Addiction-Related Gene Regulation: Risks of Exposure to Cognitive Enhancers vs. Other Psychostimulants

Abstract

1. Introduction

Cognitive enhancers, sometimes called “smart drugs” or “memory enhancers,” are substances taken with the expectation that they increase mental functions such as attention, concentration, alertness, memory, motivation, planning, and decision making (Svetlov et al., 2007; Lanni et al., 2008; Husain and Mehta, 2011). The most widely used cognitive enhancers include the psychostimulant medications methylphenidate (Ritalin, Concerta), amphetamine (Adderall), and modafinil (Provigil). The oldest of these drugs is amphetamine, which was first synthesized in 1887 and has been used in the clinic since the 1930s (Berman et al., 2009). Methylphenidate, first produced in 1944, has also been used as a medication for many decades (Leonard et al., 2004), whereas modafinil was introduced only in the early 1990s (Minzenberg and Carter, 2008).

2. Neurochemical effects of psychostimulant cognitive enhancers

2.1. Changes in monoamine transmission

Psychostimulants cause, among other effects, amplification of monoamine neurotransmission by promoting release and/or blocking reuptake of monoamines and thus prolonging their actions (Natarajan and Yamamoto, 2011; Sulzer, 2011). Psychostimulant-induced potentiation of the dopamine transmission (Di Chiara and Imperato, 1988) is considered critical to the addiction process, whereas serotonin and norepinephrine play modulatory roles (Berke and Hyman, 2000; Nestler, 2001).

Adderall is a mixture of D- and L-amphetamine salts (Berman et al., 2009) and, like cocaine, amphetamines produce elevated extracellular levels of the monoamines dopamine, norepinephrine, and serotonin (Di Chiara and Imperato, 1988; Hurd and Ungerstedt, 1989; Ritz et al., 1990; Kuczenski and Segal, 1997, 2001). Modafinil seems to primarily inhibit dopamine and norepinephrine reuptake (Madras et al., 2006; Volkow et al., 2009; Schmitt and Reith, 2011; Loland et al., 2012) but, probably indirectly, affects other neurotransmitters as well (e.g., histamine, orexin and serotonin; see Minzenberg and Carter, 2008). Better established are the effects of methylphenidate. Methylphenidate binds to and blocks the dopamine and norepinephrine transporters (Schweri et al., 1985; Gatley et al., 1996) and thus produces overflow of these two monoamines (Hurd and Ungerstedt, 1989; Kuczenski and Segal, 1997; Volkow et al., 1998; Gerasimov et al., 2000; Bymaster et al., 2002; Berridge et al., 2006). In contrast, methylphenidate has low affinity for the serotonin transporter (Pan et al., 1994; Wall et al., 1995; Gatley et al., 1996; Bymaster et al., 2002) and produces minimal or no effects on serotonin levels, even with high doses (30 mg/kg, i.p.) (Kuczenski and Segal, 1997; Segal and Kuczenski, 1999; Kankaanpaa et al., 2002).

In vivo microdialysis studies demonstrate that these psychostimulant-induced neurochemical effects are very robust in the prefrontal cortex and in parts of the basal ganglia (Figure 1), especially the striatum (dorsal striatum/caudate-putamen, ventral striatum/nucleus accumbens) (Di Chiara and Imperato, 1988; Hurd and Ungerstedt, 1989; Kuczenski and Segal, 1997, 2001; Gerasimov et al., 2000; Berridge et al., 2006). Low, clinically relevant doses of cognitive enhancers seem to preferentially boost extracellular levels of dopamine and norepinephrine in the prefrontal cortex (Berridge and Devilbiss, 2011, but see Volkow et al., 2001). Higher doses (presumably associated with abuse) predominantly affect dopamine in the striatum due to orders of magnitude higher levels of dopamine tissue content in the striatum.

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Figure 1

Schematic illustrations of cortico-basal ganglia-thalamocortical circuits. (A) Striatal sectors used for mapping gene expression and their main cortical inputs (arrows) are shown for frontal, rostral, middle, and caudal levels of the rat forebrain (for details, see Willuhn et al., 2003; Yano and Steiner, 2005a,b). Psychostimulant-induced gene regulation is maximal in the dorsal sensorimotor sectors of the middle and caudal striatum (darkest shading), which receive inputs from the medial agranular (M2), primary motor (M1), and somatosensory (SS) cortex (see Section 3.1.2). Limbic (white), associative (light grey), and sensorimotor sectors (darker grey) are indicated. The scatterplot (inset upper right) displays the association between methylphenidate-induced Zif268 expression in individual striatal sectors and Zif268 expression in their indicated cortical input regions (values averaged if more than one input). Values are differences in gene expression between animals sacrificed 40 minutes after methylphenidate administration (5 mg/kg, i.p.) and controls sacrificed immediately after drug injection, and are expressed as the percentage of maximal increase in the striatum (see Yano and Steiner, 2005a). CG, cingulate; I, insular; IL, infralimbic; I/LO, insular/lateral orbital; P, piriform; PL, prelimbic. *** p < 0.001. (B) Direct and indirect striatal output pathways in the cortico-basal ganglia-thalamocortical circuits. Direct pathway (striatonigral) neurons contain mainly D₁ dopamine receptors and the neuropeptides substance P (SP) and dynorphin (DYN), whereas neurons that give rise to the indirect pathway (striatopallidal neurons) express mostly D₂ receptors and the peptide enkephalin (ENK), in addition to their main neurotransmitter γ-aminobutyric acid (GABA). + and − denote facilitatory and inhibitory, respectively. GLU, glutamate; GPe, globus pallidus external segment; GPi, globus pallidus internal segment; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus

3. Gene regulation by amphetamine and cocaine in corticostriatal circuits

Most studies on the molecular effects of psychostimulants have focused on gene regulation in dopamine target areas, especially the striatum, which displays particularly robust changes in gene regulation after treatments with amphetamine, cocaine, and other abused drugs (Harlan and Garcia, 1998; Berke and Hyman, 2000).

