The in Vivo Neurochemical Profile of Selectively Bred High

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In vivo neurochemical profile of selectively bred highresponder and low-responder rats reveals baseline, cocaineand novelty-evoked differences in monoaminergic systems Omar S. Mabrouk, John L. Han, Jenny-Marie T Wong, Huda Akil, Robert T. Kennedy, and Shelly B Flagel ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00294 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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ACS Chemical Neuroscience

In vivo neurochemical profile of selectively bred high-responder and low-responder rats reveals baseline, cocaine- and novelty-evoked differences in monoaminergic systems 1,2

Omar S. Mabrouk, 1John L. Han, 1Jenny-Marie T. Wong, 4Huda Akil,

1,2

Robert T. Kennedy,

3,4

Shelly

B. Flagel* 1

Department of Chemistry, 2Department of Pharmacology, 3Department of Psychiatry, 4Molecular and

Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan

Author for correspondence* Shelly B. Flagel, Ph.D. Associate Professor Department of Psychiatry Molecular and Behavioral Neuroscience Institute 205 Zina Pitcher Place Ann Arbor, MI 48109 (USA) Phone: 734-936-2033 Fax: 734-647-4130 Email: [email protected]

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Abstract Relative to bred low-responder (bLR) rats, bred high-responder (bHR) rats have an exaggerated locomotor response to a novel environment, are more risk-taking, more impulsive, and more likely to exhibit compulsive drug-seeking behaviors. These phenotypic differences in addiction-related behaviors and temperament have previously been associated with differences in neurotransmitter signaling, including the mesolimbic dopamine system. In the current study, we applied advanced in vivo microdialysis sampling in the nucleus accumbens of bHRs/bLRs to assess differences in basal and stimulated neurochemical efflux more broadly. We used liquid-chromatography-mass spectrometry measurements of dialysate samples to quantify a panel of 17 neurochemicals including dopamine, norepinephrine, serotonin, histamine, glutamate, GABA, acetylcholine, adenosine, DOPAC, 3-MT, HVA, 5-HIAA, normetanephrine, taurine, serine, aspartate and glycine. We also applied a stable isotope labeling technique to assess absolute baseline concentrations of dopamine and norepinephrine in the nucleus accumbens. Finally, we investigated the role of norepinephrine tone in the nucleus accumbens on the bHR phenotype. Our findings show that bHRs have elevated basal and cocaine-evoked dopamine and norepinephrine levels in the nucleus accumbens compared to bLRs. Furthermore, norepinephrine signaling in the nucleus accumbens appeared to be an important contributor to the bHR phenotype since bilateral perfusion of the α1 adrenergic receptor antagonist terazosin (10 µM) into the nucleus accumbens abolished the bHR response to novelty. These findings are the first to demonstrate a role for norepinephrine in the bHR phenotype. They reveal a positive relationship between dopamine and norepinephrine signaling in the nucleus accumbens in mediating the exaggerated response to novelty, and point to norepinephrine signaling as a potential target in the treatment of impulse control disorders. Keywords Neurochemistry, nucleus accumbens, high-responders, mass spectrometry, norepinephrine, dopamine 2 ACS Paragon Plus Environment

