Hold-down as an alternative to unit dose in cocaine self-administration experiments: Characterization using a progressive ratio schedule.

RATIONALE: Virtually all cocaine self-administration studies have used a "unit dose" as a reinforcing stimulus; the subject is a passive recipient of an experimenter-selected dose. OBJECTIVES: The present experiments examined the consequence of requiring the subject to actively determine the dose and speed of each injection. METHODS: A two-lever procedure was used in which responding on a progressive ratio (PR) schedule provided access to cocaine on a hold down (HD) schedule. With HD, the pump is turned on for the duration that the lever is held down, thus the dose and speed of injection is determined by the behavior of the subject. The procedure allows for the evaluation of both drug taking and drug seeking responses. RESULTS: The results were qualitatively different from PR self-administration studies using unit dose. The self-administered HD dose varied across the session; the self-administered dose was found to inversely correlate with drug levels at the time of access. Importantly, the 2 L-PR-HD procedure identified a subpopulation of subjects that showed extremes in both drug seeking and drug taking. Subjects at the top end of the distribution displayed unprecedented final ratios (> 900) and rapidly self-administered very large doses (> 1.4 mg; ~ 4.2 mg/kg). Manipulation of drug-taking variables (HD access duration and concentration of drug in the pump) showed that the immediacy of a cocaine bolus, not the duration of access, is the major determinant of drug seeking. CONCLUSIONS: Incorporating a consummatory response into a PR procedure provides a unique perspective on the interactions of drug-seeking and drug-taking.

Intermittent intake of rapid cocaine injections promotes robust psychomotor sensitization, increased incentive motivation for the drug and mGlu2/3 receptor dysregulation.

The choice between smoking, injecting or swallowing a drug influences the risk of addiction, as this determines both how much drug gets into the brain and how fast. Most animal studies on addiction focus on how much drug it takes to produce pathological drug use. How fast drugs get to the brain is generally ignored. A few studies have examined the influence of the speed of drug onset, but speed varied along with cumulative intake. Here we held average cumulative intake constant and determined whether variation in the speed of cocaine onset alone predicts outcome. Two groups of rats self-administered intravenous cocaine (0.25 mg/kg/injection) during daily sessions. Cocaine was available intermittently during each session. This produces the spikes and troughs in brain levels of cocaine thought to model how addicts take the drug. To vary the speed of cocaine onset, each injection was delivered over 5 s to one group, and over 90 s to the other. Average cumulative cocaine intake was the same in the two groups. However, rapid injections promoted robust psychomotor sensitization and potentiated incentive motivation for cocaine (0.063-0.25 mg/kg/injection). This addiction-relevant phenotype was accompanied by enhanced functional activity of metabotropic glutamate group II receptors (mGluR2/3s) in the prelimbic cortex and nucleus accumbens. Pharmacological activation of mGluR2/3s with LY379268 also preferentially decreased the motivation to take cocaine in rats previously exposed to rapid drug injections. Thus, varying the speed of drug onset can be used to parse the neurobiology of addiction from that of mere drug taking.

How fast and how often: The pharmacokinetics of drug use are decisive in addiction.

How much, how often and how fast a drug reaches the brain determine the behavioural and neuroplastic changes associated with the addiction process. Despite the critical nature of these variables, the drug addiction field often ignores pharmacokinetic issues, which we argue can lead to false conclusions. First, we review the clinical data demonstrating the importance of the speed of drug onset and of intermittent patterns of drug intake in psychostimulant drug addiction. This is followed by a review of the preclinical literature demonstrating that pharmacokinetic variables play a decisive role in determining behavioural and neurobiological outcomes in animal models of addiction. This literature includes recent data highlighting the importance of intermittent, 'spiking' brain levels of drug in producing an increase in the motivation to take drug over time. Rapid drug onset and intermittent drug exposure both appear to push the addiction process forward most effectively. This has significant implications for refining animal models of addiction and for better understanding the neuroadaptations that are critical for the disorder.

CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.

UNLABELLED: Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression. IMPORTANCE: The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

Nipping cue reactivity in the bud: baclofen prevents limbic activation elicited by subliminal drug cues.