The striatum, the main input nucleus of the basal ganglia, is an important component of cortico-basal ganglia-cortical circuits (Gerfen and Bolam, 2010; Figure 1), which play a critical role in motivational, executive, and motor aspects of all goal-directed behavior and thus in addiction (Steiner, 2010). Psychostimulant-induced molecular changes in these circuits through the striatum are important for various aspects of addiction, including abnormal reward processing, habit formation, and compulsive behavior (Robbins and Everitt, 1999; Berke and Hyman, 2000; Hyman and Malenka, 2001; Gerdeman et al., 2003; Everitt and Robbins, 2005; Belin and Everitt, 2010). However, some of the most affected (dorsal) striatal circuits (Willuhn et al., 2003; Yano and Steiner, 2005a; 2005b; Unal et al., 2009) also participate in frontostriatal attentional networks and may thus be a therapeutic target in ADHD (Robbins et al., 1998; Solanto, 2002). We therefore focus on psychostimulant-induced gene regulation in corticostriatal circuits.

Microarray investigations indicate that hundreds of genes are affected by dopamine and psychostimulants in these circuits (Berke et al., 1998; McClung and Nestler, 2003; Konradi et al., 2004; Yuferov et al., 2005; Adriani et al., 2006a; Adriani et al., 2006b; Black et al., 2006; Yano and Steiner, 2007; Heiman et al., 2008). However, the vast majority of studies have assessed effects on the expression of neuropeptide transmitters and immediate-early genes (IEGs). Neuropeptides are often selectively contained in specific neuronal subtypes and thus serve as cell type markers (see Section 3.1.3), but they also modulate basal ganglia functions on several levels (e.g., Steiner, 2010). IEGs are useful as markers for cell activation due to their rapid and transient induction by neuronal activity and drug treatments (Sharp et al., 1993; Chaudhuri, 1997; Harlan and Garcia, 1998). They are thus frequently used to map drug effects in the brain.

Immediate-early genes are also of interest because of their direct involvement in neuroplasticity. Many IEGs encode transcription factors that regulate the expression of other genes (e.g., c-Fos, Zif268; Knapska and Kaczmarek, 2004). Others (e.g., Homer 1a) code for members of a family of scaffolding proteins that anchor receptors to the postsynaptic density and play a role in receptor trafficking, dendritic spine formation, and other processes of synaptic plasticity (Xiao et al., 2000; Thomas, 2002). These latter processes may be involved in the abnormal spine formation in striatal neurons produced by psychostimulant treatment (Robinson and Kolb, 1997; Ferrario et al., 2005; Jedynak et al., 2007; Kim et al., 2009).

In the following sections, we first describe the functional domains of the striatum and then present studies that illustrate psychostimulant-induced effects on gene regulation in these domains. The findings reveal which cortico-basal ganglia-cortical circuits (functional domains, cell types) are affected by these drugs and establish a cellular framework for evaluating and understanding the effects of cognitive enhancers such as methylphenidate and modafinil. We then provide examples of molecular changes induced by repeated psychostimulant treatment and discuss their potential functional significance.

3.1. Corticostriatal circuits affected

3.1.1. Functional domains of the striatum

The functional domains of the striatum are defined by their cortical inputs (Figure 1). According to current models of basal ganglia function, the basal ganglia and cortex are interconnected by several parallel anatomical circuits/loops that arise in the cortex and project in a topographical manner to the striatum and from there via the basal ganglia output nuclei and thalamus back to the cortex (Alexander et al., 1986; Albin et al., 1989; Alexander et al., 1990; Groenewegen et al., 1990; Haber, 2003; Joel and Weiner, 1994; Redgrave et al., 2010). Functionally, these circuits can be roughly categorized as limbic, associative, and sensorimotor, arising, respectively, in the limbic, associative, and sensory and motor regions of the cortex and projecting to their associated domains in all basal ganglia nuclei. The behavioral consequences of psychostimulant-induced molecular changes (or indeed of any pathological changes) in the basal ganglia are therefore dependent on the particular circuits affected. Thus there is interest in determining which circuits/functional domains in the striatum are altered by these drugs.

3.1.2. Topography of psychostimulant-induced gene regulation

Early descriptions of the regional distribution of psychostimulant-induced gene regulation in the striatum were in relatively vague terms anatomically (e.g., dorsolateral quadrant, dorsal vs. ventral). Nevertheless, the findings were clear and consistent between laboratories that these effects differ considerably between the different striatal regions. This variability is characteristic of the effects of amphetamine, cocaine (Figure 2A), and methylphenidate (Figure 2B).

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Figure 2

Psychostimulant-induced immediate-early gene expression in corticostriatal circuits as determined by in situ hybridization histochemistry. (A) Cocaine-induced c-Fos expression. Top: Film autoradiograms depict c-Fos expression in coronal sections from rostral (top), middle (center), and caudal striatal levels (bottom) in rats that received a vehicle injection (control, left halfbrain) or a cocaine injection (25 mg/kg; right halfbrain) and were sacrificed 30 minutes later (Brandon and Steiner, 2003). Striatum (S) and nucleus accumbens (NAc) are outlined in the cocaine-treated animals. Note the considerable regional differences in the c-Fos response. Bottom: Time course of cocaine (30 mg/kg)-induced c-Fos and Zif268 expression in the dorsal striatum on the middle level (mean density, expressed as percentage of maximal induction) (Steiner and Gerfen, 1998). Basal expression is indicated by broken lines. (B) Methylphenidate-induced gene expression. Top: Film autoradiograms show c-Fos (left), Zif268 (middle), and Homer 1a expression (right) at 0 minutes (control, left halfbrain) and 40 minutes (c-Fos) or 1 hour (Zif268, Homer 1a) after methylphenidate injection (MP, 5 mg/kg, i.p.; right halfbrain) (Yano and Steiner, 2005a,b). Bottom: Time course of methylphenidate (5 mg/kg)-induced expression of c-Fos, Zif268, and Homer 1a for the dorsal striatal sector on the middle level (Yano and Steiner, 2005a,b). LS, lateral shell of nucleus accumbens