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Introduction The way an individual responds to its surrounding environment may be a key determinant of psychopathology. Previous work, using a unique genetic animal model, indicates that individual differences in environmental exploration are associated with differences in stress response, risk-taking behavior, impulsivity and compulsive drug-taking behavior (1, 2, for review see 3). In the current study, we exploit this established animal model—rats that have been selectively bred based on locomotor response to novelty—to examine the neurochemical profile that might be associated with this so-called “sensation-seeking trait” (4-5). Over a decade ago, the selectively bred high-responder (bHR) and low-responder (bLR) rat lines were generated (6), modeling those that explore the most and the least, respectively, in a novel environment (7). This model provides an a priori way of predicting which rats are destined to become high- vs. low-responders, allowing us to identify the antecedent variables that might determine these traits. Locomotor response to an inescapable novel environment has previously been associated with anxiety-like behaviors and the propensity to self-administer drugs of abuse in outbred rats (7-8). It is important to note, however, that the selectively bred lines exhibit a number of other addiction-related traits (1-3) that are not necessarily apparent in outbred high- and low-responder rats (e.g. 9-10). In addition to increased exploration in a novel environment—the trait for which they were bred—bHR rats are also more aggressive, more impulsive on tests of impulsive action, and exhibit maladaptive and persistent responses to food- and cocaine-associated cues (1, 3). bHRs are also more likely to exhibit compulsive drug-seeking behavior following a prolonged cocaine self-administration paradigm and an enhanced propensity for reinstatement of drug-seeking behavior, or relapse, following abstinence (2). Taken together, relative to bLR rats, bHRs appear to be more behaviorally disinhibited, with increased susceptibility to addiction-related behaviors. 3 ACS Paragon Plus Environment


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To date, much of the work characterizing the neurobiological antecedents of the addictionrelated behaviors exhibited by bHRs and bLRs has focused on the dopaminergic system, which has previously been implicated in a number of these traits (e.g. 11-16). Namely, relative to bLRs, bHRs have lower levels of dopamine D2 receptor mRNA in the striatum (1-2), but a greater proportion of dopamine D2high , the functionally active form of the receptor; despite comparable levels of total D2 binding (1). In addition, relative to bLRs, bHR rats have more spontaneous dopamine ‘release events’ as measured using fast-scan cyclic voltammetry in the core of the nucleus accumbens (NAc) (1). In agreement with these reported differences in dopaminergic “tone”, bHRs exhibit increased psychomotor response to dopaminergic drugs, including quinpirole (1) and cocaine (17-18). In the current study, we focused our analyses on the NAc to more thoroughly examine the neurochemical profile in bHR and bLR rats. Since bHRs display exaggerated behavioral responses to novelty and drugs of abuse compared to bLRs (1-2), we hypothesized that bHRs and bLRs would exhibit differential neurochemical release patterns in the NAc under both basal and stimulated conditions. To test this hypothesis, we used in vivo microdialysis to sample the NAc in both bHRs and bLRs then used benzoyl chloride derivatization and liquid chromatography-mass spectrometry (LC-MS) to assess concentrations differences in a total of 17 neurochemicals, including neurotransmitters, neuromodulators and metabolites (19). We evaluated these neurochemical profiles at rest (Experiment 1), in response to an acute injection of cocaine (Experiment 2), and in response to a novel environment (Experiment 3). Because of the broad analytical nature of this technique, it is well-suited for determining differences between different strains or genotypes. To more accurately quantify extracellular dopamine and norepinephrine (NE) in the NAc under basal conditions we employed a novel stable isotope labeling (SIL) procedure which corrects for variance in microdialysis probe recovery (20).


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Our results demonstrate distinct neurochemical signaling patterns between bHRs and bLRs under basal conditions, in response to cocaine, and upon exposure to a novel environment. These findings identify a link between the bHR phenotype and dopamine-NE interactions in the NAc, and shed light on the neurobiological substrates underlying the behavioral traits exhibited by these selectively bred rat lines. Results and Discussion The current study aimed to test the hypothesis that bHR and bLR rats may differ in their neurochemical signaling profiles in the NAc, a brain area associated with locomotor activity and motivated behaviors including, reward-seeking and response to drugs of abuse (21-23). To achieve this, the study applied advanced LC-MS analytical techniques and video capture behavioral analysis to identify baseline and stimulus-induced neurochemical correlates of the bHR/bLR phenotypes. bHRs show exaggerated behavioral responses to cocaine including, increased rates of acquisition of self-administration (24) and greater psychomotor response to acute and repeated cocaine treatment (17-18). Likewise, outbred HR rats have hyperdopaminergic responses in the NAc in response to psychostimulants like cocaine (25). Upon exposure to a reward-paired cue, bHR rats, who tend to signtrack to such cues, have exaggerated cue-evoked dopamine events in the NAc relative to bLRs, who tend to goal-track (26). Taken together, bHRs appear to be more sensitive to stimuli that drive ventral striatal dopamine release. Despite these lines of evidence for stimulus-induced differences, basal differences in dopamine activity between bHRs and bLRs have yet to be defined. The current study took advantage of recent advances in mass spectrometry-based small molecule measurements which enabled the development of a 17-analyte neurochemical panel (19) and a SIL assay for improved extracellular quantitation (20). We applied these methods under basal conditions (SIL) or in response to stimuli



