Fluoxetine potentiates methylphenidate-induced behavioral responses: Enhanced locomotion or stereotypies and facilitated acquisition of cocaine self-administration.

The medical psychostimulant methylphenidate (MP) is used to treat attention-deficit hyperactivity disorder and recreationally as a "cognitive enhancer". MP is a dopamine reuptake inhibitor, but does not affect serotonin. Serotonin contributes to addiction-related gene regulation and behavior. Previously, we showed that enhancing serotonin action by adding a selective serotonin reuptake inhibitor, fluoxetine (FLX), to MP potentiates MP-induced gene regulation in striatum and nucleus accumbens, mimicking cocaine effects. Here, we investigated the behavioral consequences of MP+FLX treatment. Young adult male rats received MP (5 mg/kg, i.p.) or MP+FLX (5 mg/kg each) daily for 6-8 days. Behavioral effects were assessed in an open-field test during the repeated treatment. Two weeks later the motor response to a cocaine challenge (25 mg/kg) and the rate of acquisition of cocaine self-administration behavior were determined. Our results demonstrate that FLX potentiates effects of MP on open-field behavior. However, we found differential behavioral responses to MP+FLX treatment, as approximately half of the rats developed high rates of focal stereotypies (termed "MP+FLX/high reactivity" group), whereas the other half did not, and only showed increased locomotion ("MP+FLX/low reactivity" group). Two weeks later, cocaine-induced locomotion and stereotypies were positively correlated with MP+FLX-induced behavior seen at the end of the repeated MP+FLX treatment. Moreover, the MP+FLX/high reactivity group, but not the low reactivity group, showed facilitated acquisition of cocaine self-administration. These results demonstrate that repeated MP+FLX treatment can facilitate subsequent cocaine taking behavior in a subpopulation of rats. These findings suggest that MP+FLX exposure in some individuals may increase the risk for psychostimulant use later in life.

Selective Regulation of 5-HT1B Serotonin Receptor Expression in the Striatum by Dopamine Depletion and Repeated L-DOPA Treatment: Relationship to L-DOPA-Induced Dyskinesias.

Dopamine and serotonin in the basal ganglia interact in a bidirectional manner. On the one hand, serotonin (5-HT) receptors regulate the effects of dopamine agonists on several levels, ranging from molecular signaling to behavior. These interactions include 5-HT receptor-mediated facilitation of dopamine receptor-induced gene regulation in striatal output pathways, which involves the 5-HT1B receptor and others. Conversely, there is evidence that dopamine action by psychostimulants regulates 5-HT1B receptor expression in the striatum. To further investigate the effects of dopamine and agonists on 5-HT receptors, we assessed the expression of 5-HT1B and other serotonin receptor subtypes in the striatum after unilateral dopamine depletion by 6-OHDA and subsequent treatment with L-DOPA (5 mg/kg; 4 weeks). Neither dopamine depletion nor L-DOPA treatment produced significant changes in 5-HT2C, 5-HT4, or 5-HT6 receptor expression in the striatum. In contrast, the 6-OHDA lesion caused a (modest) increase in 5-HT1B mRNA levels throughout the striatum. Moreover, repeated L-DOPA treatment markedly further elevated 5-HT1B expression in the dopamine-depleted striatum, an effect that was most robust in the sensorimotor striatum. A minor L-DOPA-induced increase in 5-HT1B expression was also seen in the intact striatum. These changes in 5-HT1B expression mimicked changes in the expression of neuropeptide markers (dynorphin, enkephalin mRNA) in striatal projection neurons. After repeated L-DOPA treatment, the severity of L-DOPA-induced dyskinesias and turning behavior was positively correlated with the increase in 5-HT1B expression in the associative, but not sensorimotor, striatum ipsilateral to the lesion, suggesting that associative striatal 5-HT1B receptors may play a role in L-DOPA-induced behavioral abnormalities.

The 5-HT1B serotonin receptor regulates methylphenidate-induced gene expression in the striatum: Differential effects on immediate-early genes.