Relapse is a widely recognized and difficult to treat feature of the addictions. Substantial evidence implicates cue-triggered activation of the mesolimbic dopamine system as an important contributing factor. Even drug cues presented outside of conscious awareness (i.e., subliminally) produce robust activation within this circuitry, indicating the sensitivity and vulnerability of the brain to potentially problematic reward signals. Because pharmacological agents that prevent these early cue-induced responses could play an important role in relapse prevention, we examined whether baclofen-a GABAB receptor agonist that reduces mesolimbic dopamine release and conditioned drug responses in laboratory animals-could inhibit mesolimbic activation elicited by subliminal cocaine cues in cocaine-dependent individuals. Twenty cocaine-dependent participants were randomized to receive baclofen (60 mg/d; 20 mg t.i.d.) or placebo. Event-related BOLD fMRI and a backward-masking paradigm were used to examine the effects of baclofen on subliminal cocaine (vs neutral) cues. Sexual and aversive cues were included to examine specificity. We observed that baclofen-treated participants displayed significantly less activation in response to subliminal cocaine (vs neutral) cues, but not sexual or aversive (vs neutral) cues, than placebo-treated participants in a large interconnected bilateral cluster spanning the ventral striatum, ventral pallidum, amygdala, midbrain, and orbitofrontal cortex (voxel threshold p < 0.005; cluster corrected at p < 0.05). These results suggest that baclofen may inhibit the earliest type of drug cue-induced motivational processing-that which occurs outside of awareness-before it evolves into a less manageable state.

Reduction of the reinforcing effectiveness of cocaine by continuous D-amphetamine treatment in rats: importance of active self-administration during treatment period.

RATIONALE: Continuous administration of D-amphetamine has shown promise as a treatment for psychostimulant addiction. In rodent studies, constant infusion of D-amphetamine (5 mg/kg/day) has been shown to reduce cocaine-reinforced responding in the dose range of 0.19-0.75 mg/kg/inf. OBJECTIVES: The present study tested whether these effects were a reflection of pharmacological interactions between D-amphetamine and cocaine or if they resulted from associative learning mechanisms METHODS: After stable progressive ratio (PR) baselines were established, rats were implanted with subcutaneous osmotic minipumps filled with either D-amphetamine (5 mg/kg/day-groups 1 and 2) or saline (group 3). During the treatment period, groups 1 and 3 self-administered cocaine at a dose that was previously shown to produce the most robust effects in combination with D-amphetamine treatment (0.19 mg/kg/inf), while group 2 received passive cocaine infusions. RESULTS: In replication of previous studies, D-amphetamine treatment resulted in a significant (35 %) decrease in breakpoints relative to saline controls. By contrast, no reductions in breakpoints were observed in animals that received passive cocaine infusions during the treatment period (group 2). CONCLUSIONS: Active self-administration of cocaine during the treatment period appears to be an important factor in reducing cocaine-reinforced breakpoints. These findings suggest learning mechanisms are involved in the therapeutic effects of continuous D-amphetamine, and pharmacological interaction mechanisms such as cross-tolerance cannot completely account for the observed decreases in cocaine seeking.

Methylphenidate and cocaine self-administration produce distinct dopamine terminal alterations.

Methylphenidate (MPH) is a commonly abused psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder. MPH has a mechanism of action similar to cocaine (COC) and is commonly characterized as a dopamine transporter (DAT) blocker. While there has been extensive work aimed at understanding dopamine (DA) nerve terminal changes following COC self-administration, very little is known about the effects of MPH self-administration on the DA system. We used fast scan cyclic voltammetry in nucleus accumbens core slices from animals with a 5-day self-administration history of 40 injections/day of either MPH (0.56 mg/kg) or COC (1.5 mg/kg) to explore alterations in baseline DA release and uptake kinetics as well as alterations in the interaction of each compound with the DAT. Although MPH and COC have similar behavioral effects, the consequences of self-administration on DA system parameters were found to be divergent. We show that COC self-administration reduced DAT levels and maximal rates of DA uptake, as well as reducing electrically stimulated release, suggesting decreased DA terminal function. In contrast, MPH self-administration increased DAT levels, DA uptake rates and DA release, suggesting enhanced terminal function, which was supported by findings of increased metabolite/DA tissue content ratios. Tyrosine hydroxylase messenger RNA, protein and phosphorylation levels were also assessed in both groups. Additionally, COC self-administration reduced COC-induced DAT inhibition, while MPH self-administration increased MPH-induced DAT inhibition, suggesting opposite pharmacodynamic effects of these two drugs. These findings suggest that the factors governing DA system adaptations are more complicated than simple DA uptake blockade.

The group II metabotropic glutamate receptor agonist, LY379268, decreases methamphetamine self-administration in rats.

