Prefrontal cortical and striatal transcriptional responses to the reinforcing effect of repeated methylphenidate treatment in the spontaneously hypertensive rat, animal model of attention-deficit/hyperactivity disorder (ADHD)

Central nervous system (CNS) stimulants (e.g. methylphenidate and amphetamines) are recommended as first line medications for attention-deficit/hyperactivity disorder (ADHD [1]. Among the psychostimulants, methylphenidate is the most commonly prescribed [1]. Methylphenidate blocks the dopamine transporter (DAT), the key mechanism responsible for the removal of extracellular dopamine (DA), thereby elevating extracellular DA levels in various limbic, striatal, cortical, cerebellar terminal fields and increasing DA signaling and duration of DA response [2]. Neuroimaging studies showed that therapeutic doses of methylphenidate increased DA levels in the striatum and nucleus accumbens (NAc), a mechanism purportedly thought to explain methylphenidate-induced improvement of ADHD symptoms [3, 4]. However, in light of the observation that the effects of methylphenidate are similar to those exerted by other drugs affecting the CNS (e.g. addictive drugs such as cocaine or methamphetamine), the abuse potential of methylphenidate has been suggested [5].

The repeated use of stimulants can elicit adverse effects in behavior and induce drug-related behaviors such as sensitization, tolerance or dependence [6–8]. Therefore, the propensity of long-term methylphenidate treatment to induce drug addiction or dependence in individuals with ADHD has also been implied. A number of studies have investigated effects of methylphenidate treatment on subsequent abuse of other addictive drugs [9–13]. Unfortunately, however, only a few studies have sought to determine whether the repeated treatment of methylphenidate will result in the abuse of the drug itself. Furthermore, the long-term effects of repeated methylphenidate use in behavior and gene expression remain far from understood [14]. In particular, there is a dearth of information on the potential neuronal correlates of long-term recreational methylphenidate use or abuse in individuals with ADHD [8, 14].

Preclinical studies in animal models have provided an avenue for identifying the potential molecular neuropathobiology of drug-induced neuroadaptations [15]. Moreover, the use of microarray expression profiling has aided us in capturing neuroadaptations involving complex changes in gene expression that may underlie the development of drug addiction [15, 16]. The early microarray studies have shown that repeated methylphenidate treatment even at a clinically relevant dose produced changes in gene regulation in cortical and striatal neurons which are similar to those of cocaine and amphetamine, indicating addiction liability of the drug [14]. While identification of these gene sets represents a significant step, it is likely that there are other changes that have not been identified, especially those that result from the behavioral or cognitive processes associated with methylphenidate use, in addition to the direct pharmacological effects of the drug [17–19]. Indeed, by integrating methylphenidate-induced gene expression changes with outcomes in “appropriate” animal models of drug addiction (e.g. conditioned place preference [CPP] tests and drug self-administration), we identified a subset of neuronal development genes, which may also mediate the reinforcing effect of methylphenidate [19]. We suggested significant clinical implications of these findings on the abuse of methylphenidate among “healthy” individuals, as our studies [19] were conducted in Wistar rats, strain used to represent the “normal” heterogeneous population. However, the previous findings could not be generalized to individuals with ADHD who are also abusing methylphenidate, as experiments were not conducted in “appropriate” ADHD animal models.

To address this issue at the preclinical level and to shed some light on the potential molecular consequences of long-term recreational methylphenidate use or abuse in individuals with ADHD, we analyzed the differentially expressed genes (DEGs) in the brain of methylphenidate-pretreated Spontaneously Hypertensive rats (SHRs), which showed CPP to and self-administration of methylphenidate. The SHR, relative to the normotensive Wistar Kyoto rat strain, exhibits good face, construct and predictive validity mimicking the behavioral characteristics observed in ADHD, and is considered as the most widely used and validated animal model for this disorder [20, 21]. Abnormalities both in the genetic and neurotransmitter functions, such as those seen in ADHD were also observed in the SHR [22, 23]. Moreover, earlier studies have found increased reactivity of the SHR to stimulants, opioids, alcohol and other addictive drugs in comparison with other strains, indicating that the SHR, in general, may be used to investigate the relationship between ADHD and drug addiction [24]. Most studies on the molecular effects of stimulants have focused on gene regulation in DA target areas such as the striatum [25, 26]. In this regard, genome-wide transcriptional analyses were performed in the striatum because this brain region controls reward sensitivity, motor function and habit learning [27]. Furthermore, prefrontal cortices (PFC) were also included in these analyses because they include regions involved in processes relevant to drug addiction such as compulsive drug taking, and maintenance of behavioral sensitization [28, 29].

