact dlCPu function for cocaine-seeking after abstinence.

P-glycoprotein inhibition potentiates the behavioural and neurochemical actions of risperidone in rats.

Antipsychotic drugs are the mainstay pharmacotherapy for schizophrenia and related psychiatric disorders. While the metabolic pathways of antipsychotic drugs have been well defined, the role of drug transporters in the disposition and effects of antipsychotic drugs has not been systematically explored. P-glycoprotein has ubiquitous expression in brain endothelial cells and plays a protective role by effluxing substrates for elimination and by limiting their accumulation in the central nervous system. Risperidone and several other antipsychotic drugs are substrates of P-glycoprotein. Increased antipsychotic drug entry into the brain via blockade of the P-glycoprotein transporter may facilitate the amount of available drug to its targets, particularly dopamine receptors. By increasing available antipsychotic drug concentrations, P-glycoprotein inhibition offers a novel means of enhanced drug delivery. This study evaluated whether selective P-glycoprotein transporter inhibition would increase the effects of risperidone on relevant indices of behaviour (catalepsy and locomotion) and neurochemistry (dopamine release and metabolism as measured by in-vivo microdialysis). We administered the P-glycoprotein inhibitor, PSC 833 (100 mg/kg p.o.), to rats prior to administration of risperidone at varying doses (0.01-4.0 mg/kg s.c.). P-glycoprotein inhibition significantly increased risperidone-induced cataleptic effects, blockade of amphetamine-induced locomotion, and effects on dopamine turnover as seen by increased striatal dopamine metabolite levels. These results provide functional evidence concordant with prior data for increased brain levels of risperidone following PSC 833 treatment.

Extended methamphetamine self-administration in rats results in a selective reduction of dopamine transporter levels in the prefrontal cortex and dorsal striatum not accompanied by marked monoaminergic depletion.

Chronic abuse of methamphetamine leads to cognitive dysfunction and high rates of relapse, paralleled by significant changes of brain dopamine and serotonin neurotransmission. Previously, we found that rats with extended access to methamphetamine self-administration displayed enhanced methamphetamine-primed reinstatement of drug-seeking and cognitive deficits relative to limited access animals. The present study investigated whether extended access to methamphetamine self-administration produced abnormalities in dopamine and serotonin systems in rat forebrain. Rats self-administered methamphetamine (0.02-mg/i.v. infusion) during daily 1-h sessions for 7 to 10 days, followed by either short- (1-h) or long-access (6-h) self-administration for 12 to 14 days. Lever responding was extinguished for 2 weeks before either reinstatement testing or rapid decapitation and tissue dissection. Tissue levels of monoamine transporters and markers of methamphetamine-induced toxicity were analyzed in several forebrain areas. Long-access methamphetamine self-administration resulted in escalation of daily drug intake ( approximately 7 mg/kg/day) and enhanced drug-primed reinstatement compared with the short-access group. Furthermore, long-, but not short-access to self-administered methamphetamine resulted in persistent decreases in dopamine transporter (DAT) protein levels in the prefrontal cortex and dorsal striatum. In contrast, only minor alterations in the tissue levels of dopamine or its metabolites were found, and no changes in markers specific for dopamine terminals or glial cell activation were detected. Our findings suggest that persistent methamphetamine seeking is associated with region-selective changes in DAT levels without accompanying monoaminergic neurotoxicity. Greater understanding of the neuroadaptations underlying persistent methamphetamine seeking and cognitive deficits could yield targets suitable for future therapeutic interventions.

Neuronal pentraxins modulate cocaine-induced neuroadaptations.

Neuronal pentraxins (NPs) function in the extracellular matrix to bind alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Three NPs have been described, neuronal activity-regulated pentraxin (Narp), which is regulated as an immediate early gene, NP1, and neuronal pentraxin receptor (NPR). Narp and NP1 enhance synaptogenesis and glutamate signaling by clustering AMPA receptors, whereas NPR contributes to removing AMPA receptors during group I metabotropic glutamate receptor-dependent long-term depression. Here, we examine mice with genetic deletions [knockout (KO)] of each NP to assess their contributions to cocaine-induced neuroplasticity. Consistent with a shared AMPA receptor clustering function for Narp and NP1, deletion of either NP caused similar behavioral alterations. Thus, although both Narp and NP1 deletion promoted cocaine-induced place preference, NPR deletion was without effect. In addition, although Narp and NP1 KO showed reduced time in the center of a novel environment, NPR KO mice spent more time in the center. Finally, although Narp and NP1 KO mice showed blunted locomotion after AMPA microinjection into the accumbens 3 weeks after discontinuing repeated cocaine injections, the AMPA response was augmented in NPR KO. Likewise, endogenous glutamate release elicited less motor activity in Narp KO mice. Consistent with reduced AMPA responsiveness after chronic cocaine in Narp KO mice, glutamate receptor 1 was reduced in the PSD fraction of Narp KO mice withdrawn from cocaine. These data indicate that NPs differentially contribute to cocaine-induced plasticity in a manner that parallels their actions in synaptic plasticity.

Integrin expression is altered after acute and chronic cocaine.

Cocaine addiction is associated with an increase in actin cycling and alterations in dendritic spines in the nucleus accumbens. Both actin polymerization and spine morphology are regulated in part by beta-(beta) integrins. Mice were administered acute or daily injections of cocaine or saline for 7 days. After 3 weeks of withdrawal, the level of beta-integrins in the postsynaptic density enriched subfraction from nucleus accumbens tissue was quantified by immunoblotting at 0, 30 or 120min following an a cocaine challenge injection. After chronic treatment and withdrawal the basal level of beta1-integrin was increased while beta3-integrin was unaltered. However, following a cocaine challenge in chronic cocaine, but not saline-treated animals, beta3-integrin was transiently up-regulated while beta1-integrin was transiently downregulated. These data demonstrate a bidirectional regulation of beta-integrins by chronic cocaine treatment that may contribute to cocaine-induced changes in actin cycling and dendrite morphology.