Graybiel and colleagues (1990) first showed that c-Fos induction by acute cocaine treatment, although widespread in the striatum, had a distinctive topography. It was most pronounced in the dorsal central portion of the sensorimotor striatum and was fairly limited (or absent) in parts of the ventral (limbic) striatum, including the nucleus accumbens (Figure 2A). Other early studies confirmed this general pattern for c-Fos and other genes (e.g., Young et al., 1991; Hope et al., 1992; Moratalla et al., 1992; Bhat and Baraban, 1993; Steiner and Gerfen, 1993; Johansson et al., 1994; see Harlan and Garcia, 1998 for a review of the early work).

Many investigators found an overall similar (dorsal-ventral) distribution for amphetamine effects (Graybiel et al., 1990; Moratalla et al., 1992; Wang et al., 1994a; Badiani et al., 1998; Adams et al., 2001), but there are also differences between amphetamine and cocaine effects, for example, in their distribution across the striatal patch/matrix compartments (Harlan and Garcia, 1998). In contrast to the rather uniform gene induction by cocaine in terms of patch/matrix distribution, the IEG response to amphetamine appears reduced in the matrix relative to that in the patches (striosomes) (Graybiel et al., 1990; Graybiel et al., 2000). This finding was confirmed by many (e.g., Moratalla et al., 1992; Nguyen et al., 1992; Wang et al., 1995) but not all (Johansson et al., 1994; Wang et al., 1994b; Jaber et al., 1995) subsequent studies.

To better relate psychostimulant-induced molecular changes to specific corticostriatal circuits/functional domains in the rat, we mapped striatal gene regulation using 23 sampling areas (sectors)—based largely on their predominant cortical inputs—on three rostrocaudal levels (Figure 1A) (Willuhn et al., 2003; Yano and Steiner, 2005a, b; Cotterly et al., 2007; Unal et al., 2009). These studies revealed the following patterns (see Steiner, 2010 for review):

1. The most robust cocaine-induced changes in gene regulation occur in sensorimotor sectors of the middle and caudal striatum (Figure 2A) (e.g., Steiner and Gerfen, 1993; Willuhn et al., 2003; Unal et al., 2009). A similar regional distribution has been shown for amphetamine (e.g., Badiani et al., 1998).

2. Within the sensorimotor striatum, maximal changes occur in the dorsal sectors (approximately the dorsal third) (Figure 2A). These sectors are unique in that they receive the densest input from the medial agranular cortex (M2; Figure 1A) (Reep et al., 2003) in addition to convergent inputs from the somatosensory (or visual) and primary motor cortex (cf. Willuhn et al., 2003). Surrounding tissue that is, to a lesser extent, also targeted by medial agranular projections also shows robust changes in gene expression. The rat medial agranular cortex has mixed prefrontal/premotor features (Reep et al., 1987; Passingham et al., 1988; Preuss, 1995; Reep et al., 2003; Uylings et al., 2003) and can therefore be considered a prefrontal/motor interface. Our findings thus indicate that sensorimotor striatal circuits under the influence of medial agranular (prefrontal/premotor) input are particularly prone to psychostimulant-induced neuroplasticity.

3. Medial and rostral striatal sectors (associative sectors) were affected to a lesser degree (Figure 2A). These sectors receive inputs from prefrontal regions including the cingulate, prelimbic, and orbital cortex (Figure 1A) (e.g., Berendse et al., 1992).

4. On all three rostrocaudal levels, minimal or no changes in gene regulation were seen in ventral striatal sectors (Figure 2A) that receive inputs mostly from the dorsal agranular insular cortex (Figure 1A) (e.g., Berendse et al., 1992).

5. Psychostimulant-induced molecular changes in the nucleus accumbens are well appreciated in the addiction literature (e.g., Graybiel et al., 1990; Hope et al., 1992; Hope et al., 1994; for reviews, see Berke and Hyman, 2000; Nestler, 2001) because they are implicated in motivational (reward) processes (Pierce and Kalivas, 1997). However, consistent with the earlier literature (see above), our studies show that gene regulation effects of cocaine in the nucleus accumbens (Figure 2A) are modest compared with those in the sensorimotor striatum (Steiner and Gerfen, 1993; Willuhn et al., 2003; Unal et al., 2009). This effect reflects the finding that cocaine strongly activates only a small proportion of sparsely distributed neurons in the nucleus accumbens (as well as in the most rostral striatum) (Mattson et al., 2008). The nucleus accumbens shell appears more affected than the core, and the most robust effects were seen in the lateral part of the shell (Unal et al., 2009), which also receives medial agranular input (Reep et al., 1987) in addition to inputs from the ventral agranular insular cortex (Berendse et al., 1992) and other limbic areas (e.g., McGeorge and Faull, 1989; Brog et al., 1993; Wright and Groenewegen, 1996). The functional significance of these lateral shell effects is not known, but it is of interest to note that the insular cortex, one of the input regions of that part of the nucleus accumbens, is associated with craving in drug addiction (Naqvi et al., 2007), which often drives relapse.

In summary, amphetamine and cocaine produce changes in gene regulation in limbic striatal regions, but these are relatively modest. These changes are likely involved in altered reward processing in addiction (e.g., Belin and Everitt, 2010). Studies that compared effects in different striatal regions demonstrate that more robust drug-induced changes in gene regulation occur in the sensorimotor striatum. These molecular changes probably mediate the functional changes seen in these regions as the addiction disorder progresses (Porrino et al., 2007). Behaviorally, sensorimotor striatal changes may be responsible for habitual and compulsive aspects of drug taking (Berke and Hyman, 2000; Gerdeman et al., 2003; Everitt and Robbins, 2005; Belin and Everitt, 2010), and are likely also important for relapse to drug seeking after abstinence (Vanderschuren et al., 2005; Fuchs et al., 2006; See et al., 2007).

