Bryan F. Singer

Amphetamine-Induced Changes in Dendritic Morphology in Rat Forebrain Correspond to Associative Drug Conditioning Rather than Nonassociative Drug Sensitization

BACKGROUND Systemic exposure to amphetamine (AMPH) leads to a number of long-lasting neuroadaptations including changes in dendritic morphology in rat forebrain. It remains unknown whether these changes relate to associative drug conditioning or to nonassociative drug sensitization, two forms of plasticity produced by systemic exposure to AMPH. METHODS We compared the behavioral, neuronal, and morphologic consequences of exposing rats to intraperitoneal (IP) AMPH to those of exposure to AMPH applied to the ventral tegmental area (VTA), infusions that sensitize AMPH-induced locomotion and nucleus accumbens (NAcc) DA overflow but do not produce drug conditioning. RESULTS Both IP and VTA AMPH exposure sensitized locomotion and NAcc DA overflow, but only IP AMPH exposure produced conditioned locomotion. Importantly, whereas IP AMPH exposure increased spine density and dendritic length and branching in the NAcc, exposure to VTA AMPH produced the opposite effects. A similar differentiation of effects was observed in cortical areas. CONCLUSIONS Together these findings suggest that the morphological changes seen following IP AMPH exposure reflect associative drug conditioning rather than nonassociative drug sensitization. The decreases observed in the NAcc of VTA AMPH exposed rats may reflect the inability of these infusions to support conditioning.

Transient Overexpression of α-Ca2+/Calmodulin-Dependent Protein Kinase II in the Nucleus Accumbens Shell Enhances Behavioral Responding to Amphetamine

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to contribute to the expression of psychostimulant sensitization by regulating dopamine (DA) overflow from DA neuron terminals in the nucleus accumbens (NAcc). The present experiments explored the contribution of CaMKII in NAcc neurons postsynaptic to these terminals where it is known to participate in a number of signaling pathways that regulate responding to psychostimulant drugs. Exposure to amphetamine transiently increased αCaMKII levels in the shell but not the core of the NAcc. Thus, HSV (herpes simplex viral) vectors were used to transiently overexpress αCaMKII in NAcc neurons in drug-naive rats, and behavioral responding to amphetamine was assessed. Transiently overexpressing αCaMKII in the NAcc shell led to long-lasting enhancement of amphetamine-induced locomotion and self-administration manifested when αCaMKII levels were elevated and persisting long after they had returned to baseline. Enhanced locomotion was not observed after infection in the NAcc core or sites adjacent to the NAcc. Transient elevation of NAcc shell αCaMKII levels also enhanced locomotor responding to NAcc AMPA and increased phosphorylation levels of GluR1 (Ser831), a CaMKII site, both soon and long after infection. Similar increases in pGluR1 (Ser831) were observed both soon and long after exposure to amphetamine. These results indicate that the transient increase in αCaMKII observed in neurons of the NAcc shell after viral-mediated gene transfer and likely exposure to amphetamine leads to neuroadaptations in AMPA receptor signaling in this site that may contribute to the long-lasting maintenance of behavioral and incentive sensitization by psychostimulant drugs like amphetamine.

Rapid dopamine transmission within the nucleus accumbens: Dramatic difference between morphine and oxycodone delivery

While most drugs of abuse increase dopamine neurotransmission, rapid neurochemical measurements show that different drugs evoke distinct dopamine release patterns within the nucleus accumbens. Rapid changes in dopamine concentration following psychostimulant administration have been well studied; however, such changes have never been examined following opioid delivery. Here, we provide novel measures of rapid dopamine release following intravenous infusion of two opioids, morphine and oxycodone, in drug‐naïve rats using fast‐scan cyclic voltammetry and rapid (1 min) microdialysis coupled with high‐performance liquid chromatography ‐ tandem mass spectrometry (HPLC‐MS). In addition to measuring rapid dopamine transmission, microdialysis HPLC‐MS measures changes in GABA, glutamate, monoamines, monoamine metabolites and several other neurotransmitters. Although both opioids increased dopamine release in the nucleus accumbens, their patterns of drug‐evoked dopamine transmission differed dramatically. Oxycodone evoked a robust and stable increase in dopamine concentration and a robust increase in the frequency and amplitude of phasic dopamine release events. Conversely, morphine evoked a brief (~ 1 min) increase in dopamine that was coincident with a surge in GABA concentration and then both transmitters returned to baseline levels. Thus, by providing rapid measures of neurotransmission, this study reveals previously unknown differences in opioid‐induced neurotransmitter signaling. Investigating these differences may be essential for understanding how these two drugs of abuse could differentially usurp motivational circuitry and powerfully influence behavior.

