The use of laser capture microdissection to identify specific pathways and mechanisms involved in impulsive choice in rats.

BACKGROUND: Microinjections, lesions, viral-mediated gene transfer, or designer receptors exclusively activated by designer drugs (DREADDs) can identify brain signaling pathways and their pharmacology in research animals. Genetically modified animals are used for more precise assessment of neural circuits. However, only a few of the gene-based pathway modifications are available for use in outbred rat strains. NEW METHOD: Behaviorally characterized Sprague-Dawley rats undergo tract tracing through microinjection of fluorospheres, followed by laser capture microdissection (LCM) and qPCR for detecting mRNA of pathway-associated gene products. Correlations between mRNA expression and behavior identify specific involvement of pharmacologically relevant molecules within cells of interest. Here, we examined this methodology in an impulsive choice paradigm and targeted projections from the orbital and medial prefrontal cortex. RESULTS: In this proof of concept study, we demonstrate relationships between measures of impulsive choice with distinct neurotransmitter receptor expression in cell populations from four different signaling pathways. COMPARISONS WITH EXISTING METHODS: Combining behavior, tract tracing, LCM, and gene expression profiling provides more cellular selectivity than localized lesions and DREADDs, and greater pharmacological specificity than microinjections and viral-mediated gene transfer due to targeting identified neurons. Furthermore, the assessment of inter-individual pathways provides insight into the complex nature of underlying mechanisms involved in typical and atypical behavior. CONCLUSIONS: The novel combination of behavior, tract tracing, LCM, and single gene or potential whole genome transcriptome analysis allows for a more targeted understanding of the interconnection of neural circuitry with behavior, and holds promise to identify more specific drug targets that are relevant to behavioral phenotypes.

Early life stress and later peer distress on depressive behavior in adolescent female rats: Effects of a novel intervention on GABA and D2 receptors.

Early life adversity (ELA) increases the risk of depression during adolescence that may result from a decline in parvalbumin (PVB) secondary to increased neuroinflammation. In this study, we investigated depressive-like behavior following exposure to two different types of stressors that are relevant for their developmental period: 1) chronic ELA (maternal separation; MS) and 2) an acute emotional stressor during adolescence (witnessing their peers receive multiple shocks; WIT), and their interaction. We also determined whether reducing inflammation by cyclooxygenase-2 (COX-2) inhibition would prevent the onset of depressive-like behavior. Female Sprague-Dawley rat pups underwent MS for four-hours/day or received typical care (CON) between postnatal days (P) 2 and P20. A COX-2 inhibitor (COX-2I) or vehicle was administered every other day between P30 and P38. Subjects were tested for learned helplessness to assess depressive-like behavior at P40 (adolescence). MS females demonstrated increased escape latency and decreased PVB in the prefrontal cortex (PFC) and dorsal raphe that were attenuated by COX-2I intervention. Helplessness was also associated with an increase in D2 receptors in the accumbens. In contrast, WIT elevated escape latency in CON, but reduced latency in MS females. Furthermore, COX-2I intervention decreased escape latency in both CON and MS after WIT. WIT reduced PVB levels in the basolateral amygdala and increased PFC levels to CON levels. Our data suggest that decreased PVB in the PFC is important for the expression of depressive-like behavior and suggest that COX-2I intervention may provide a novel prevention for depression.

Preventative treatment in an animal model of ADHD: Behavioral and biochemical effects of methylphenidate and its interactions with ovarian hormones in female rats.

Clinical and preclinical studies on attention deficit hyperactivity disorder (ADHD) show that juvenile males that are exposed to methylphenidate (MPH) show reduced risk for substance use later in life. In contrast, little is known about whether females have the same enduring treatment response to stimulants and how gonadal hormones influence their behavior later in life. Females received either a sham or 6-hydroxydopamine (6-OHDA) microinjection in the prefrontal cortex (PFC) at postnatal day (P)10. Subjects were then treated with Vehicle or MPH (2mg/kg, p.o.) between P20-35 and tested during late adolescence/young adulthood (P60); half of these subjects underwent ovariectomy at P55 to determine hormonal influences. Females with 6-OHDA were depleted of PFC dopamine by 61% and demonstrated increased impulsive choice (delayed discounting) and preferences for cocaine-associated environments relative to control females. Both MPH and ovariectomy reduced impulsive choice and cocaine preferences in 6-OHDA females, but had no enduring effect in Sham females. Ovariectomy itself did not significantly affect impulsivity. Juvenile MPH interacted strongly with 6-OHDA to increase D4, D5, Alpha-1A, Alpha-2A, and 5-HT-1A mRNA receptor expression in the PFC. MPH alone effected D1 mRNA, while 6-OHDA increased BDNF; all markers were decreased by ovariectomy. Together, these data suggest that 6-OHDA changes in dopamine are not only relevant for ADHD-like behaviors, but their long-term modulation by treatment and the influence of cyclical differences in menstrual cycle.