3.1.3. Striatal cell types

The main cell type of the striatum is the medium-sized spiny projection neuron (“medium spiny neuron”); in the rat, interneurons account for less than 3% of striatal neurons (Oorschot, 2010, 2013). Colocalization studies indicate that psychostimulants affect gene regulation in projection neurons but have minimal or no effect in interneurons (Berretta et al., 1992). For example, cholinergic interneurons showed cocaine-induced IEG expression only in the ventromedial striatum and the medial shell of the nucleus accumbens but not in the core or in the dorsolateral striatum (Berlanga et al., 2003).

Striatal projection neurons are divided into two subtypes that are intermingled and approximately equal in number and that give rise to two different striatal output pathways (Figure 1B). The “direct pathway” (striatonigral neurons) connects the striatum directly to the basal ganglia output nuclei (substantia nigra pars reticulata, entopeduncular nucleus/internal pallidum); the “indirect pathway” begins with the striatopallidal neurons and projects to the output nuclei indirectly via the globus pallidus (external pallidum) and subthalamic nucleus (Gerfen and Bolam, 2010).

Both subtypes of striatal projection neurons use γ-aminobutyric acid as their main neurotransmitter, but they differ in a number of receptors and neuropeptides they express (Steiner and Gerfen, 1998; Heiman et al., 2008). Striatonigral neurons contain predominantly the D₁ receptor subtype and the neuropeptides substance P and dynorphin (Figure 1B), whereas striatopallidal neurons mostly express the D₂ receptor and the neuropeptide enkephalin. (Because of this differential receptor/neuropeptide distribution, these neurons are sometimes referred to as D₁ or D₂ neurons, respectively, and these neuropeptides often serve as markers to differentiate effects of drug treatments between these striatal output pathways).

These two striatal output pathways have opposite effects on basal ganglia output and motor control. According to current models of basal ganglia function (Figure 1B), activity in the direct pathway inhibits basal ganglia output, thus disinhibiting thalamocortical (and brainstem) activity (Chevalier and Deniau, 1990) and facilitating behavior, whereas activity in the indirect pathway (i.e., in striatopallidal neurons) results in disinhibition of basal ganglia output, thus arresting behavior (Albin et al., 1989; DeLong, 1990; Redgrave et al., 2010). It is thought that activity in the direct pathway (the “Go pathway”) functions to initiate (or facilitate selection of) motor programs, whereas activity in the indirect pathway (the “Stop pathway”) interrupts motor programs and/or suppresses unwanted (or incompatible) movement. (For an elegant recent demonstration of this oppositional movement control, see Kravitz et al., 2010.) Although these concepts were initially derived from anatomical findings in the dorsal/sensorimotor striatum (Alexander et al., 1986; Albin et al., 1989), recent results confirmed such antagonistic functions of these two pathways for cocaine-induced behavior mediated by the dorsal striatum (Ferguson et al., 2011) and reward processes mediated by the limbic/ventral striatum (Lobo et al., 2010).

Given the differential functional roles of the two subtypes of striatal projection neurons, it was of considerable interest to determine whether psychostimulants alter gene regulation in both subtypes or whether one is preferentially affected. Early clues were obtained from drug effects on the expression of the neuropeptides that are differentially localized in these neurons and thus serve as cell type markers (Steiner and Gerfen, 1998). Many studies showed that amphetamine and cocaine robustly induce expression of substance P and dynorphin (e.g., Hanson et al., 1987; Sivam, 1989; Hurd and Herkenham, 1992; Hurd and Herkenham, 1993; Steiner and Gerfen, 1993; Daunais and McGinty, 1994; Wang and McGinty, 1995a; Drago et al., 1996; Adams et al., 2001; Frankel et al., 2008), which are contained in striatonigral neurons. In contrast, enkephalin expression (in striatopallidal neurons) is only modestly affected by psychostimulants (Steiner and Gerfen, 1993; Jaber et al., 1995; Wang and McGinty, 1996a; Spangler et al., 1997; Mathieu-Kia and Besson, 1998). (It should be noted, however, that enkephalin expression is readily induced by glutamate receptor stimulation or D₂ receptor blockade [e.g., Steiner and Gerfen, 1999].)

Colocalization studies using neuropeptide messenger RNAs (mRNAs) or tract tracers as markers confirmed this differential gene regulation for IEGs as well. Amphetamine and cocaine induce IEGs predominantly in striatonigral neurons (Berretta et al., 1992; Cenci et al., 1992; Johansson et al., 1994; Jaber et al., 1995; Kosofsky et al., 1995; Badiani et al., 1999). However, depending on the treatment conditions (i.e., with enough cortical activation/glutamate input; see Steiner, 2010), some IEG induction also occurs in striatopallidal neurons (e.g., Jaber et al., 1995; Badiani et al., 1999; Uslaner et al., 2001; Ferguson and Robinson, 2004).

3.1.4. Dopamine receptor subtypes

The differential effects on striatonigral versus striatopallidal neurons are likely based on the differential distribution of dopamine receptor subtypes between the two projection neuron subtypes (Figure 1B): As mentioned above, D₁ receptors are predominantly expressed in striatonigral neurons, and D₂ receptors mostly in striatopallidal neurons (Gerfen et al., 1990; Le Moine et al., 1990; Le Moine et al., 1991; Curran and Watson, 1995; Le Moine and Bloch, 1995). Numerous studies show that D₁ receptor stimulation and resulting activation of second messenger signaling cascades (Bronson and Konradi, 2010; Caboche et al., 2010) are critical for psychostimulant-induced gene regulation in striatal neurons. Thus, IEG expression induced by amphetamine and cocaine is eliminated either by systemic or intrastriatal administration of D₁ receptor antagonists (Graybiel et al., 1990; Young et al., 1991; Moratalla et al., 1992; Cole et al., 1992; Steiner and Gerfen, 1995) or by targeted deletion of the D₁ receptor (D₁ receptor knockouts) (Drago et al., 1996; Moratalla et al., 1996b; Zhang et al., 2004).