The Role of AMPAR Trafficking Mediated by Neuronal Pentraxins in Cocaine-induced Neuroadaptations.

Glutamate AMPA receptors (AMPAR) in the nucleus accumbens have an important role in cocaine-induced neuroadaptations. Neuronal pentraxins function in the extracellular matrix to bind AMPAR. Three neuronal pentraxins have been described Narp, NP1 and NPR. Narp and NP1 cluster AMPA receptors, while NPR contributes to removing AMPA receptors during mGluR-dependent long-term depression. We recently demonstrated that each pentraxin contributes to cocaine-induced neuroadaptations in a way that is consistent with its role in AMPAR clustering and trafficking. Thus, Narp and NP1 deletion promoted cocaine-induced place preference and showed blunted AMPA induced locomotion after cocaine withdrawal. In contrast NPR deletion augmented the AMPA response and was without effect on place preference. Consistent with reduced AMPA responsiveness after chronic cocaine in Narp KO mice, GluR1 was reduced in the postsynaptic density (PSD) fraction of Narp KO mice withdrawn from cocaine. These findings will be discussed in light of recent data showing that rats withdrawn from cocaine have marked deficits in developing long-term potentiation and long-term depression.

Long-term consequences of methamphetamine exposure in young adults are exacerbated in glial cell line-derived neurotrophic factor heterozygous mice.

Methamphetamine abuse in young adults has long-term deleterious effects on brain function that are associated with damage to monoaminergic neurons. Administration of glial cell line-derived neurotrophic factor (GDNF) protects dopamine neurons from the toxic effects of methamphetamine in animal models. Therefore, we hypothesized that a partial GDNF gene deletion would increase the susceptibility of mice to methamphetamine neurotoxicity during young adulthood and possibly increase age-related deterioration of behavior and dopamine function. Two weeks after a methamphetamine binge (4 x 10 mg/kg, i.p., at 2 h intervals), GDNF(+/-) mice had a significantly greater reduction of tyrosine hydroxylase immunoreactivity in the medial striatum, a proportionally greater depletion of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum, and a greater increase in activated microglia in the substantia nigra than wild-type mice. At 12 months of age, methamphetamine-treated GDNF(+/-) mice exhibited less motor activity and lower levels of tyrosine hydroxylase-immunoreactivity, dopamine, DOPAC, and serotonin than wild-type mice. Greater striatal dopamine transporter activity in GDNF(+/-) mice may underlie their differential response to methamphetamine. These data suggest the possibility that methamphetamine use in young adults, when combined with lower levels of GDNF throughout life, may precipitate the appearance of parkinsonian-like behaviors during aging.

A glutamate-dopamine interaction in the persistent enhanced response to amphetamine in nucleus accumbens core but not shell following a single restraint stress.

The administration of psychostimulant drugs or stress can elicit a sensitized response to the stimulating and reinforcing properties of the drug. We previously demonstrated that a single restraint stress session enhanced d-amphetamine (d-AMPH)-induced locomotion the day after the stress session, which lasted up to 8 days. The present experiments were designed to identify the contribution of major dopamine (DA) brain areas in the short- and long-lasting enhancement of d-AMPH-induced locomotion following a single stress, and to test the involvement of N-methyl-D-aspartate (NMDA) receptors in that phenomena. To achieve our goal, 24 h and 8 days after a 2-h restraint stress session either with or without a NMDA receptor blockade, we measured locomotor activity and DA overflow in nucleus accumbens (NAcc) core and shell and caudate putamen (CPu) following a d-AMPH injection (0.5 mg/kg i.p.). The stimulant effect of d-AMPH on DA overflow was enhanced in all nuclei at 24 h after a single stress, while at 8 days the enhanced responsiveness was maintained only in the NAcc core. When the rats were administered with MK-801 (0.1 mg/kg i.p.) 30 min before restraint stress, the d-AMPH-induced enhancement on locomotor activity and DA neurotransmission was prevented in all studied brain areas at both times. These findings show that a glutamate-dopamine link is underlying the short- and long- term d-AMPH-induced enhancement on DA and locomotor activity following stress. The persistent glutamate-dependent DA enhancement in NAcc core highlights the relevance of this region in the long-term proactive effects of stress on vulnerability to drug abuse.

Nrf2 gene deletion fails to alter psychostimulant-induced behavior or neurotoxicity.

The transcription factor NF-E2-related factor (Nrf2) regulates the induction of phase 2 detoxifying enzymes by oxidative stress, including synthesis of the catalytic subunit (xCT) of the heterodimeric cystine-glutamate exchanger (system xc-). Repeated cocaine treatment in rats causes persistent neuroadaptations in glutamate neurotransmission in the nucleus accumbens that result, in part, from reduced activity of system xc-. Since in vitro under- or over-expression of Nrf2 regulates system xc- activity and xCT content, it was hypothesized that in vivo deletion of the Nrf2 gene would: 1) decrease system xc- activity, 2) produce a behavioral phenotype resembling that elicited by chronic cocaine administration, and 3) enhance dopamine depletion after methamphetamine-induced oxidative stress. In all three experiments no genotypic difference was measured between mice sustaining homozygous Nrf2 gene deletion and wild-type littermates. Thus, while Nrf2 is a transcriptional regulator of xCT and capable of protecting cells from oxidative stress, following Nrf2 gene deletion this role can be partially compensated by other mechanisms and methamphetamine-induced oxidative stress and dopamine toxicity does not significantly involve Nrf2.