Left thoracotomy surgical approach for chronic instrumentation in dogs and monkeys providing high-quality electrocardiogram signals.

INTRODUCTION: Assessment of cardiovascular parameters, including the electrocardiogram (ECG) is required by the regulatory guidelines. In safety pharmacology studies, this is typically done using chronically implanted radiotelemetry devices in non-rodent species. METHODS: We compared ECG signal quality from ten male beagle dogs and 10 male cynomolgus monkeys with telemetry transmitters implanted using two surgical approaches: i) epicardial ECG lead placement via single incision, left side thoracotomy or ii) subcutaneous ECG lead placement via laparotomy. In addition, epicardial leads and semi-automated scoring were used in combination to detect changes in ECG values caused by moxifloxacin. Telemetry-instrumented male beagle dogs (n=8) and male cynomolgus monkeys (n=8) were given moxifloxacin at 10, 30, or 100 mg/kg (dogs) and 10, 50, or 175 mg/kg (monkeys) as a single dose by oral gavage. RESULTS: ECG signals were of excellent quality with epicardial lead placement, and human activity in the room did not significantly alter signal quality. Administration of moxifloxacin was associated with prolongation of QTc interval, in both dogs and monkeys in a dose-dependent pattern. Dogs given 30 mg/kg and 100 mg/kg, the maximum QTcf interval prolongations were 22 ms (+9%, 8 h postdose) and 60 ms (+24%, 15 h postdose). In monkeys given 50 and 175 mg/kg, the QTcb interval was significantly prolonged from 1 to 6h postdose, and QTcb interval prolongation persisted in monkeys given 175 mg/kg through 19 h postdose. In monkeys given 175 mg/kg, the maximum QTcb interval prolongation was 43 ms (+12.9%, 16 h postdose). DISCUSSION: The present study demonstrated that placing leads directly on the epicardium drastically diminishes signal disruption due to room disturbances and subsequent animal excitement. This novel surgical model demonstrated adequate sensitivity to detect changes in ECG parameters, specifically QTc interval prolongation in both the dog and monkey.

Contributions of nucleus accumbens core and shell GluR1 containing AMPA receptors in AMPA- and cocaine-primed reinstatement of cocaine-seeking behavior.

Glutamate signaling in the nucleus accumbens influences reinstatement of previously extinguished cocaine-seeking behavior in rats. Whether or not region specific glutamate signaling in the nucleus accumbens contributes to reinstatement of cocaine-seeking behavior is not known. We investigated whether directly stimulating ionotropic glutamate receptors (GluRs) within the nucleus accumbens core or shell would differentially influence renewed cocaine-seeking behavior following extinction training. We also tested the hypothesis that GluR1 subunit (GluR1) containing alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors in the nucleus accumbens core and not the shell regulate reinstatement of previously extinguished cocaine-seeking behavior. Microinjection of AMPA into the nucleus accumbens shell and the nucleus accumbens core dose-dependently elicited significant cocaine-seeking behavior. Administration of antisense oligonucleotides (AS) directed against GluR1 subunit mRNA into the core and shell disrupted AMPA- and cocaine-primed reinstatement--with the most pronounced effects seen in the nucleus accumbens shell. These results demonstrate that GluRs in the nucleus accumbens core and shell influence AMPA- and cocaine-primed reinstatement, yet the nucleus accumbens shell exerts a prepotency over the nucleus accumbens core.

Concurrent access to sucrose pellets decreases methamphetamine-seeking behavior in Lewis rats.

Investigation of the role of choice between use of drugs of abuse and pursuit of alternative non-drug reinforcers is receiving greater attention. An understanding of the determinants influencing choice between drugs and alternative reinforcers will eventually lead to an understanding to the neural substrates of the drug altered brain. We investigated the impact of concurrent access to sucrose pellets on methamphetamine self-administration and self-regulated reinstatement of methamphetamine seeking following extinction training in Lewis rats. Our results from the self-administration experiment show that rats with concurrent access to sucrose self-administered significantly less methamphetamine compared to the methamphetamine only group. For our extinction/reinstatement experiment, concurrent access to sucrose during self-regulated methamphetamine reinstatement reduced methamphetamine intake and non-reinforced methamphetamine-seeking behavior in rats compared to rats that received access to just methamphetamine. These findings indicate that concurrent access to alternative reinforcers during various stages of methamphetamine-seeking behavior robustly decreased methamphetamine intake and serves as a valid rodent choice paradigm.