D₂ receptors also affect gene regulation in striatal neurons. In contrast to D₁ receptors, however, stimulation of D₂ receptors inhibits gene expression in striatopallidal neurons (e.g., Gerfen et al., 1990; Le Moine et al., 1997; Pinna et al., 1997), whereas blockade of D₂ receptors (e.g., by antipsychotic drugs) increases gene expression in these neurons (e.g., Steiner and Gerfen, 1998). This difference in effect presumably reflects the fact that D₂ receptors inhibit second messenger signaling, as opposed to the stimulatory action of D₁ receptors (Bronson and Konradi, 2010). However, stimulation of D₂ plus D₁ receptors potentiates D₁ receptor–mediated gene regulation in striatonigral neurons (D₁–D₂ receptor synergy; e.g., Paul et al., 1992; LaHoste et al., 1993; Gerfen et al., 1995). Consistent with this observation, a full gene response to psychostimulants requires combined stimulation of D₁ and D₂ receptors (Ruskin and Marshall, 1994). This interaction between D₁ and D₂ receptors is thought to be mediated by cholinergic interneurons (Wang and McGinty, 1996b; Pisani et al., 2007)—for example, via a D₂ receptor–mediated inhibition of inhibitory cholinergic input to striatonigral neurons (Wang and McGinty, 1996b).

In addition, D₃ receptors modify such molecular effects. These receptors are predominantly present in ventral striatal regions where they are partly coexpressed with D₁ receptors in striatonigral neurons (Le Moine and Bloch, 1996; Schwartz et al., 1998). Because they also exert opposite (inhibitory) effects on second messenger signaling (Zhang et al., 2004), D₃ receptors dampen gene induction by D₁ receptor stimulation (Carta et al., 2000; Zhang et al., 2004).

In summary, these findings demonstrate that (1) amphetamine- and cocaine-induced changes in gene regulation in the striatum occur preferentially (but not exclusively) in direct pathway (striatonigral) neurons (see also Lobo and Nestler, 2011), and (2) D₁ receptors (and their downstream signaling cascades; Caboche et al., 2010) are critical for these molecular changes.

4. Gene regulation by oral Adderall

The above reviewed effects of amphetamine in animal studies were mostly obtained with intraperitoneal (i.p.) or subcutaneous (s.c.) administration of relatively high doses (~3–10 mg/kg). How relevant are these findings for therapeutic use of Adderall, which involves lower doses and predominantly oral administration?

Psychostimulant effects on gene regulation are dose-dependent. Higher doses produce greater increases in gene expression across a wide range of doses (e.g., Steiner and Gerfen, 1993; Wang and McGinty, 1995b, 1997; Brandon and Steiner, 2003; Chase et al., 2003; Chase et al., 2005a; Yano and Steiner, 2005b). [Occasionally, very high doses have been found to result in attenuated expression (Wang and McGinty, 1995b, 1997), similar to other neuronal effects (e.g., Hanson et al., 2002), possibly due to receptor inactivation (internalization) by the high dose or other mechanisms.]

Gene regulation is also under the control of the drug delivery rate. For example, fast intravenous (i.v.) delivery of a certain cocaine dose (2 mg/kg) produced greater c-Fos induction in the striatum than slower delivery of the same dose (Samaha et al., 2004; Samaha and Robinson, 2005). Similarly, with repeated treatment (self-administration model), faster drug delivery produced more robust blunting of c-Fos inducibility (Wakabayashi et al., 2010). These enhanced neuronal changes were associated with indices of a greater addiction liability (greater escalation of drug intake and propensity to relapse; Wakabayashi et al., 2010).

Conversely, oral (or intragastric) administration of drugs produces slower (and lower) uptake (Swanson and Volkow, 2003; Kuczenski and Segal, 2005; Yano and Steiner, 2007), which would thus be expected to produce less molecular changes. Few studies have investigated the molecular effects of oral amphetamine administration in a therapeutic dose range. Researchers recently used a model with prepubertal rats to assess whether a low dose (1.6 mg/kg) of orally (p.o.) administered Adderall (mix of D- and L-amphetamine), which resulted in amphetamine levels in the blood close to those of children treated with amphetamine, caused changes in c-Fos expression in corticostriatal circuits (Allen et al., 2010). The results showed that despite the low dose and oral route, acute Adderall administration produced significant c-Fos induction in the striatum and cortex (Allen et al., 2010). Moreover, repeated treatment (1.6 mg/kg, p.o., once daily for 14 days) resulted in blunting of c-Fos inducibility in these brain regions (Allen et al., 2010). An earlier study in adult cats reported that 1 mg/kg (p.o.) of amphetamine induced c-Fos in the cortex and striatum (Lin et al., 1996). Consistent with these findings, another study in young rats showed that repeated treatment with a low dose of amphetamine (0.5 mg/kg, s.c., twice daily for 13 days), which resulted in amphetamine plasma concentrations corresponding to the clinical range used in the treatment of ADHD, produced altered dendritic architecture in the prefrontal cortex (Diaz Heijtz et al., 2003.

In summary, the findings obtained with faster administration and higher doses of amphetamine (and cocaine) may be more relevant for abuse of psychostimulants. But the results described above indicate that therapeutically relevant amphetamine doses and routes of administration can produce qualitatively similar molecular changes in neurons of corticostriatal circuits (Carrey and Wilkinson, 2011).