To model recreational methylphenidate use in individuals with ADHD, we subjected SHRs to repeated methylphenidate treatment (7 days, twice daily) at a dose (5 mg/kg, i.p.) beyond the clinically-relevant range (0.5-2 mg/kg, i.p.), and tested them for subsequent methylphenidate CPP and drug self-administration. In order to shed light on the potential molecular consequences of long-term recreational methylphenidate use or abuse in individuals with ADHD, we analyzed differentially expressed genes (DEGs) in the PFC and striatum of methylphenidate-pretreated SHRs, which showed methylphenidate reinforcement (i.e. CPP to and self-administration of methylphenidate). The findings from behavioral studies revealed methylphenidate CPP and acquisition of methylphenidate self-administration in methylphenidate-pretreated SHRs. Genome-wide analyses in the PFC and striatum of these rats showed that methylphenidate alters the expression of a number of genes involved in many functional systems. We discuss herein some of these important DEGs whose functions have already been characterized, and hence a role in methylphenidate reinforcement in SHRs can be extrapolated.

Previous studies showed that methylphenidate pretreatment at the 5 mg/kg dosage (i.p.) enhanced psychomotor response to cocaine in other rat strains (e.g. Sprague–Dawley rats and Wistar rats) [10]. Moreover, pretreatment at various dosages of methylphenidate also increased vulnerability to cocaine as well as to other addictive drugs [8, 10, 11]. We have previously reported enhancement of CPP response to methylphenidate in methylphenidate-pretreated (5 mg/kg, ip., 7 days, twice daily) Wistar rats [19] indicating the occurrence of behavioral sensitization, a phenomenon associated with enhanced behavioral response to repeated treatment of addictive drugs [37]. Drug-induced sensitization has been thought to underlie certain aspects of addiction; in particular, the neuroadaptations that accompany this phenomenon have been suggested to mediate the maintenance and reinstatement of addiction [37]. We observed in this study that methylphenidate pretreatment produced reinforcing effects in SHRs, however, we also found comparable CPP scores and similar rates of methylphenidate self-administration in both saline- and methylphenidate-pretreated rats (cohort 1 and 2 rats, respectively). Investigating the mechanism that underlies differential behavioral responses of strains to the reinforcing effects of repeated methylphenidate treatment is beyond the scope of this study [for review, however, see[38]]. Nevertheless, the present and the previous findings [19] appear to simulate the reported similarity in the rates of drug abuse or dependence to psychoactive substances in both ADHD and non-ADHD controls [39].

In general, our microarray studies revealed transcriptional changes associated with pharmacological effects of the drug or consequential to effects of chronic methylphenidate exposure (e.g. genes related with apoptosis, inflammation, etc.), although these analyses also found some DEGs whose expression may have been changed in response to the cognitive or behavioral processes associated with drug use (e.g. genes involved in neuronal and/or synaptic plasticity as well as in neuronal development) [[17, 19], for review see[18]]. In the PFC, methylphenidate differentially altered the expression of genes involved in apoptosis (e.g. S100a9, Angptl4, Nfkbia), transcription (Cebpb,Per3), and neuronal plasticity (Homer1, Jam2, Asap1). qRT-PCR validated differential expression patterns of S100a9, a gene that encodes S100a9, which together with S100a8 exerts broad apoptotic activities [40]. Psychostimulants, including methylphenidate [41], activate the production of superoxides [42], inflammatory cells (astrocytes and microglia), and they in turn have been shown to release numerous pro-inflammatory factors and cytokines [43]. Moreover, psychostimulant-induced increases in synaptic and cytosolic DA levels have been shown to be neurotoxic due to DA-induced production of reactive oxygen species (ROS), reactive nitrogen species, hydrogen peroxide and dopamine quinones [44–46]. Therefore, enhanced expression of apoptosis-related genes in the PFC (as well as in the striatum) of cohort 2 SHRs is consistent with the observed neurotoxic effects of long-term psychostimulant exposure. Additionally, these findings may also reflect other previously described neurotoxic effects of chronic methylphenidate treatment such as decreased antioxidant defenses promoting peripheral oxidative adaptation [47], altered Na⁺, K⁺-ATPase activity in the cerebrum thereby affecting cellular excitability [48], downregulation of activity regulated cytoskeletal gene expression interfering with long-term potentiation and consolidation of long-term memory [49], as well as drug-induced cognitive impairment on spatial reference and working memory tasks [50].

Of note, we observed a larger number of DEGs in the striatum when compared with the PFC in cohort 2 SHRs coinciding with the findings of our previous study [19], and corroborating the previous assumption on the crucial role of the striatum during compulsive drug use or abuse [51]. Interestingly, a majority of the DEGs in the striatum are downregulated genes associated with important cellular functions such as cell adhesion (e.g. Pcdh10, Ctbbd1, Itgb6), transcription (e.g. Notch3, Nsbp1, Sik1), mitochondrion organization (e.g. Prps18c, Letm1, Uqcrc2) and ubiquitin-mediated proteolysis (e.g. Nedd4, Usp27x, Ube2d2). The expression patterns of representative genes (e.g. Pcdh10, Ctnd1, Uqcrc2, Nedd4) from the different functional categories have been validated by confirmatory qRT-PCR analyses. However, it remains to be established whether changes in expression levels of these genes influenced methylphenidate reinforcement in drug-treated SHRs. Moreover, further studies are warranted to determine the contribution of these altered transcripts in the molecular mechanism of long-term recreational methylphenidate use or abuse in individuals with ADHD.