Black agouti (ACI) rats show greater drug- and cue-induced reinstatement of methamphetamine-seeking behavior than Fischer 344 and Lewis rats.

Fischer 344 (F344) and Lewis (LEW) rats differ in methamphetamine self-administration (SA) and methamphetamine-induced reinstatement of previously extinguished behavior. We sought to determine whether genetic background also influences methamphetamine reinforcement efficacy, conditioned reinstatement, and methamphetamine-primed reinstatement of responding in F344, LEW, and Black Agouti (ACI) rats. We implanted rats with jugular catheters and trained them to self-administer methamphetamine (0.06 mg/kg/infusion) under a progressive ratio (PR) schedule of reinforcement during daily 2-h SA sessions. A compound stimulus (light+tone; LT) was paired with each infusion. Dose-dependent intake was determined for each rat. Rats then entered the extinction phase of the experiment where responding resulted in no programmed consequences. Following extinction sessions, rats underwent conditioned reinstatement testing. For conditioned reinstatement, rats received response-contingent presentations of the LT and no methamphetamine. Last, methamphetamine-primed reinstatement test sessions where conducted where subjects received experimenter delivered infusions of methamphetamine (0.06, 0.12, or 0.24 mg/kg). The strains did not differ in PR responding across the doses tested. The ACI rats demonstrated the highest behavioral output during extinction training, conditioned- and methamphetamine-primed reinstatement of previously extinguished behavior compared to the other strains. These data suggest that genetic background differentially influences extinction, conditioned reinstatement and methamphetamine-primed reinstatement in rats.

Dopamine D4 receptor knockout mice exhibit neurochemical changes consistent with decreased dopamine release.

Dopamine D4 receptor (D4R) knockout mice (D4R-/-) provided for unique neurochemical studies designed to understand D4R contributions to dopamine (DA) regulation. In this study, post-mortem brain tissue content of DA did not differ between D4R+/+ and D4R-/- mice in the striatum (Str) or nucleus accumbens core (NAc). However, there was a significant decrease (82%) in the content of 3,4-dihydoxyphenylacetic acid (DOPAC), a major metabolite of DA, in the NAc of D4R-/- mice. Microdialysis studies performed in a region of brain spanning of the dorsal Str and NAc showed lower baseline levels of DA and a significant reduction in KCl-evoked overflow of DA in the D4R-/- mice. Baseline extracellular levels of DOPAC and homovanillic acid were also significantly lower in the D4R-/- mice. In vivo chronoamperometric recordings of KCl-evoked release of DA also showed decreased release of DA in the Str and NAc of the D4R-/- mice. These studies demonstrate a role of D4Rs in presynaptic DA regulation and support the hypothesis that alterations in D4Rs may lead to diminished DA function.

Does response-contingent access to cocaine reinstate previously extinguished cocaine-seeking behavior in C57BL/6J mice?

Inbred strains of mice are valuable tools for determining the impact of genes and the environment on behavior. However, use of mice in intravenous (iv) cocaine self-administration (SA) extinction/reinstatement paradigms has yielded mixed results. Mice do demonstrate significant conditioned reinstatement but do not significantly reinstate previously extinguished cocaine-seeking behavior when passively primed with cocaine. We tested the hypothesis that C57BL/6J (B6) mice would reinstate previously extinguished cocaine-seeking behavior when provided with response-contingent access to conditioned cues and fixed doses of cocaine. Male B6 mice were implanted with jugular catheters and trained to lever press for cocaine infusions. Each infusion was paired with a compound stimulus (light and tone; LT). Following 14 days of SA, subjects underwent extinction training--responding resulted in no programmed consequences. After at least 5 extinction sessions, cue-primed reinstatement was tested (LT-test). For the LT-test, mice received response-contingent presentations of the LT. After the LT-test, subjects returned to extinction training. Once responding decreased to extinction criteria, cocaine priming began. During cocaine priming, mice had response-contingent access to saline, 1.05, 3.5, or 17.5 mg/kg cocaine. Response-contingent presentations of the LT significantly reinstated cocaine-seeking behavior in the mice. Response-contingent access to cocaine dose-dependently reinstated responding. Our results suggest that response-contingent access to cocaine is a robust method for modeling cocaine craving and relapse in mice.