5. Gene regulation by methylphenidate

Methylphenidate, widely used in the treatment of ADHD and other mental disorders, is also popular as a cognitive enhancer (see Introduction). Although methylphenidate has been effective in the clinic for several decades, assessment of its molecular impacts began only about 10 years ago (Yano and Steiner, 2007). Because both clinical and recreational exposure to methylphenidate occurs predominantly in children and adolescents, preclinical studies often focus on the effects in prepubertal/adolescent animals (Yano and Steiner, 2007; Carrey and Wilkinson, 2011; Marco et al., 2011).

Early microarray studies in adolescent rats showed that acute and repeated treatment with 2 mg/kg (i.p.) of methylphenidate altered the expression of more than 2,000 genes in the striatum (Adriani et al., 2006a,b). Similar to other psychostimulants (Section 3), methylphenidate affected genes that encode transcription factors, neurotransmitter receptors, ion channels, postsynaptic density proteins, and other signaling-related molecules as well as many other classes (e.g., molecules involved in cell migration, survival, maturation, and other forms of neuroplasticity) (Adriani et al., 2006a,b; see also Yano and Steiner, 2007; Carrey and Wilkinson, 2011; Marco et al., 2011). Some of the molecular changes persisted well past the termination of the drug treatment, into the adulthood of the animals (Adriani et al., 2006a,b; for similarly long-lasting changes, see Chase et al., 2007; Warren et al., 2011).

Most of these wide-ranging effects will have to be confirmed in follow-up studies; but the effects on the expression of transcription factors/IEGs and neuropeptides in corticostriatal circuits are well established. We summarize these findings here for comparison with the effects of amphetamine and cocaine described above.

5.1. Regulation of immediate-early genes and neuropeptides

The first demonstration of gene regulation by methylphenidate was through oral administration in adult cats (Lin et al., 1996). This study showed that 2.5 mg/kg (p.o.) induced c-Fos expression in many brain areas, including the cortex and striatum. The regional patterns were described as “highly similar” to those produced by 1 mg/kg (p.o.) of amphetamine (these doses were compared because of their similar effects on wakefulness). Importantly, the c-Fos expression patterns of both drugs were different from those induced by modafinil (5 mg/kg, p.o.), also chosen for a similar waking effect (Lin et al., 1996). This comparison indicates that these regional patterns reflect the pharmacological targets of these drugs rather than their waking effect. Acute c-Fos induction by methylphenidate in the striatum (Figure 2B) was confirmed by many subsequent studies in both mice (Penner et al., 2002; Trinh et al., 2003; Hawken et al., 2004) and rats (Brandon and Steiner, 2003; Chase et al., 2003; Chase et al., 2005b; Yano and Steiner, 2005b).

Examples of other IEGs induced in the striatum include the transcription factors Zif268 (Figure 2B) (Brandon and Steiner, 2003; Yano and Steiner, 2005a) and FosB (Chase et al., 2005a) as well as the effector IEGs Arc (Chase et al., 2007; Banerjee et al., 2009) and Homer 1a (Figure 2B) (Yano and Steiner, 2005a; Adriani et al., 2006a; Cotterly et al., 2007).

A few studies show that methylphenidate also affects neuropeptide markers in striatal output neurons. These studies indicate that methylphenidate increases substance P expression (striatonigral neurons) (Figure 3) in a manner similar to other psychostimulants, whereas the opioid peptides dynorphin (striatonigral neurons) and enkephalin (striatopallidal neurons) appear to be less affected (Yano and Steiner, 2007).

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Figure 3

Methylphenidate-induced neuropeptide expression. Top: Film autoradiograms depict substance P expression in the middle striatum at 0 minutes (control, left halfbrain) and 1 hour after injection of methylphenidate (MP, 5 mg/kg, i.p.; right halfbrain). Bottom: Time course of methylphenidate (5 mg/kg)-induced expression of substance P (SP), dynorphin (DYN), and enkephalin (ENK) (in percentage of basal expression) for the dorsal striatal sector on the middle level (Yano and Steiner, 2005b). Note that substance P expression increased in 13 of the 23 striatal sectors, whereas dynorphin and enkephalin expression significantly increased in only two and one sector, respectively (values in area of maximal increase are shown here; see Yano and Steiner, 2005b, for details). ** p < 0.01, * p < 0.05 versus 0 minutes.

We directly compared methylphenidate effects on the expression of these genes by monitoring their mRNA levels between 20 minutes and 24 hours after acute injection of methylphenidate (2–10 mg/kg, i.p., adult rats; Yano and Steiner, 2005b) (Figure 3). Similar to the effects of cocaine/amphetamine (see Section 3.1.3), we found that substance P expression increased in many striatal sectors in a dose-dependent and very robust manner, with elevated mRNA levels present within 20 minutes and lasting for more than 3 hours. Conversely, for dynorphin mRNA, we detected a statistically significant, if modest, increase only in two sectors at 1 hour (Figure 3) (Yano and Steiner, 2005b). This latter finding contrasts with studies on amphetamine and cocaine effects, which showed that significantly increased dynorphin mRNA levels are present within 30 minutes (Willuhn et al., 2003), are prominent at 2 to 3 hours (Hurd and Herkenham, 1992; Smith and McGinty, 1994), and last 18 to 30 hours (Smith and McGinty, 1994; Wang et al., 1995) after acute drug administration.

Enkephalin, which is strongly induced, for example, by D2 receptor antagonists (Steiner and Gerfen, 1998), is only moderately affected by acute cocaine and amphetamine treatments (Hurd and Herkenham, 1992; Steiner and Gerfen, 1993; Wang and McGinty, 1995, 1996a). Acute methylphenidate did not produce consistent effects on enkephalin expression (Yano and Steiner, 2005b).