Unpredictable saccharin reinforcement enhances locomotor responding to amphetamine

Drug-naïve, non-deprived rats were trained to lever press for saccharin under fixed-ratio (FR) or variable-ratio (VR) schedules of reinforcement. Rats trained on the VR schedule in which saccharin reinforcement was not predicted by a fixed number of lever presses subsequently showed an enhanced locomotor response to a threshold amphetamine challenge injection (0.5mg/kg IP) administered 2 weeks following the last saccharin session. This finding suggests that chronic exposure to gambling-like conditions of uncertain reinforcement can induce neuroadaptations in brain reward systems that are similar to those produced by repeated psychostimulant exposure and may lead to the development of addictive behaviors.

Rats that sign-track are resistant to Pavlovian but not instrumental extinction

Individuals vary in the extent to which they attribute incentive salience to a discrete cue (conditioned stimulus; CS) that predicts reward delivery (unconditioned stimulus; US), which results in some individuals approaching and interacting with the CS (sign-trackers; STs) more than others (goal-trackers; GTs). Here we asked how periods of non-reinforcement influence conditioned responding in STs vs. GTs, in both Pavlovian and instrumental tasks. After classifying rats as STs or GTs by pairing a retractable lever (the CS) with the delivery of a food pellet (US), we introduced periods of non-reinforcement, first by simply withholding the US (i.e., extinction training; experiment 1), then by signaling alternating periods of reward (R) and non-reward (NR) within the same session (experiments 2 and 3). We also examined how alternating R and NR periods influenced instrumental responding for food (experiment 4). STs and GTs did not differ in their ability to discriminate between R and NR periods in the instrumental task. However, in Pavlovian settings STs and GTs responded to periods of non-reward very differently. Relative to STs, GTs very rapidly modified their behavior in response to periods of non-reward, showing much faster extinction and better and faster discrimination between R and NR conditions. These results highlight differences between Pavlovian and instrumental extinction learning, and suggest that if a Pavlovian CS is strongly attributed with incentive salience, as in STs, it may continue to bias attention toward it, and to facilitate persistent and relatively inflexible responding, even when it is no longer followed by reward.

Individual Variation in Incentive Salience Attribution and Accumbens Dopamine Transporter Expression and Function

Cues (conditioned stimuli; CSs) associated with rewards can come to motivate behavior, but there is considerable individual variation in their ability to do so. For example, a lever‐CS that predicts food reward becomes attractive and wanted, and elicits reward‐seeking behavior, to a greater extent in some rats (‘sign‐trackers’; STs) than others (‘goal‐trackers’; GTs). Variation in dopamine (DA) neurotransmission in the nucleus accumbens (NAc) core is thought to contribute to such individual variation. Given that the DA transporter (DAT) exerts powerful regulation over DA signaling, we characterized the expression and function of the DAT in the accumbens of STs and GTs. STs showed greater DAT surface expression in ventral striatal synaptosomes than GTs, and ex vivo fast‐scan cyclic voltammetry recordings of electrically evoked DA release confirmed enhanced DAT function in STs, as indicated by faster DA uptake, specifically in the NAc core. Consistent with this, systemic amphetamine (AMPH) produced greater inhibition of DA uptake in STs than in GTs. Furthermore, injection of AMPH directly into the NAc core enhanced lever‐directed approach in STs, presumably by amplifying the incentive value of the CS, but had no effect on goal‐tracking behavior. On the other hand, there were no differences between STs and GTs in electrically‐evoked DA release in slices, or in total ventral striatal DA content. We conclude that greater DAT surface expression may facilitate the attribution of incentive salience to discrete reward cues. Investigating this variability in animal sub‐populations may help explain why some people abuse drugs while others do not.

Transient viral-mediated overexpression of α-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence

Calcium/calmodulin‐dependent protein kinase II (CaMKII) activity is necessary for the long‐lasting expression of locomotor sensitization and enhanced drug‐taking observed in rats previously exposed to psychostimulants. Exposure to these drugs also transiently increases αCaMKII levels in the nucleus accumbens (NAcc), an effect that, when mimicked by transient viral‐mediated overexpression of αCaMKII in NAcc shell neurons, leads to long‐lasting enhancement in locomotor responding to amphetamine and NAcc α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA). The present experiments characterized the dopamine (DA) dependence of the functional AMPA receptor upregulation observed long after transient overexpression of αCaMKII. Rats infected with herpes simplex virus‐αCaMKII in the NAcc shell showed a transient increase in αCaMKII levels that peaked at 4 days post‐infection and returned to baseline 8 days later. When challenged with AMPA (0.8 nmol/side) in the NAcc shell at 20 days post‐infection, these rats showed enhanced locomotion compared with controls. This sensitized locomotor response was blocked when AMPA was coinfused with either the DA type‐1 receptor antagonist SCH23390 (0.8 nmol/side) or the protein kinase A inhibitor Rp‐cAMPS (80 nmol/side). Neither SCH23390 nor Rp‐cAMPS produced locomotor effects when infused by itself into the NAcc shell. Furthermore, these antagonists did not block the acute non‐sensitized locomotor response to AMPA observed in control rats. These findings show that transient viral‐mediated overexpression of αCaMKII in neurons of the NAcc shell leads to long‐lasting functional upregulation of AMPA receptors that is DA type‐1 receptor and protein kinase A dependent. Thus, transient increases in levels of αCaMKII in the NAcc shell produce long‐lasting changes in the way that DA and glutamate interact in this site to generate locomotor behavior.