BACKGROUND: Given the problems associated with the escalation in methamphetamine (METH) use, the identification of more effective treatment strategies is essential. Group II metabotropic glutamate receptors (mGluRs) have been suggested to be a novel therapeutic target for psychostimulant addiction. We sought to test the ability of the selective group II mGluR agonist LY379268 to reduce METH self-administration in rats. METHODS: Rats were trained to self-administer METH on a progressive ratio (PR) schedule. Animals were then switched to fixed ratio responding and given daily extended access (6 h/day) to METH self-administration for 14 days. Rats were then re-tested on the PR schedule. The effect of LY379268 on METH-reinforced PR responding was determined before and after 14 days of extended access. To test for non-specific effects, a separate group of animals received LY379268 prior to a sucrose pellet-reinforced PR schedule. RESULTS: Animals escalated their daily intake of METH during extended access. PR responding did not change as a function of extended access. LY379268 significantly attenuated METH reinforced responding, both before and after extended access. The degree of attenuation did not change as a function of extended access. LY379268 had no effect on sucrose pellet-reinforced responding at any dose. CONCLUSIONS: LY379268 selectively reduced the motivation to self-administer METH. In contrast to data with other compounds, the sensitivity to the effects of LY379268 did not change following extended access to METH self-administration. Group II mGluR agonists, therefore, may represent a relatively new class of compounds for the development of pharmacotherapies for METH addiction.

Paradoxical tolerance to cocaine after initial supersensitivity in drug-use-prone animals.

There is great interest in outlining biological factors and behavioral characteristics that either predispose or predict vulnerability to substance use disorders. Response to an inescapable novel environment has been shown to predict a "drug-use-prone" phenotype that is defined by rapid acquisition of cocaine self-administration. Here, we showed that response to novelty can also predict the neurochemical and behavioral effects of acute and repeated cocaine in rats. We used cocaine self-administration under a fixed-ratio 1 schedule followed by fast-scan cyclic voltammetry in brain slices to measure subsecond dopamine (DA) release and uptake parameters in drug-use-prone and -resistant phenotypes. Despite no significant differences in stimulated release and uptake, animals with high responses to a novel environment had DA transporters that were more sensitive to cocaine-induced uptake inhibition, which corresponded to greater locomotor activating effects of cocaine. These animals also acquired cocaine self-administration more rapidly and, after 5 days of extended access cocaine self-administration, high-responding animals showed robust tolerance to DA uptake inhibition by cocaine. The effects of cocaine remained unchanged in animals with low novelty responses. Similarly, the rate of acquisition was negatively correlated with DA uptake inhibition by cocaine after self-administration. Thus, we showed that tolerance to the cocaine-induced inhibition of DA uptake coexists with a behavioral phenotype that is defined by increased preoccupation with cocaine as measured by rapid acquisition and early high intake.

Conflation of cocaine seeking and cocaine taking responses in IV self-administration experiments in rats: methodological and interpretational considerations.

IV drug self-administration is a special case of an operant task. In most operant experiments, the instrumental response that completes the schedule requirement is separate and distinct from the consumptive response (e.g. eating or drinking) that follows the delivery of the reinforcing stimulus. In most IV self-administration studies drug seeking and drug taking responses are conflated. The instrumental lever press or nose poke is also a consumptive response. The conflation of these two response classes has important implications for interpretation of the data as they are differentially regulated by dose and price. The types of pharmacological pretreatments that affect appetitive responses are not necessarily the same as those that affect consumptive responses suggesting that the neurobiology of the two response classes are to some extent controlled by different mechanisms. This review discusses how schedules of reinforcement and behavioral economic analyses can be used to assess the regulation of drug seeking and drug taking separately. New methods are described that allow the examination of appetitive or consumptive responding in isolation and provide subjects with greater control over the self-administered dose. These procedures provide novel insights into the regulation of drug intake. Cocaine intake patterns that result in large, intermittent spikes in cocaine levels are shown to produce increases in appetitive responding (i.e. drug seeking). The mechanisms that control drug intake should be considered distinct from appetitive and motivational processes and should be taken into consideration in future IV self-administration studies.

Temporal pattern of cocaine intake determines tolerance vs sensitization of cocaine effects at the dopamine transporter.