The decrease in Pcdh10 gene expression is both a novel and interesting observation considering the role of cell adhesion genes in maintaining neuronal and synaptic connections, and also in neuronal development [52]. Pcdh10, a member of the protocadherin gene family of cell adhesion molecules (CAMs), codes for a cadherin-related neuronal receptor involved in the establishment and function of specific cell-cell connections in the brain [53]. Furthermore, mutant analysis has demonstrated that loss of Pcdh10 can influence different aspects of development and post-natal life [48]. Whereas prior to this study there has been no experimental literature implicating the role of Pcdh10 in drug abuse, the involvement of this gene in a neurodevelopmental disorder, autism spectrum disorder, has already been reported [54]. Hence, downregulation of Pcdh10 gene expression observed in this study is an index of altered cell-to-cell communication, which may also signify disturbances in synaptic or neuronal plasticity within the striatal complex.

Altered gene expression of Uqcrc2 has also been confirmed by qRT-PCR. The Uqcrc2 gene is a component of the mitochondrial respiratory complex III, which codes for an enzyme involved in the electron transport chain. Furthermore, dysregulation of the expression of this gene has been implicated in some neuropsychiatric disorders such as bipolar disorder [55]. A previous study reported inhibition of mitochondrial respiratory chain complex III in the striatum as well as other brain regions of adult Wistar rats exposed to chronic (28 days) treatment of methylphenidate [56]. Downregulation of mitochondrial respiratory complex III (as well that of other complexes) has been viewed as a compensatory response in order to maintain energy homeostasis through enhancement of other metabolic systems [56]. Alternatively, it has been assumed to result from neurotoxic effects of methylphenidate via the generation of ROS [56]. Further studies are required to examine this point in detail. Nevertheless, the decrease in Uqcrc2 expression as observed in this study extends the previous findings [56], and indicates that methylphenidate treatment alters brain mitochondrial functions. These findings also suggest disruptions in brain energy metabolism in methylphenidate-treated and reinforced SHRs, although additional studies are required to confirm this.

Nedd4 gene expression has also been decreased in the striatum of cohort 2 SHR. Nedd4 belongs to a family of ubiquitin ligases, which has been shown to play key roles in both trafficking and degradation of proteins. In particular, Nedd4 has been hypothesized to maintain target protein at the plasma membrane by balancing steady state insertion and retrieval of proteins critical to the development and neuronal function in the brain [57]. Although further studies are required, downregulation of Nedd4 in this study may indicate disruption in the removal of misfolded or unwanted proteins causing their accumulation. The pathological consequences of this altered process in the context of long-term methylphenidate abuse are still unknown, although accumulation of unnecessary proteins resulting from a defective ubiquitin dependent proteolysis has been shown to contribute to aggregation events, a pathogenic mechanism in several neurobehavioral and neurodegenerative disorders [58, 59]. Furthermore, in view of the important roles of ubiquitin system in synaptic growth and function, downregulation of Nedd4 may also affect synaptic growth and plasticity, contributing to altered brain function.

Altered expression of genes associated with apoptosis, cell adhesion, mitochondria organization, ubiquitin-mediated proteolysis, etc. in the PFC and/or striatum of methylphenidate-treated SHRs which showed methylphenidate CPP and self-administration indicate methylphenidate-induced neurotoxicity, alterations in synaptic and neuronal plasticity, energy metabolism and ubiquitin-dependent protein degradation. In addition, these changes may also reflect cognitive impairment associated with chronic methylphenidate use as demonstrated in preclinical studies [e.g.[49, 50]]. Future studies should be performed to determine the link between changes in the expression of these genes and influence on methylphenidate reinforcement in SHRs. Moreover, additional studies are warranted to determine the clinical significance of the findings of this study with regard to methylphenidate abuse or addiction in individuals with ADHD. Nevertheless, this study presents new research directions and interesting topics for further investigation. For instance, it would be worthwhile to examine molecular changes that occur in the brains of methylphenidate-pretreated and methylphenidate-reinforced WKY. Providing answers to this query will establish whether the reported gene expression changes are exclusive to SHR and not possibly due to inbreeding. Furthermore, investigating gene expression changes in methylphenidate-pretreated animals which showed less methylphenidate CPP and self-administration will also provide important information not only on the mechanisms of methylphenidate abuse in ADHD individuals, but also on protection against methylphenidate addiction via development of novel targets for the treatment of methylphenidate or psychostimulant addiction. Along these lines, complementary findings can be obtained by comparing gene expression patterns associated with adolescent exposure to methylphenidate and atomoxetine, another widely-used ADHD drug without stimulant-like effects and assumed to be devoid of abuse liability, in view results from a previous study which showed differential effects of these drugs in SHRs [60].