Drug-Paired Contextual Stimuli Increase Dendritic Spine Dynamics in Select Nucleus Accumbens Neurons

Repeated exposure to amphetamine leads to both associative conditioning and nonassociative sensitization. Here we assessed the contribution of neuronal ensembles in the nucleus accumbens (NAcc) to these behaviors. Animals exposed to amphetamine IP or in the ventral tegmental area (VTA) showed a sensitized locomotor response when challenged with amphetamine weeks later. Both exposure routes also increased ΔFosB levels in the NAcc. Further characterization of these ΔFosB+ neurons, however, revealed that amphetamine had no effect on dendritic spine density or size, indicating that these neurons do not undergo changes in dendritic spine morphology that accompany the expression of nonassociative sensitization. Additional experiments determined how neurons in the NAcc contribute to the expression of associative conditioning. A discrimination learning procedure was used to expose rats to IP or VTA amphetamine either Paired or Unpaired with an open field. As expected, compared with Controls, Paired rats administered IP amphetamine subsequently showed a conditioned locomotor response when challenged with saline in the open field, an effect accompanied by an increase in c-Fos+ neurons in the medial NAcc. Further characterization of these c-Fos+ cells revealed that Paired rats showed an increase in the density of dendritic spines and the frequency of medium-sized spines in the NAcc. In contrast, Paired rats previously exposed to VTA amphetamine showed neither conditioned locomotion nor conditioned c-Fos+ expression. Together, these results suggest a role for c-Fos+ neurons in the medial NAcc and rapid changes in the morphology of their dendritic spines in the expression of conditioning evoked by amphetamine-paired contextual stimuli.

Neuronal and psychological underpinnings of pathological gambling.