The dopamine transporter (DAT) is responsible for terminating dopamine (DA) signaling and is the primary site of cocaine's reinforcing actions. Cocaine self-administration has been shown previously to result in changes in cocaine potency at the DAT. To determine whether the DAT changes associated with self-administration are due to differences in intake levels or temporal patterns of cocaine-induced DAT inhibition, we manipulated cocaine access to produce either continuous or intermittent elevations in cocaine brain levels. Long-access (LgA, 6 h) and short-access (ShA, 2 h) continuous self-administration produced similar temporal profiles of cocaine intake that were sustained throughout the session; however, LgA had greater intake. ShA and intermittent-access (IntA, 6 h) produced the same intake, but different temporal profiles, with 'spiking' brain levels in IntA compared with constant levels in ShA. IntA consisted of 5-min access periods alternating with 25-min timeouts, which resulted in bursts of high responding followed by periods of no responding. DA release and uptake, as well as the potency of cocaine for DAT inhibition, were assessed by voltammetry in the nucleus accumbens slices following control, IntA, ShA, and LgA self-administration. Continuous-access protocols (LgA and ShA) did not change DA parameters, but the 'spiking' protocol (IntA) increased both release and uptake of DA. In addition, high continuous intake (LgA) produced tolerance to cocaine, while 'spiking' (IntA) produced sensitization, relative to ShA and naive controls. Thus, intake and pattern can both influence cocaine potency, and tolerance seems to be produced by high intake, while sensitization is produced by intermittent temporal patterns of intake.

Striatal CB1 and D2 receptors regulate expression of each other, CRIP1A and δ opioid systems.

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1 R) and D2 dopamine (D2 R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1 R or D2 R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1 R and D2 R knockdown reduced striatal dopaminergic-stimulated [(35) S]GTPγS binding, and D2 R knockdown reduced pallidal WIN55212-2-stimulated [(35) S]GTPγS binding. Decreased D2 R and CB1 R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1 Rs or D2 Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [(35) S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1 R or D2 R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.

Examination of behavioral strategies regulating cocaine intake in rats.

RATIONALE: It has long been observed that rats self-administer psychostimulants in a highly regular pattern. The inverse relationship between dose and rate of drug intake has been interpreted as a titration phenomenon wherein brain-cocaine levels are maintained within a range. Most studies examining this phenomenon have used fixed, unit doses in which case the only titration strategy available to the animal is to adjust inter-infusion intervals. OBJECTIVES: In this study, we examined whether selection of dose size could also be a factor in regulation of intake. We used a schedule of reinforcement, under which the dose can vary through a wide range and is determined by the behavior of the animal. METHODS: Rats self-administered cocaine using a behaviorally dependent dosing schedule of reinforcement, under which the size of each dose was determined by the length of time the lever was held down. The concentration of cocaine was changed across sessions. RESULTS: Total pump-time self-administered decreased by 56 % following each doubling of the concentration, which led to an average 11 % increase in total intake. Similarly, estimated brain levels of cocaine increased by 12 % for each doubling of concentration. These adjustments were the result of manipulation of both the size and spacing of infusions. CONCLUSIONS: In agreement with previous studies, the regular pattern of intake appears to be the result of a titration mechanism in which animals maintain brain levels of cocaine above some threshold. Compensatory regulation appeared to involve both the selection of dose size and inter-infusion intervals.

Cocaine self-administration produces pharmacodynamic tolerance: differential effects on the potency of dopamine transporter blockers, releasers, and methylphenidate.

The dopamine transporter (DAT) is the primary site of action for psychostimulant drugs such as cocaine, methylphenidate, and amphetamine. Our previous work demonstrated a reduced ability of cocaine to inhibit the DAT following high-dose cocaine self-administration (SA), corresponding to a reduced ability of cocaine to increase extracellular dopamine. However, this effect had only been demonstrated for cocaine. Thus, the current investigations sought to understand the extent to which cocaine SA (1.5 mg/kg/inf × 40 inf/day × 5 days) altered the ability of different dopamine uptake blockers and releasers to inhibit dopamine uptake, measured using fast-scan cyclic voltammetry in rat brain slices. We demonstrated that, similar to cocaine, the DAT blockers nomifensine and bupropion were less effective at inhibiting dopamine uptake following cocaine SA. The potencies of amphetamine-like dopamine releasers such as 3,4-methylenedioxymethamphetamine, methamphetamine, amphetamine, and phentermine, as well as a non-amphetamine releaser, 4-benzylpiperidine, were all unaffected. Finally, methylphenidate, which blocks dopamine uptake like cocaine while being structurally similar to amphetamine, shared characteristics of both, resembling an uptake blocker at low concentrations and a releaser at high concentrations. Combined, these experiments demonstrate that after high-dose cocaine SA, there is cross-tolerance of the DAT to other uptake blockers, but not releasers. The reduced ability of psychostimulants to inhibit dopamine uptake following cocaine SA appears to be contingent upon their functional interaction with the DAT as a pure blocker or releaser rather than their structural similarity to cocaine. Further, methylphenidate's interaction with the DAT is unique and concentration-dependent.