When the party is over: depressive-like states in rats following termination of cortical D1 receptor overexpression.

RATIONALE: Increased activity of prefrontal D1 dopamine receptors (D1R) is involved in reward-related behavior found in bipolar disorder and drug addiction. While the effects of elevated D1R are known, depressive-like behaviors also occur in these disorders after reward-seeking ends. OBJECTIVES: The goal is to characterize how termination of D1R overexpression influences depressive-like behaviors. METHODS: An inducible (Tet.On), lentiviral vector was used to manipulate the expression of the DRD1 gene in glutamate neurons within the prefrontal cortex in male, adult rats. Sexual activity and sucrose preference were studied in both D1R elevated ON and relatively reduced OFF states. Following termination of the D1R ON state, depressive-like behavior was determined in the OFF state. Expression of the transcriptional regulator, cyclic AMP-responsive element-binding protein (CREB), was used as an indication of downstream effects in the nucleus accumbens (NA). RESULTS: ON D1R expression increased sexual activity that returned to baseline in the OFF state. Sucrose preferences increased ~6 % in ON state but fell 11 % below control levels when OFF. Consistent with a depressive-like phenotype, D1R OFF decreased activity by 40 %, impaired the ability to control (43 %) and motivation to escape shock (27 % more impaired) relative to dsRed OFF. CREB increased 29 % in the NA in the D1R OFF state relative to the ON state. CONCLUSIONS: This novel approach demonstrates that elevated D1R expression increased hedonic behavior, whereas the termination of D1R overexpression often resulted in depressive-like behavior. These observations support a role for D1R expression cycling in bipolar-associated behaviors and addiction.

The developmental inter-relationships between activity, novelty preferences, and delay discounting in male and female rats.

Increased locomotion, novelty-seeking, and impulsivity are risk factors associated with substance use. In this study, the inter-relationships between activity, novelty preferences, and delay discounting, a measure of impulsivity, were examined across three stages: juvenile/early adolescence (postnatal Day [P] 15, 19, and 42 for activity, novelty, and impulsivity, respectively), adolescent/late adolescent (P28, 32, 73), and adult (P90, 94, 137) in male and female rats. Our estimates of impulsive choice, where animals were trained to criterion, revealed an age × sex interaction where early adolescent females had the lowest levels of impulsivity. The relationships of activity and novelty to impulsivity significantly changed across age within each sex. Early adolescent males with high activity, but low novelty preferences, were more impulsive; however, low activity and high novelty preferences were related to high impulsivity in adult males. Female activity gradually increased across age, but did not show a strong relationship with impulsivity. Novelty preferences are moderately related to impulsivity into adulthood in females. These data show that males and females have different developmental trajectories for these behaviors. Males show greater sensation-seeking (e.g., activity) and risky behavior (e.g., novelty preferences) earlier in life, whereas these behaviors emerge during adolescence in females.

Developmental emergence of an obsessive-compulsive phenotype and binge behavior in rats.