Although pathological gambling (PG) is a prevalent disease, its neurobiological and psychological underpinnings are not well characterized. As legal gambling increases in prominence in a growing number of casinos as well as on the internet, the potential for a rise in PG diagnoses warrants investigation of the disorder. The recent reclassification of PG as a behavioral addiction in the DSM-5 raises the possibility that similar cognitive and motivational phenotypes may underlie both gambling and substance use disorders. Indeed, in this Research Topic, Zack et al. (2014) tested the hypothesis that exposure to reward unpredictability can recruit brain dopamine (DA) systems in a similar way to chronic exposure to drugs of abuse (see also Singer et al., 2012). Over the years a variety of models have proposed that alterations in DA signaling may mediate the transition from drug use to dependence; similarly, the hypothesis that aberrant DA responses may influence the transition from recreational, to problematic, and finally PG has only recently begun to be tested. The collection of articles in this Research Topic highlights the complexity of PG and posits several theories of how dopaminergic signaling may contribute to behavioral maladaptations that contribute to PG. In this Research Topic, Paglieri et al. (2014) report a growing incidence of PG with a lack of effective treatments. As described by Goudriaan et al. (2014) (this Research Topic), PG is thought to result from “diminished cognitive control over the urge to engage in addictive behaviors” that manifests in the inability to control desire to gamble despite negative consequences. PG is characterized by several cognitive dysfunctions, including increased impulsivity and cognitive interference. Similar to drug addictions, gambling behavior is powerfully modulated by exposure to gambling-related conditioned stimuli. In this Research Topic both Anselme and Robinson (2013) as well as Linnet (2014) describe the supporting role of gambling-related cues in this behavioral addiction. Anselme and Robinson (2013) present a series of findings suggesting that surprising non-rewards enhance incentive salience attribution to conditioned cues in conditioning procedures as well as during gambling episodes. They discuss a possible evolutionary origin of this counterintuitive process. Linnet (2014) reviews the contribution of DA signaling to incentive salience and reward prediction. Noting the research demonstrating brain activation during gambling tasks despite the possibility of a loss, he suggests a role for DA dysfunction in reward “wanting” and anticipation. Ventral striatal activation is thought to be critical for the attribution of incentive salience to reward-related cues. In this Research Topic, Lawrence and Brooks (2014) found that healthy individuals who are more likely to display disinhibitory personality traits, such as financial extravagance and irresponsibility, show increased capacity for ventral striatal DA synthesis. Thus it is possible that individual variation in DA signaling due to genetics or environmental factors may influence PG. Porchet et al. (2013) (this Research Topic) also investigated whether physiological and cognitive responses observed during the performance of gambling tasks could be altered in recreational gamblers with pharmacological manipulations. As the commentary from Zack (2013) suggests, the Porchet et al. (2013) results may reflect important differences in neurobiological function between recreational and pathological gamblers. This hypothesis, along with the results of Lawrence and Brooks (2014) demonstrating increased DA capacity in individuals thought to be more prone to gambling, illustrates the complexity of PG as a disease and the need to sample different populations with different techniques and behavioral tasks. Two papers in this Research Topic suggest a role for cortisol in modulating incentive motivation in the ventral striatum. Li et al. (2014) demonstrate an imbalanced sensitivity to monetary vs. non-monetary incentives in the ventral striatum of pathological gamblers. They show that cortisol levels in PG positively correlate with ventral striatal responses to monetary cues. van den Bos et al. (2013) provide further evidence for the importance of cortisol by highlighting the strong positive correlation observed in men between salivary cortisol levels and risk-taking measures. This was a significant contrast to the weak negative correlation seen in women. Their findings highlight important gender differences in how stress hormones affect risky-decision making, and by extension, the role of stress in gambling. In this Research Topic, Clark and Dagher (2014) provide a review of the literature investigating the relationship between DA agonists and impulse control disorders in Parkinsons patients, and how this relates to potential gains and losses within a decision-making framework. They provide the beginnings of a hypothetical model of how DA agonist treatments affect value and risk assessments. While a variety of research suggests that dopaminergic treatments for Parkinsons disease may affect PG, few have probed whether individuals with Huntingtons disease (HD) display gambling-related phenotypes. Kalkhoven et al. (2014) (this Research Topic) show that HD patients exhibit symptoms of behavioral disinhibition similar to those observed in PG. However, HD patients do not typically develop problem gambling. Based on neurobehavioral evidence, these authors suggest why HD patients are unlikely to start gambling but have a higher chance of developing PG if they encounter a situation that promotes such behavior. The investigation of neural mechanisms underlying PG is currently at an early stage. As emphasized by Potenza (2013) in this Research Topic, while previous research and the present findings suggest that DA may underlie gambling-related behaviors, other neurotransmitters and signaling pathways may also play vital roles in the emergence of the disease. Individual variation in PG populations (e.g., differing levels of impulsivity, compulsivity, decision making, and DA pathology) has produced discrepancies in the PG literature, warranting a systematic approach to investigating the disease in the future. Paglieri et al. (2014) also suggest the need for greater methodological integration of animal studies (rodents and primates) to better understand the mechanisms underlying PG. In particular, Tedford et al. (2014) note in this Research Topic that gambling activity involves costs/benefits decision-making and that intracranial self-stimulation provides experimental advantages over traditional reinforcement methods used to model PG in animals. Finally, Paglieri et al. (2014) suggest that computational modeling, already used to account for other psychiatric diseases, might be applied to PG as well. Taken together, this collection of articles suggests novel avenues for future research of PG to improve treatment options for the disease.

Inhibiting cyclin-dependent kinase 5 in the nucleus accumbens enhances the expression of amphetamine-induced locomotor conditioning.

When psychostimulant drugs like amphetamine are administered repeatedly in the presence of a contextual stimulus complex, long-lasting associations form between the unconditioned effects of the drug and the contextual stimuli. Here we assessed the role played by the proline-directed serine/threonine kinase cyclin-dependent kinase 5 (Cdk5) in the nucleus accumbens (NAcc) on the expression of the conditioned locomotion normally observed when rats are returned to a context previously paired with amphetamine. Infusing the Cdk5 inhibitor roscovitine (40nmol/0.5μl/side) into the NAcc 30-min before the test for conditioning significantly enhanced the conditioned locomotor response observed in rats previously administered amphetamine in the test environment. This effect was specific to the expression of a conditioned response as inhibiting Cdk5 produced no effect in control rats previously administered saline or previously administered amphetamine elsewhere. As inhibiting Cdk5 during exposure to amphetamine has been found to block the accrual of locomotor conditioning, the present results suggest distinct roles for NAcc Cdk5 in the induction and expression of excitatory conditioning by amphetamine.