Cocaine self-administration in rats: hold-down procedures.

For decades, researchers have used animal self-administration models to examine the effects drugs of abuse have on physiology and behavior. Sophisticated self-administration procedures have been developed to model many different aspects of drug addiction. The hold-down procedure provides animals with control over the amount of each injection. Holding the lever down turns the syringe pump on and subsequently releasing the lever turns the pump off. In this way, animals can hold the lever down for any duration of time thereby self-administering any dose on a continuous spectrum. This procedure eliminates some of the ambiguity in translating results from effects only observed at one unit dose and allows examination of which dose the animal "prefers" at different times.

Cocaine self-administration in rats: discrete trials procedures.

A discrete trials procedure involves splitting up a self-administration session so that there are multiple distinct trials and inter-trial-intervals. This schedule is well suited to be used over 24 h periods which allows insight into diurnal variability in self-administration behavior. DT is also well suited for investigations using pretreatments for increasing or decreasing both high and low probability behavior.

Cocaine self-administration in rats: threshold procedures.

Cocaine self-administration provides a methodology allowing researchers to study changes in distinct aspects of drug-taking behavior that model behaviors observed in drug addicts. Traditionally, self-administration schedules were designed to independently study changes in drug-taking behaviors (e.g., rate of responding, reinforcing efficacy, etc.). The threshold self-administration procedure was developed to measure two distinct dependent measures within the same experimental session that are important in the study of drug addiction: the maximal price an animal expends to self-administer cocaine and an animal's preferred level of cocaine consumption when available at a low behavioral cost.

Brain-cocaine concentrations determine the dose self-administered by rats on a novel behaviorally dependent dosing schedule.

A novel behaviorally dependent dosing (BDD) schedule was used to examine the relationship between doses of cocaine self-administered by rats and brain drug levels within a session. The BDD schedule used a hold-down response to activate a syringe pump. The length of time the lever was held down determined the duration that the syringe pump was activated. In the first experiment, rats self-administered cocaine for daily 3 h sessions and brain levels of cocaine were modeled using well-established parameters. Although analysis revealed that rats self-administered doses within a predicted range, one extremely large dose was consistently observed at the beginning of each session when brain levels of cocaine were low. In the second experiment, we introduced a range of timeout periods (10-25 min) in order to produce variability in brain-cocaine concentrations. Animals self-administered larger doses immediately following each timeout period and the dose size was inversely correlated with the length of the timeout. These results show that the dose of cocaine that rats self-administer within a session is inversely related to the amount of drug on board.

A novel IV cocaine self-administration procedure in rats: differential effects of dopamine, serotonin, and GABA drug pre-treatments on cocaine consumption and maximal price paid.

RATIONALE: Behavior occurring during cocaine self-administration can be classified as either consummatory or appetitive. These two concepts are usually addressed independently using separate reinforcement schedules. For example, appetitive behavior can be assessed with a progressive ratio schedule, whereas consummatory behavior is typically measured using a fixed ratio schedule. OBJECTIVES: Depending on the schedule used, it is often difficult to determine whether a particular drug pretreatment is affecting self-administration through an effect on appetitive responding, consummatory responding, or perhaps both. In the present study, we tested the effect of pretreating rats with four different drugs on appetitive and consummatory behaviors. MATERIALS AND METHODS: We recently developed a technique that provides an independent assessment of both behavioral concepts within the same experimental session. In this threshold procedure, rats are offered a descending series of 11 unit doses (422-1.3 µg/injection) during consecutive timed intervals under a fixed-ratio schedule. Consummatory behavior can be analyzed by assessing intake at high unit doses; an estimate of appetitive responding can be determined from responding occurring at the threshold dose. Applying behavioral economics to these data provides dependent measures of consumption when minimally constrained by price and the maximal price paid (P (max)) for cocaine. RESULTS: Haloperidol increased cocaine consumption when minimally constrained by price but decreased P (max). In contrast, D: -amphetamine increased P (max). Fluoxetine decreased P (max) and consumption when minimally constrained by price. Baclofen selectively decreased P (max). CONCLUSIONS: These data suggest that drug pretreatments can alter consummatory and appetitive behavior differently because each concept involves distinct neural mechanisms.