RATIONALE: Obsessive-compulsive disorder (OCD) gradually emerges and reaches clinical significance during early adulthood. Whether a predisposition for OCD manifests as binge eating disorder earlier during adolescence is proposed. OBJECTIVES: To further characterize how OCD-like behaviors increase across maturation and to determine whether an OCD-like predisposition increases the likelihood of binge eating during adolescence. METHODS: Male and female Sprague-Dawley rats were injected with the tricyclic antidepressant clomipramine (CMI, 15 mg/kg) or saline vehicle twice daily between postnatal days 9-15. Both groups were tested for perseverative (spontaneous alternation) and anxiety-like (elevated plus maze; marble burying) behaviors during juvenility (day 28), adolescence (day 60), and adulthood (day 90). Both motivations to eat sucrose pellets and binge eating on fat were investigated. RESULTS: Sex- and age-dependent increases in anxiety-like and perseverative behavior were observed in CMI subjects. Differences in consummatory behaviors emerged during late adolescence, while no significant differences in alternation or anxiety-like behaviors were detected between CMI and vehicle animals until adulthood. Adolescent CMI females consumed more sucrose pellets in 30 min relative to vehicle females, whereas adolescent CMI males consumed approximately half as much as vehicle males. Sucrose consumption did not differ between groups in adulthood. Adolescent CMI rats demonstrated more fat bingeing than vehicles, independent of sex. CONCLUSIONS: OCD-like behaviors are emerging during adolescence, but sucrose consumption and fat bingeing in CMI-treated animals significantly precedes the appearance of anxiety and perseveration. This OCD-like phenotype emerges fully during adulthood, suggesting that eating may likely serve as a coping strategy in these animals.

Extinction and reinstatement to cocaine-associated cues in male and female juvenile rats and the role of D1 dopamine receptor.

Extinction of behaviors in response to drug-associated cues and prevention of reinstatement are integral for addiction treatment, and can reverse or ameliorate the harmful consequences of drug use. The mechanisms controlling extinction and reinstatement involve prefrontal cortical dopamine receptors, which change in expression and activity during the juvenile and adolescent transitions until they mature in adulthood. Little is known about the role that PFC D1 dopamine receptors play in extinction of drug-paired associations early in life. We used extinction of place preferences for cocaine in juvenile male and female rats following genetic, cell-specific overexpression of D1 on glutamatergic cells in the PFC. All subjects needed to demonstrate cocaine preferences for inclusion in the extinction studies. Here, male juveniles with a preference to 10 mg/kg cocaine took longer to extinguish preferences compared to both male adults and female juveniles. Female juveniles extinguished more rapidly than male juveniles at 20 mg/kg cocaine. Overexpression of D1 in juvenile males significantly facilitated extinction relative to juvenile male controls, whereas D1 prolonged expression of extinction in adults overexpressing D1 and adolescents who naturally have elevated D1 expression. These data suggest that an immature D1 profile in juveniles prevented the learning of new associations, and D1 overexpression may provide sufficient activity to facilitate extinction learning. D1 overexpression reduced reinstatement to a priming dose of cocaine in juvenile males. Together, these data show D1 expression may re-program motivational circuitry to facilitate extinction learning during juvenility that is normally unavailable to juveniles and that sex differences exist.

Cocaine-conditioned odor cues without chronic exposure: Implications for the development of addiction vulnerability.

Adolescents are highly vulnerable to addiction and are four times more likely to become addicted at first exposure than at any other age. The dopamine D1 receptor, which is typically overexpressed in the normal adolescent prefrontal cortex, is involved in drug cue responses and is associated with relapse in animal models. In human drug addicts, imaging methods have detected increased activation in response to drug cues in reward- and habit-associated brain regions. These same methods can be applied more quantitatively to rodent models. Here, changes in neuronal activation in response to cocaine-conditioned cues were observed using functional magnetic resonance imaging in juvenile rats that were made to over-express either D1 receptors or green fluorescent protein by viral-mediated transduction. Reduced activation was observed in the amygdala and dopamine cell body regions in the low cue-preferring/control juvenile rats in response to cocaine cues. In contrast, increased activation was observed in the dorsal striatum, nucleus accumbens, prefrontal cortex, and dopamine cell bodies in high cue-preferring/D1 juveniles. The increase in cue salience that is mediated by increased D1 receptor density, rather than excessive cocaine experience, appears to underlie the transition from aversion to reward in cue-induced neural response and may form the basis for habit-forming vulnerability.

Sex-dependent changes in ADHD-like behaviors in juvenile rats following cortical dopamine depletion.