Neurotensin is another neuropeptide expressed in striatal output pathways; it is contained in both types of projection neurons, and its expression is also regulated by D1, D2 and glutamate receptors (see Hanson et al., 1992; Alburges et al., 2011). Its apparent interactions with the mesolimbic and mesostriatal dopamine systems suggest that neurotensin may influence the addictive properties of psychostimulants (cf. Alburges et al., 2011). Both cocaine and amphetamine treatments produce increased neurotensin expression (e.g., Letter et al., 1987; Hanson et al., 1989; Gygi et al., 1994), and a recent study shows that methylphenidate increases neurotensin expression as well (Alburges et al., 2011).

Overall, the molecular effects of methylphenidate described above typically emerged with doses of ≥2 mg/kg (i.p. or s.c.), and juvenile/adolescent rodents tended to be more sensitive than adults, as is true with other psychostimulants (Yano and Steiner, 2007; Carrey and Wilkinson, 2011).

6. Gene regulation by modafinil

Modafinil is a relatively novel agent that promotes wakefulness and is thus widely used to treat excessive daytime sleepiness associated with narcolepsy and other sleep disorders, but it is also gaining popularity as a cognitive enhancer (see Introduction). Its effects on gene regulation have been described in only a handful of studies.

To our knowledge, the first study to indicate such molecular effects showed increased expression of glutamine synthetase, an enzyme involved in brain metabolism, after a single injection of modafinil in rats (Touret et al., 1994). More recently, a gene microarray study identified several molecule classes affected by modafinil, including transcription factors such as c-Fos (Hasan et al., 2009), and thus suggested that modafinil exposure may alter various neuronal processes. However, most studies to date have assessed only c-Fos expression (Fos immunoreactivity) as a marker to identify neuronal systems involved in the regulation of sleep and wakefulness.

The 1996 study by Lin and colleagues showed that in adult cats modafinil (5 mg/kg, p.o.) produced minor c-Fos induction in striatum and cortex (i.e., considerably less than induced by methylphenidate [2.5 mg/kg, p.o.] and amphetamine [1 mg/kg, p.o.], despite causing similar wakefulness), but induced pronounced c-Fos expression in the hypothalamus and other brain regions (Lin et al., 1996). A study in rats (300 mg/kg, i.p.; Engber et al., 1998) confirmed these effects.

More recent work found c-Fos induction by modafinil (75–300 mg/kg, i.p.) in various nuclei from the hypothalamus to the brainstem, but also described considerable induction in the cortex and striatum in mice (Willie et al., 2005; Hasan et al., 2009) and rats (Scammell et al., 2000; Fiocchi et al., 2009). Based on findings with other psychostimulants, which typically show correlated regulation of several genes (Steiner and Gerfen, 1993; Willuhn et al., 2003; Yano and Steiner, 2005a,b; Unal et al., 2009), it is likely that modafinil alters the expression of various genes in concert in these brain regions.

Little is known about regional variations in the cortex and striatum or about the cell types and receptors involved. Given that less than 3% of striatal neurons are interneurons (Oorschot, 2010), it is clear that striatal c-Fos induction in these studies also predominantly occurred in projection neurons. In most studies, c-Fos induction in the dorsal striatum was abundant, while the nucleus accumbens showed only modest (Willie et al., 2005) or no induction (Scammell et al., 2000; Fiocchi et al., 2009). A recent mapping study in rats used a modafinil dose (10 mg/kg, i.v.) that produced clinically relevant plasma levels and found the most robust increase in c-Fos expression in the dorsomedial striatum, with a significant c-Fos response also in the nucleus accumbens shell (but not core) and the cingulate cortex (Gozzi et al., 2012).

Based on the mechanisms underlying gene regulation by other dopamine-enhancing drugs, it can be assumed that dopamine receptors are also important for modafinil-induced gene regulation, although this remains to be determined. However, in support of this notion, recent studies showed that D₁ (and D₂) receptors are important for modafinil-induced increases in motivation and arousal (Qu et al., 2008; Young and Geyer, 2010). Future studies will have to elucidate which corticostriatal circuits are affected and identify the mechanisms underlying these molecular changes.

In summary, knowledge on the molecular effects of modafinil in corticostriatal circuits is currently limited, but early findings suggest that this psychostimulant may have the potential to produce effects that are qualitatively similar to those of cocaine, amphetamine, and methylphenidate.

7. Drug interactions: SSRI antidepressants potentiate methylphenidate-induced gene regulation

As discussed, drug-induced gene regulation in neurons critically depends on the neurochemical effects of the drug. In this section we address an aspect of drug treatments that is often overlooked in the assessment of addiction liability: drug interactions based on the neurochemical effects.

If a combination drug treatment results in altered net neurochemical effects, modified gene regulation and thus presumably addiction liability should be expected. Such drug interactions in gene regulation have recently been shown for methylphenidate and certain prescription medications that modify serotonin transmission. Methylphenidate alone increases dopamine overflow but does not affect serotonin (e.g., Kuczenski and Segal, 1997; Borycz et al., 2008; see Section 2.1) and appears to have a reduced propensity to produce neuroadaptations compared with cocaine and amphetamine. In contrast to methylphenidate, cocaine and amphetamine elevate extracellular serotonin levels as well (Yano and Steiner, 2007). Would a combination treatment of methylphenidate with a drug that enhances serotonin action therefore produce more cocaine-/amphetamine-like gene regulation?

A host of findings support this possibility. For example, studies have shown that serotonin contributes significantly to various behavioral effects of cocaine (for reviews, see Filip et al., 2005; Muller and Huston, 2006; Carey et al., 2008). Similarly, whereas dopamine is critical for cocaine-induced gene regulation in the striatum (see Section 3.1.4), serotonin facilitates such effects (Bhat and Baraban, 1993). Thus, attenuation of the serotonin transmission by transmitter depletion (Bhat and Baraban, 1993), receptor antagonism (Lucas et al., 1997; Castanon et al., 2000), or receptor deletion (Lucas et al., 1997) reduces IEG induction by cocaine in the striatum. Conversely, direct and indirect serotonin receptor agonists increase the expression of IEGs (Li and Rowland, 1993; Torres and Rivier, 1994; Wirtshafter and Cook, 1998; Gardier et al., 2000) and other genes (e.g., Mijnster et al., 1998; Morris et al., 1988; Walker et al., 1996) in the striatum.