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known to evoke dopamine release (23), including exposure to a novel environment and cocaine administration.

Experiment 1: Baseline neurochemical differences between awake bHRs and bLRs To determine whether bHRs and bLRs differ in their NAc neurochemical profiles at rest, microdialysis was performed in both phenotypes and samples were analyzed for 17 neurochemicals, including dopamine, norepinephrine, serotonin, histamine, glutamate, GABA, acetylcholine, adenosine, DOPAC, 3-MT, HVA, 5-HIAA, normetanephrine, taurine, serine, aspartate and glycine (Table 1). Results indicate that only dopamine in dialysate samples was significantly different between phenotypes (T2.45, dF15, p=0.027; q=0.096), with elevated levels in bHRs (1.42 nM, n=10) compared to bLRs (0.87 nM, n=7; see Table 1). Since previous studies have identified a relationship between locomotor activity and accumbal dopamine and NE (27), we exploited the same bHR and bLR rats used above to estimate absolute extracellular levels of these transmitters in the NAc. To do so, we used a recently developed SIL technique which accounts for variability in probe recovery (Fig 1) (20). This required the addition of isotopically labeled dopamine (13C6-dopamine; 5 nM) and NE (d6-NE; 5 nM) in the perfusate of the dialysates collected under baseline conditions. The average extraction fraction (Ed) taken at baseline for dopamine was 0.34 ± 0.03 for bHRs and 0.35 ± 0.02 for bLRs. Applying these Ed values generated absolute baseline dopamine concentrations of 5.09 ± 0.38 nM (n=10) and 2.76 ± 0.48 nM (n=7) for bHRs and bLRs, respectively (Fig 1). The average Ed taken at baseline for NE was 0.24 ± 0.02 for bHRs and 0.36 ± 0.03 for bLRs. Applying these Ed values generated absolute NE concentrations of 2.20 ± 0.29 nM (n=10) and 1.01 ± 0.20 nM (n=7) for bHRs and bLRs, respectively (Fig 1). Similar to the data without SIL correction, dopamine was significantly higher in bHRs compared to bLRs (T3.86, dF15, 6 ACS Paragon Plus Environment

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p=0.002). The analysis also showed that bHRs had significantly higher basal NE levels in NAc compared to bLRs (T3.05, dF15, p=0.008). These results are consistent with previous work in outbred HRs (e.g. 28-29). In particular, outbred HRs have been shown to have higher basal firing rates in the VTA (14), which is congruent with bHRs showing increased spontaneous dopamine events in the NAc (1). Ed for catecholamines has been shown to be relatively insensitive to release inhibition, metabolism and synthesis, but strongly affected by uptake inhibition (30). Thus, under baseline conditions, there appears to be disrupted catecholamine uptake in bHRs, which may contribute to the behavioral phenotype of these animals. Experiment 2: Behavioral and neurochemical effects of cocaine in bHR and bLR rats In order to assess behavioral differences in a dopamine-stimulated state, we treated bHR (n=6) and bLR rats (n=8) with a single injection of cocaine (15 mg/kg i.p.) (Fig 2). To determine phenotypic differences in cocaine-mediated behavioral responses relative to baseline activity, we performed a twoway repeated measure (RM) ANOVA analysis across all time points (-60 to 60 min; Fig. 2). There was a significant effect of time (F23,288=8.83, p

The in Vivo Neurochemical Profile of Selectively Bred High

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