Reduced cortical dopamine levels have been observed in individuals with attention deficit hyperactivity disorder (ADHD). Global dopamine depletions by 6-hydroxydopamine (6-OHDA; with noradrenergic protection) in neonatal rats produces locomotor hyperactivity, with less known about how cortical depletion modulates risky behaviors. Here, we determined the effect of a medial prefrontal cortex (PFC) 6-OHDA depletions (30-60%) or sham microinjection at postnatal day 11 on behavior in male and female juvenile rats. Separate groups were studied for delay discounting (impulsive choice), novelty-preference, and preferences for cues and environments associated with cocaine (10, 20, and 40 mg/kg), their extinction, and reinstatement with place conditioning. Because PFC D1 receptors play a role in these behaviors, confocal microscopy was used to measure D1-immunoreactive projections to the nucleus accumbens core. Both 6-OHDA males and females increased delay discounting relative to sham controls, although only 6-OHDA females increased novelty preferences. Preferences for cocaine-associated environments, their extinction, and reinstatement with a priming dose of cocaine were reduced in 6-OHDA subjects overall. However, impulsive choice at 5s positively correlated with preferences for cocaine-associated environments in 6-OHDA subjects, but not sham controls. As possible compensation for low dopamine levels, D1-immunoreactivity on traced neurons increased in 6-OHDA females; dopamine levels did not remain low in adolescent 6-OHDA males and D1 did not change. We believe that these modest depletions restricted to the PFC demonstrate the role of dopamine, and not norepinephrine, in understanding these behaviors in other animal models where cortical dopamine is reduced during development.

Viral over-expression of D1 dopamine receptors in the prefrontal cortex increase high-risk behaviors in adults: comparison with adolescents.

RATIONALE: Adolescents are often described as "lacking brakes" resulting in an increase in several behaviors associated with risk for addiction. Prefrontal cortex dopamine and cortico-limbic interaction play an important role in addiction, and we have previously shown that the dopamine D1 receptor is elevated on prelimbic prefrontal output neurons in adolescent rats. We hypothesized that a constellation of risk-related behaviors is mediated by prefrontal output neuron expression of D1. OBJECTIVES: We aimed to determine the role of the dopamine D1 receptor in behavioral and neural correlates of risk for addiction that are often observed in adolescents. Therefore, high-risk behaviors as well as subcortical D2 receptor expression were investigated in adult animals with experimentally elevated D1 on prefrontal glutamatergic neurons. METHODS: A lentiviral vector that selectively expressed the D1 receptor within glutamate neurons was injected in the prelimbic prefrontal cortex of adult male rats. Place conditioning to cocaine, alcohol, and nicotine, as well as delay discounting, novelty preferences, anxiety, cocaine self-administration, and sucrose preferences were assessed. RESULTS: Virally mediated D1 over-expression in adults leads to stronger drug-cue associations and greater consumption of sweet solutions, elevates bias towards immediate satisfaction rather than delaying gratification, decreases anxiety, and causes rats to work harder for and take more cocaine. Furthermore, elevated cortical D1 reduces D2 receptors in the accumbens (a putative risk marker). CONCLUSIONS: Together, these data suggest a common mechanism for increased motivational drive to seek and consume substances with hedonic value, consistent with adolescent addictive processes.

Depressive-like behavior in adolescents after maternal separation: sex differences, controllability, and GABA.

Exposure to adversity during development is an identified risk factor for depression later in life. In humans, early adversity accelerates the onset of depressive symptoms, which manifest during adolescence. Animal studies have used maternal separation as a model of early adversity to produce adult depressive-like behaviors, but have yet to examine these behaviors during adolescence. Moreover, the nature of depressive-like behaviors has not been well characterized in this model. Here, we used the triadic model of learned helplessness to understand controllability, helplessness, and motivational factors following maternal separation in male and female adolescent rats. We found sex-dependent changes in the effects of separation, with males demonstrating loss of controllability in an escapable shock condition, whereas females demonstrated motivational impairment in a no-shock condition. The effect, however, did not endure as adult females were no longer helpless. Reductions in parvalbumin, a GABAergic marker, in the prefrontal cortex of separated subjects relative to age-matched controls were evident and paralleled depressive-like behavior. Understanding the risk factors for depression, the nature of depressive-like behaviors, and their unique sex dependency may ultimately provide insight into improved treatments.