We therefore investigated whether enhancing serotonin transmission by an SSRI (selective serotonin reuptake inhibitor) antidepressant in conjunction with methylphenidate treatment would modify methylphenidate-induced gene regulation. Our results show that this is indeed the case: adding an SSRI (fluoxetine or citalopram) to methylphenidate treatment potentiates acute induction of IEGs (Steiner et al., 2010; Van Waes et al., 2010), and substance P and dynorphin (but not enkephalin) (Van Waes et al., 2012a) in the striatum (Figure 4). Moreover, repeated treatment with the methylphenidate+SSRI combination produced potentiated blunting of IEG inducibility and increased dynorphin expression (Van Waes et al., 2012b). This SSRI potentiation of methylphenidate-induced gene regulation was present in most striatal sectors (Figure 4B) but was maximal in the lateral sensorimotor striatum (Van Waes et al., 2010; Van Waes et al., 2012a), mimicking cocaine effects. Behaviorally, these SSRIs potentiated methylphenidate-induced locomotion (Borycz et al., 2008) and stereotypies (Van Waes et al., 2010), and produced other behavioral changes (e.g., enhanced sensitivity to cocaine reward and stress-eliciting situations; Warren et al., 2011; see Section 8.1).

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Figure 4

Fluoxetine potentiates methylphenidate-induced gene regulation. (A) Film autoradiograms depict expression of Zif268 in the middle striatum for rats that received a single injection of vehicle (V), methylphenidate (MP, 5 mg/kg), fluoxetine (FLX, 5 mg/kg), or a combination of methylphenidate plus fluoxetine (Van Waes et al., 2010). (B) Association between the potentiation of Zif268 expression (at 40 minutes; Van Waes et al., 2010) and that of substance P expression (at 90 minutes after drug injection; Van Waes et al., 2012a) in the 23 striatal sectors (open diamonds, sensorimotor; full circles, non-sensorimotor; data expressed as percentage of maximal increase). Potentiation is the difference between MP+FLX and MP groups. The potentiation was most robust in sectors of the sensorimotor striatum (open diamonds). *** p < 0.001

The potential significance of these findings relates to the medical use of methylphenidate and SSRIs. SSRIs such as fluoxetine are among the first-line treatments for several depressive and anxiety disorders (Petersen et al., 2002) and are given to millions of patients in the United States alone every year. As discussed, methylphenidate is used both in the treatment of conditions such as ADHD (Biederman et al., 2007; Swanson and Volkow, 2008) and as a recreational drug and cognitive enhancer (Greely et al., 2008; Kollins, 2008; Wilens et al., 2008). The rate of accidental coexposure due to such overlapping drug use/treatments is unclear, but combination therapies of methylphenidate and an SSRI are indicated for several conditions, including ADHD and anxiety/depression comorbidity (Safer et al., 2003; Bhatara et al., 2004; Kollins, 2008). Methylphenidate is also combined with SSRIs as augmentation therapy in major depressive disorder (e.g., Nelson, 2007; Ishii et al., 2008; Ravindran et al., 2008), as acceleration treatment for SSRIs (e.g., Lavretsky et al., 2003), and as treatment for sexual dysfunction related to SSRIs (e.g., Csoka et al., 2008).

Further studies are necessary to determine how much methylphenidate-SSRI coexposure occurs due to clinical administration or as a result of uncontrolled cognitive enhancer use by patients on SSRIs and whether such coexposure enhances the addiction liability of methylphenidate, as the potentiated gene regulation effects might suggest.

8. Behavioral consequences and clinical considerations

While the potential benefits and ethics of cognitive enhancer use are being debated (e.g., Farah et al., 2004; Greely et al., 2008; Chatterjee, 2009; Harris, 2009; Outram, 2010; Hyman, 2011), developmental neurobiologists and addiction researchers warn that the long-term consequences of protracted use of these psychostimulants, especially during brain development, are hardly understood (e.g., Carlezon and Konradi, 2004; Andersen, 2005; Swanson and Volkow, 2008; Berman et al., 2009) (for reviews of neurobehavioral effects of SSRI exposure during development, see, e.g., Oberlander et al., 2009; Olivier et al., 2011). What are the known behavioral consequences of exposure to psychostimulant cognitive enhancers?

9. Conclusions

The findings we have summarized show that psychostimulants such as cocaine and amphetamine produce changes in gene regulation in specific corticostriatal circuits. These effects are most robust in sensorimotor circuits (which are implicated in habit formation and compulsive aspects of drug taking), less pronounced in associative circuits, and more modest in limbic circuits (where they are thought to contribute to altered reward processing in addiction). At the cellular level, psychostimulants alter predominantly neurons of the direct striatal output pathway (“Go pathway”), while the indirect pathway is less affected, and those changes appear to depend on the treatment context (i.e., arousal and associated changes in excitatory striatal input). Overall, these findings support the notion that action selection and initiation are compromised in addiction.

Comparative studies on cognitive enhancers (Adderall, methylphenidate, modafinil) show that they can induce largely similar molecular changes in corticostriatal circuits. Moreover, the same functional domains, cell types, neurotransmitters, and receptors appear to be affected. These effects were mostly explored with high drug doses, but qualitatively similar molecular changes were evident with drug treatments that mimicked medical treatments (oral administration and low drug plasma levels), although it is not clear whether these molecular changes are robust enough to alter behavior. Drug levels associated with cognitive enhancer abuse, however, are likely higher and, with protracted use, likely contribute to molecular changes that increase the addiction liability of these drugs. Further research is necessary to resolve these questions.

Acknowledgments

Abbreviations

Footnotes

References