Drug combinations that include a psychostimulant such as methylphenidate (Ritalin) and a selective serotonin reuptake inhibitor such as fluoxetine are indicated in several medical conditions. Co-exposure to these drugs also occurs with "cognitive enhancer" use by individuals treated with selective serotonin reuptake inhibitors. Methylphenidate, a dopamine reuptake inhibitor, by itself produces some addiction-related gene regulation in the striatum. We have demonstrated that co-administration of selective serotonin reuptake inhibitors potentiates these methylphenidate-induced molecular effects, thus producing a more "cocaine-like" profile. There is evidence that the 5-HT1B serotonin receptor subtype mediates some of the cocaine-induced gene regulation. We thus investigated whether the 5-HT1B receptor also modifies methylphenidate-induced gene regulation, by assessing effects of a selective 5-HT1B receptor agonist (CP94253) on immediate-early gene markers ( Zif268, c- Fos, Homer1a) in adolescent male rats. Gene expression was measured by in situ hybridization histochemistry. Our results show that CP94253 (3, 10 mg/kg) produced a dose-dependent potentiation of methylphenidate (5 mg/kg)-induced expression of Zif268 and c- Fos. This potentiation was widespread in the striatum and was maximal in lateral (sensorimotor) sectors, thus mimicking the effects seen after cocaine alone, or co-administration of fluoxetine. However, in contrast to fluoxetine, this 5-HT1B agonist did not influence methylphenidate-induced expression of Homer1a. CP94253 also potentiated methylphenidate-induced locomotor activity. These findings indicate that stimulation of the 5-HT1B receptor can enhance methylphenidate (dopamine)-induced gene regulation. This receptor may thus participate in the potentiation induced by fluoxetine (serotonin) and may serve as a pharmacological target to attenuate methylphenidate + selective serotonin reuptake inhibitor-induced "cocaine-like" effects.

Fluoxetine potentiation of methylphenidate-induced gene regulation in striatal output pathways: potential role for 5-HT1B receptor.

Drug combinations that include the psychostimulant methylphenidate plus a selective serotonin reuptake inhibitor (SSRI) such as fluoxetine are increasingly used in children and adolescents. For example, this combination is indicated in the treatment of attention-deficit/hyperactivity disorder and depression comorbidity and other mental disorders. Such co-exposure also occurs in patients on SSRIs who use methylphenidate as a cognitive enhancer. The neurobiological consequences of these drug combinations are poorly understood. Methylphenidate alone can produce gene regulation effects that mimic addiction-related gene regulation by cocaine, consistent with its moderate addiction liability. We have previously shown that combining SSRIs with methylphenidate potentiates methylphenidate-induced gene regulation in the striatum. The present study investigated which striatal output pathways are affected by the methylphenidate + fluoxetine combination, by assessing effects on pathway-specific neuropeptide markers, and which serotonin receptor subtypes may mediate these effects. Our results demonstrate that a 5-day repeated treatment with fluoxetine (5 mg/kg) potentiates methylphenidate (5 mg/kg)-induced expression of both dynorphin (direct pathway marker) and enkephalin (indirect pathway). These changes were accompanied by correlated increases in the expression of the 5-HT1B, but not 5-HT2C, serotonin receptor in the same striatal regions. A further study showed that the 5-HT1B receptor agonist CP94253 (3-10 mg/kg) mimics the fluoxetine potentiation of methylphenidate-induced gene regulation. These findings suggest a role for the 5-HT1B receptor in the fluoxetine effects on striatal gene regulation. Given that 5-HT1B receptors are known to facilitate addiction-related gene regulation and behavior, our results suggest that SSRIs may enhance the addiction liability of methylphenidate by increasing 5-HT1B receptor signaling.

Potentiated gene regulation by methylphenidate plus fluoxetine treatment: Long-term gene blunting (Zif268, Homer1a) and behavioral correlates.