Dopamine D2 receptors mediate reversal learning in male C57BL/6J mice.

Dopamine is critical for directing goal-oriented behavior. We investigated dopamine D2 receptor involvement in reversal learning and reinforcement efficacy in mice lacking functional dopamine D2 receptors and their heterozygous and wild-type littermates. Mice discriminated between two odors to receive a food reinforcer. One odor signaled a reinforcer (S+); the other odor signaled no reinforcer (S-). After mice learned the S+/S- relationship, we inverted the reinforcement contingencies. The necessary number of trials to relearn the new reinforcement contingencies served as our index of reversal learning. Mice lacking functional dopamine D2 receptors repeatedly failed to inhibit previously reinforced responses during reversal trials. In a separate experiment, mice responded for reinforcers on a progressive ratio schedule of reinforcement. Mice lacking functional dopamine D2 receptors earned significantly fewer reinforcers than did heterozygous mice. Our results suggest that dopamine D2 receptors regulate reversal learning and influence the reinforcing efficacy of natural rewards.

Differences in extinction responding and reinstatement of methamphetamine-seeking behavior between Fischer 344 and Lewis rats.

Fischer 344 (F344) and Lewis (LEW) rats differ in a number of self-administration behaviors. Whether or not these strains differ in methamphetamine-primed reinstatement of extinguished responding is unknown. F344 and LEW rats were trained to self-administer intravenous (i.v.) methamphetamine (0.06 mg/kg) during daily 2-h limited access sessions for 14 days. Following methamphetamine self-administration, subjects underwent a minimum of 6 extinction sessions where responding on the previously active lever resulted in no programmed consequences. Following extinction sessions, we evaluated strain and dose dependency of methamphetamine-primed (0.06, 0.12, or 0.24 mg/kg/i.v.) reinstatement of responding. All subjects received each dose once. Dosing order was determined by utilizing a within-subjects Latin square design. We found partial strain differences in daily methamphetamine self-administration. In addition, F344 rats responded significantly more during the first extinction session compared LEW rats. Last, the LEW rats demonstrated a heightened propensity to reinstate responding following methamphetamine priming injections compared to F344 rats. Our results suggest that genetic background influences differences in methamphetamine-seeking behaviors in rats.

Different patterns of pharmacological reinstatement of cocaine-seeking behavior between Fischer 344 and Lewis rats.

RATIONALE: Fischer 344 (F344) and Lewis (LEW) rats differ in cocaine self-administration behaviors. Whether or not these inbred strains of rats differ in pharmacological reinstatement of cocaine-seeking behavior is unknown. OBJECTIVES: The purpose of the present study was to determine if inbred strains of rats demonstrate differences in alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and cocaine-induced reinstatement of previously extinguished cocaine-seeking behavior. METHODS: F344 and LEW rats received indwelling jugular catheters, bilateral guide cannula aimed at the nucleus accumbens core, and were trained to lever press for 0.5 mg/kg intravenous cocaine during 2-h self-administration sessions. Following 14 sessions, rats underwent extinction sessions, where previously reinforced lever pressing resulted in no programmed consequences. Just prior to beginning extinction session 7, rats received an intracranial infusion of saline. Lever pressing was not reinforced. During subsequent extinction sessions, rats received AMPA injections (0.2, 0.4, or 0.6 nM). Dosing order was determined by a within-subject Latin square design. At least two extinction sessions separated each AMPA session. Rats then underwent cocaine-induced reinstatement test sessions (lever pressing was not reinforced). Rats received passive intravenous cocaine (0.0, 0.5, 1.0, or 2.0 mg/kg) after being placed in the experimental chamber. At least two extinction sessions separated each cocaine-prime session, and subjects were tested at each dose according to a within-subjects Latin square design. RESULTS: LEW rats demonstrated blunted maximal responding to AMPA-induced reinstatement and heightened sensitivity to cocaine-induced reinstatement compared with F344 rats. CONCLUSIONS: This study demonstrates that inbred strains differ in pharmacological reinstatement of cocaine-seeking behavior.