Use of psychostimulants such as methylphenidate (Ritalin) in medical treatments and as cognitive enhancers in the healthy is increasing. Methylphenidate produces some addiction-related gene regulation in animal models. Recent findings show that combining selective serotonin reuptake inhibitor (SSRI) antidepressants such as fluoxetine with methylphenidate potentiates methylphenidate-induced gene regulation. We investigated the endurance of such abnormal gene regulation by assessing an established marker for altered gene regulation after drug treatments - blunting (repression) of immediate-early gene (IEG) inducibility - 14 days after repeated methylphenidate+fluoxetine treatment in adolescent rats. Thus, we measured the effects of a 6-day repeated treatment with methylphenidate (5 mg/kg), fluoxetine (5 mg/kg) or their combination on the inducibility (by cocaine) of neuroplasticity-related IEGs (Zif268, Homer1a) in the striatum, by in situ hybridization histochemistry. Repeated methylphenidate treatment alone produced modest gene blunting, while fluoxetine alone had no effect. In contrast, fluoxetine given in conjunction with methylphenidate produced pronounced potentiation of methylphenidate-induced blunting for both genes. This potentiation was seen in many functional domains of the striatum, but was most robust in the lateral, sensorimotor striatum. These enduring molecular changes were associated with potentiated induction of behavioral stereotypies in an open-field test. For illicit psychostimulants, blunting of gene expression is considered part of the molecular basis of addiction. Our results thus suggest that SSRIs such as fluoxetine may increase the addiction liability of methylphenidate. Key words: cognitive enhancer, dopamine, serotonin, gene expression, psychostimulant, SSRI antidepressant, striatum.

Fluoxetine potentiation of methylphenidate-induced neuropeptide expression in the striatum occurs selectively in direct pathway (striatonigral) neurons.

Concomitant therapies combining psychostimulants such as methylphenidate and selective serotonin reuptake inhibitors (SSRIs) are used to treat several mental disorders, including attention-deficit hyperactivity disorder/depression comorbidity. The neurobiological consequences of these drug combinations are poorly understood. Methylphenidate alone induces gene regulation that mimics partly effects of cocaine, consistent with some addiction liability. We previously showed that the SSRI fluoxetine potentiates methylphenidate-induced gene regulation in the striatum. The present study investigated which striatal output pathways are affected by the methylphenidate + fluoxetine combination, by assessing effects on pathway-specific neuropeptide markers. Results demonstrate that fluoxetine (5 mg/kg) potentiates methylphenidate (5 mg/kg)-induced expression of substance P and dynorphin, markers for direct pathway neurons. In contrast, no drug effects on the indirect pathway marker enkephalin were found. Because methylphenidate alone has minimal effects on dynorphin, the potentiation of dynorphin induction represents a more cocaine-like effect for the drug combination. On the other hand, the lack of an effect on enkephalin suggests a greater selectivity for the direct pathway compared with psychostimulants such as cocaine. Overall, the fluoxetine potentiation of gene regulation by methylphenidate occurs preferentially in sensorimotor striatal circuits, similar to other addictive psychostimulants. These results suggest that SSRIs may enhance the addiction liability of methylphenidate.

CB1 Cannabinoid Receptor Expression in the Striatum: Association with Corticostriatal Circuits and Developmental Regulation.

Corticostriatal circuits mediate various aspects of goal-directed behavior and are critically important for basal ganglia-related disorders. Activity in these circuits is regulated by the endocannabinoid system via stimulation of CB1 cannabinoid receptors. CB1 receptors are highly expressed in projection neurons and select interneurons of the striatum, but expression levels vary considerably between different striatal regions (functional domains). We investigated CB1 receptor expression within specific corticostriatal circuits by mapping CB1 mRNA levels in striatal sectors defined by their cortical inputs in rats. We also assessed changes in CB1 expression in the striatum during development. Our results show that CB1 expression is highest in juveniles (P25) and then progressively decreases toward adolescent (P40) and adult (P70) levels. At every age, CB1 receptors are predominantly expressed in sensorimotor striatal sectors, with considerably lower expression in associative and limbic sectors. Moreover, for most corticostriatal circuits there is an inverse relationship between cortical and striatal expression levels. Thus, striatal sectors with high CB1 expression (sensorimotor sectors) tend to receive inputs from cortical areas with low expression, while striatal sectors with low expression (associative/limbic sectors) receive inputs from cortical regions with higher expression (medial prefrontal cortex). In so far as CB1 mRNA levels reflect receptor function, our findings suggest differential CB1 signaling between different developmental stages and between sensorimotor and associative/limbic circuits. The regional distribution of CB1 receptor expression in the striatum further suggests that, in sensorimotor sectors, CB1 receptors mostly regulate GABA inputs from local axon collaterals of projection neurons, whereas in associative/limbic sectors, CB1 regulation of GABA inputs from interneurons and glutamate inputs may be more important.

Differential developmental trajectories for CB1 cannabinoid receptor expression in limbic/associative and sensorimotor cortical areas.

Cannabis use during adolescence is associated with an increased risk for schizophrenia and other disorders. The neuronal basis is unclear, but prefrontal cortical mechanisms have been implicated. Here, we investigated developmental changes in the endocannabinoid system by assessing expression and function of the CB1 cannabinoid receptor in prefrontal and other cortical areas in juvenile (postnatal day 25, P25), adolescent (P40), and adult (P70) rats. Overall, the expression of CB1 receptors in the cortex is highest in juveniles and drops thereafter toward adult levels. However, CB1 receptor expression follows distinct developmental trajectories in different cortical areas. The most pronounced and progressive decrease in CB1 expression was observed in medial prefrontal and other limbic/associative regions. In contrast, major changes in sensorimotor cortices occurred only after P40. We also assessed electrophysiological measures of CB1 receptor function and found that CB1-dependent inhibition of synaptic transmission in the prefrontal cortex follows the same developmental trajectory as observed for receptor expression. Together, these findings indicate that CB1 receptor-mediated signaling decreases during development but is differentially regulated in limbic/associative vs. sensorimotor systems. Therefore, cannabis use during adolescence likely differentially affects limbic/associative and sensorimotor cortical circuits.

Long-lasting dysregulation of gene expression in corticostriatal circuits after repeated cocaine treatment in adult rats: effects on zif 268 and homer 1a.

Human imaging studies show that psychostimulants such as cocaine produce functional changes in several areas of cortex and striatum. These may reflect neuronal changes related to addiction. We employed gene markers (zif 268 and homer 1a) that offer a high anatomical resolution to map cocaine-induced changes in 22 cortical areas and 23 functionally related striatal sectors, in order to determine the corticostriatal circuits altered by repeated cocaine exposure (25 mg/kg, 5 days). Effects were investigated 1 day and 21 days after repeated treatment to assess their longevity. Repeated cocaine treatment increased basal expression of zif 268 predominantly in sensorimotor areas of the cortex. This effect endured for 3 weeks in some areas. These changes were accompanied by attenuated gene induction by a cocaine challenge. In the insular cortex, the cocaine challenge produced a decrease in zif 268 expression after the 21-day, but not 1-day, withdrawal period. In the striatum, cocaine also affected mostly sensorimotor sectors. Repeated cocaine resulted in blunted inducibility of both zif 268 and homer 1a, changes that were still very robust 3 weeks later. Thus, our findings demonstrate that cocaine produces robust and long-lasting changes in gene regulation predominantly in sensorimotor corticostriatal circuits. These neuronal changes were associated with behavioral stereotypies, which are thought to reflect dysfunction in sensorimotor corticostriatal circuits. Future studies will have to elucidate the role of such neuronal changes in psychostimulant addiction.

Dysregulation of gene induction in corticostriatal circuits after repeated methylphenidate treatment in adolescent rats: differential effects on zif 268 and homer 1a.

Psychostimulants and other dopamine agonists produce molecular changes in neurons of cortico-basal ganglia-cortical circuits, and such neuronal changes are implicated in behavioural disorders. Methylphenidate, a psychostimulant that causes dopamine overflow (among other effects), alters gene regulation in neurons of the striatum. The present study compared the effects of acute and repeated methylphenidate treatment on cortical and striatal gene regulation in adolescent rats. Changes in the expression of the immediate-early genes zif 268 and homer 1a were mapped in 23 striatal sectors and 22 cortical areas that provide input to these striatal sectors, in order to determine whether specific corticostriatal circuits were affected by these treatments. Acute administration of methylphenidate (5 mg/kg, i.p.) produced modest zif 268 induction in cortical areas. These cortical zif 268 responses were correlated in magnitude with zif 268 induction in functionally related striatal sectors. In contrast, after repeated methylphenidate treatment (10 mg/kg, 7 days), cortical and striatal gene induction were dissociated. In these animals, the methylphenidate challenge (5 mg/kg) produced significantly greater gene induction (zif 268 and homer 1a) in the cortex. This enhanced response was widespread but regionally selective, as it occurred predominantly in premotor, motor and somatosensory cortical areas. At the same time, striatal gene induction was partly suppressed (zif 268) or unchanged (homer 1a). Thus, repeated methylphenidate treatment disrupted the normally coordinated gene activation patterns in cortical and striatal nodes of corticostriatal circuits. This drug-induced dissociation in cortical and striatal functioning was associated with enhanced levels of behavioural stereotypies, suggesting disrupted motor switching function.