Increased AMPA GluR1 receptor subunit labeling on the plasma membrane of dendrites in the basolateral amygdala of rats self-administering morphine.

Glutamate-dependent synaptic plasticity is emerging as an important neural substrate of addiction. These drug-dependent neural adaptations may occur within brain systems that mediate reward, emotion, and cognitive function such as the amygdala complex. Modification of glutamate receptor targeting may be a key mechanism mediating neural plasticity; however, evidence for alteration of amygdala AMPA receptor localization in response to drug self-administration is lacking. High-resolution immunogold electron microscopic immunocytochemistry was used to compare surface and intracellular labeling of the calcium sensitive AMPA GluR1 receptor subunit in the basolateral (BLA) and central (CeA) nuclei of the amygdala in rats self-administering escalating doses of morphine or saline. Morphine self-administration was associated with regionally diverse effects on dendritic GluR1 targeting in the BLA and CeA. In the BLA of morphine self-administering animals, there was a significant increase in the proportion of immunogold particles for GluR1 on the plasma membrane of dendrites, particularly in association with extrasynaptic sites, which was most prominent in large (2-4 microm) profiles. In contrast, there were no significant differences in surface or intracellular immunogold labeling in the CeA between morphine self-administering and control animals. In both amygdala regions, GluR1 and the micro-opioid receptor, the major cellular target of morphine, were only infrequently colocalized. These results indicate that GluR1 targeting is a dynamic process that can be differentially affected in distinct amygdala regions in response to chronic self-administration of morphine. Homeostatic adaptations in the subcellular localization of calcium sensitive AMPA receptors within the BLA may be an important neural substrate for alterations in reward, autonomic function, and behavioral processes associated with opiate addiction.

Decreased plasma membrane targeting of NMDA-NR1 receptor subunit in dendrites of medial nucleus tractus solitarius neurons in rats self-administering morphine.

Opioid abuse is associated with repeated administration and escalation of dose that can result in profound adaptations in homeostatic processes. Potential cellular mechanisms and neural sites mediating opiate-dependent adaptations may involve NMDA-dependent synaptic plasticity within brain areas participating in behaviors related to consumption of natural reinforcers, as well as affective-autonomic integration, notably the medial nucleus tractus solitarius (mNTS). NMDA-dependent synaptic plasticity may be mediated by changes in the intracellular and surface targeting of NMDA receptors, particularly in postsynaptic sites including spines or small distal dendrites. High-resolution immunogold electron microscopic immunocytochemistry combined with morphometry were used to measure changes in targeting of the NMDA-NR1 (NR1) receptor subunit between intracellular and plasmalemmal sites in dendrites of neurons of the intermediate mNTS of rats self-administering escalating doses of morphine (EMSA). In control and EMSA rats, the density of plasmalemmal and cytosolic gold particles was inversely related to profile size. Collapsed across all NR1-labeled dendrites, rats self-administering morphine had a lower number of plasmalemmal gold particles per unit surface area (7.1 +/- 0.8 vs. 14.4 +/- 1 per 100 microm), but had a higher number of intracellular gold particles per unit cross-sectional area (169 +/- 6.1 vs. 148 +/- 5.1 per 100 microm2) compared to saline self-administering rats. Morphometric analysis showed that the decrease in plasma membrane labeling of NR1 was most robust in small dendritic profiles (<1 microm), where there was a reciprocal increase in the density of intracellular particles. These results indicate that the plasmalemmal distribution of the essential NR1 subunits in distal sites may prominently contribute to NMDA receptor-dependent modulation of neural circuitry regulating homeostatic processes, and targeting of these proteins can be prominently affected by morphine self-administration.

Dopamine D4 receptor-deficient mice, congenic on the C57BL/6J background, are hypersensitive to amphetamine.

Mice lacking the dopamine D4 receptor subtype (D4R-/-) are supersensitive to methamphetamine and cocaine. We sought to expand and refine earlier experiments performed on F2 generation D4R-/- mice by lengthening the behavioral session, utilizing an N10 D4R-/- incipient congenic C57BL/6J line (D4R-/- mice backcrossed with wildtype C57BL/6J mice for 10 successive generations), and investigating whether dopamine D4Rs are necessary for the expression of behavioral sensitization to amphetamine. The D4R-/- mice demonstrated an enhanced and dose-dependent increase in amphetamine-stimulated activity compared to wildtype mice following acute administrations of amphetamine. For the behavioral sensitization experiments, separate groups of mice received either repeated administrations of the same dose of amphetamine or a subthreshold dose of amphetamine (2 mg/kg) 28 days following pretreatment with either saline, 1.0, 3.0, or 10.0 mg/kg amphetamine. The D4R-/- mice displayed an enhanced dose-dependent sensitized response to repeated amphetamine administrations compared to their wildtype littermates in both behavioral sensitization paradigms. Our present results further support the importance of dopamine D4Rs in psychostimulant-mediated locomotion and neural plasticity.

3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone.

Thyroxine (T(4)) is the predominant form of thyroid hormone (TH). Hyperthyroidism, a condition associated with excess TH, is characterized by increases in metabolic rate, core body temperature and cardiac performance. In target tissues, T(4) is enzymatically deiodinated to 3,5,3'-triiodothyronine (T(3)), a high-affinity ligand for the nuclear TH receptors TR alpha and TR beta, whose activation controls normal vertebrate development and physiology. T(3)-modulated transcription of target genes via activation of TR alpha and TR beta is a slow process, the effects of which manifest over hours and days. Although rapidly occurring effects of TH have been documented, the molecules that mediate these non-genomic effects remain obscure. Here we report the discovery of 3-iodothyronamine (T(1)AM), a naturally occurring derivative of TH that in vitro is a potent agonist of the G protein-coupled trace amine receptor TAR1. Administering T(1)AM in vivo induces profound hypothermia and bradycardia within minutes. T(1)AM treatment also rapidly reduces cardiac output in an ex vivo working heart preparation. These results suggest the existence of a new signaling pathway, stimulation of which leads to rapid physiological and behavioral consequences that are opposite those associated with excess TH.

Dopamine D2 receptors mediate two-odor discrimination and reversal learning in C57BL/6 mice.

BACKGROUND: Dopamine modulation of neuronal signaling in the frontal cortex, midbrain, and striatum is essential for processing and integrating diverse external sensory stimuli and attaching salience to environmental cues that signal causal relationships, thereby guiding goal-directed, adaptable behaviors. At the cellular level, dopamine signaling is mediated through D1-like or D2-like receptors. Although a role for D1-like receptors in a variety of goal-directed behaviors has been identified, an explicit involvement of D2 receptors has not been clearly established. To determine whether dopamine D2 receptor-mediated signaling contributes to associative and reversal learning, we compared C57Bl/6J mice that completely lack functional dopamine D2 receptors to wild-type mice with respect to their ability to attach appropriate salience to external stimuli (stimulus discrimination) and disengage from inappropriate behavioral strategies when reinforcement contingencies change (e.g. reversal learning). RESULTS: Mildly food-deprived female wild-type and dopamine D2 receptor deficient mice rapidly learned to retrieve and consume visible food reinforcers from a small plastic dish. Furthermore, both genotypes readily learned to dig through the same dish filled with sterile sand in order to locate a buried food pellet. However, the dopamine D2 receptor deficient mice required significantly more trials than wild-type mice to discriminate between two dishes, each filled with a different scented sand, and to associate one of the two odors with the presence of a reinforcer (food). In addition, the dopamine D2 receptor deficient mice repeatedly fail to alter their response patterns during reversal trials where the reinforcement rules were inverted. CONCLUSIONS: Inbred C57Bl/6J mice that develop in the complete absence of functional dopamine D2 receptors are capable of olfaction but display an impaired ability to acquire odor-driven reinforcement contingencies. Furthermore, the ability of dopamine D2 receptor deficient mice to adjust their responding to a previously reinforced stimulus when unexpected outcomes are encountered is significantly impaired. These findings suggest that signaling mediated by the dopamine D2 receptor is important for regulating associative and reversal learning and may have implications for the treatment of human attention disorders.