Vladimir L. Tsibulsky

Priming threshold: a novel quantitative measure of the reinstatement of cocaine self-administration

The intravenous injection of cocaine has been reported to reliably reinstate (prime) the self-administration of cocaine in animals. We report herein that there is a cocaine priming threshold in rats trained to self-administer cocaine. The cocaine priming threshold is defined as the minimum level of cocaine in the body that will reinstate maintained cocaine self-administration. The mean cocaine priming threshold in rats was calculated to be approximately 186 to 212 microg kg(-1). Therefore, any injection, series of injections or continuous infusion that result in a level of cocaine equivalent to that produced by a single intravenous injection of this range of doses, will reinstate cocaine self-administration. The priming threshold was significantly increased by the D(1) dopamine receptor antagonist SCH23390 (10 microg kg(-1), i.v.), indicating a role for dopaminergic neurotransmission. The priming threshold, but not the inter-injection interval of maintained self-administration, was increased following withdrawal from a 7-day infusion of D-amphetamine. In addition, there was no correlation between the cocaine priming threshold and the inter-injection intervals of maintained cocaine self-administration. Therefore, the mechanisms underlying the reinstatement of cocaine self-administration are distinct from the mechanisms underlying the maintenance of cocaine self-administration and they are differentially regulated. It is possible that the priming threshold may represent a distinct target for medications development.

Mixed D2/5-HT2A Antagonism of Cocaine-Induced Facilitation of Brain Stimulation Reward

Previous behavioral, neurochemical and neurophysiological experiments have shown that selective 5-HT2A and mixed D2/5-HT2A antagonists can attenuate some, but not all, responses to amphetamine. The generality of these findings were determined in the present experiment by assessing the effect of mixed D2/5-HT2A antagonists on cocaine-induced facilitation of ventral tegmental area self-stimulation in rats. Although amphetamine and cocaine influence activity in monoaminergic neurons through different mechanisms, our previous research has shown that selective D2 and 5-HT2A antagonists have similar effects on behavioral responses to these psychostimulants. Therefore, we expected a similar pattern of results using mixed D2/5-HT2A antagonists. As shown previously, cocaine decreased self-stimulation threshold in a dose-dependent manner. Haloperidol and the mixed D2/5-HT2A antagonists risperidone and MDL 28, 133A antagonized cocaine-induced facilitation of self-stimulation, but only at doses that increased baseline self-stimulation threshold. There was a significant correlation (r = 0.87, p < 0.001) between antagonist-induced change in baseline threshold and attenuation of cocaines effect on threshold. Taken together, the results of this and previous experiments support the importance of D2 receptors in the mechanisms of brain stimulation reward. 5-HT2A receptors appear not to be involved in mediation of both brain stimulation reward and amphetamine- and cocaine-induced facilitation of brain stimulation reward.

Transient amelioration of the sensitization of cocaine-induced behaviors in rats by the induction of tolerance

Intermittent administration of cocaine produced a progressive increase in the stereotypy response of rats to a challenge dose of cocaine (7.5 mg/kg, i.p.). Continuous infusion of cocaine (80 mg/kg per day) via osmotic pumps for 7 days into the sensitized rats produced tolerance to the behavioral responses to the challenge dose of cocaine 1 day after the removal of the pumps. Therefore, tolerance can mask the expression of behavioral sensitization in rats. However, by 10 days after the removal of the pumps, the behavioral tolerance was reversed and the rats again displayed a sensitized response to cocaine. Therefore, the tolerance to cocaine was temporary while the underlying sensitization persisted. The development of tolerance did not alter the underlying sensitization demonstrating that these represent independent phenomena. The relationship between sensitization and tolerance observed in these studies may provide a model relevant to the progress in humans of addiction to psychomotor stimulants.

Characterization of the distribution of the cocaine priming threshold and the effect of SCH23390.

The cocaine-induced reinstatement (priming) of cocaine self-administration occurs when the cumulative concentration of cocaine reaches a threshold level that we have previously termed the cocaine priming threshold. The present studies used a modified procedure to measure the cocaine priming threshold over 4-8-month periods in individual rats. The values for the priming threshold varied between days but there was no evidence of a systematic change in the priming threshold over time, indicating that neither tolerance nor sensitization occurred. The frequency distribution of the priming threshold was significantly different from a normal distribution but was not significantly different from a log-normal distribution. Therefore, the geometric mean with its associated variance estimates, but not the arithmetic mean, appropriately describe the distribution of the cocaine priming threshold. The estimate of the geometric mean value of the priming threshold for this group of Sprague-Dawley rats was 284 (CI(95): 234-344) microg/kg of cocaine. The log-normal distribution of equieffective doses of cocaine is typical of agonist-induced pharmacological responses. In the presence of the D(1) dopamine receptor-selective antagonist SCH23390, the geometric means of the cocaine priming threshold were significantly increased in a dose-dependent manner, implying a role for D(1) dopamine receptors in the priming response. This technique provides a quantitative method for the measurement of antagonist-induced increases in the cocaine priming threshold.

Competitive dopamine receptor antagonists increase the equiactive cocaine concentration during self‐administration

Competitive dopamine receptor antagonists increase the rate of cocaine self‐administration. As the rate of self‐administration at a particular unit dose is determined by the satiety threshold and the elimination half‐life (t1/2) of cocaine, we investigated whether dopamine receptor antagonists altered these parameters in rats. The plasma cocaine concentration at the time of each self‐administration was constant during a session demonstrating that this satiety threshold concentration represents an equiactive cocaine concentration. The plasma cocaine concentration at the time of self‐administration was increased by SCH23390, consistent with pharmacological theory. In rats trained to reliably self‐administer cocaine, SCH23390 had no effect on the plasma steady‐state cocaine concentration produced by constant infusions of cocaine. Therefore, this antagonist had no effect on cocaine t1/2 at a dose that accelerated cocaine self‐administration. A constant cocaine infusion at a rate that maintained steady state concentrations above the satiety threshold stopped self‐administration. SCH23390, or the D2 dopamine receptor antagonist (−)eticlopride, reinstated self‐administration in the presence of the constant cocaine infusion. This is consistent with SCH23390 and eticlopride raising the satiety threshold above the steady state level produced by the constant cocaine infusion. It is concluded that the antagonist‐induced acceleration of cocaine self‐administration is the result of a pharmacokinetic/pharmacodynamic interaction whereby the rate of cocaine elimination is faster at the higher concentrations, as dictated by first‐order kinetics, so that cocaine levels decline more rapidly to the elevated satiety threshold. This results in the decreased interinjection intervals. Synapse, 2010.

Using the self-administration of apomorphine and cocaine to measure the pharmacodynamic potencies and pharmacokinetics of competitive dopamine receptor antagonists

Competitive dopamine receptor antagonists accelerate psychomotor stimulant self-administration. According to pharmacological theory of competitive antagonism antagonists raise the equiactive agonist concentration. In the self-administration paradigm this is assumed to be the satiety threshold or C(min). The magnitude of the proportional increase in satiety threshold (agonist concentration ratio) as a function of antagonist dose should reflect the antagonist pharmacodynamic potency. The time course of this effect should reflect the rate of change of antagonist occupancy of receptors and, therefore, antagonist concentration, i.e. pharmacokinetics. Rats self-administered apomorphine or cocaine at a stable rate and were then injected i.v. with one of four competitive D₁-like or D₂-like dopamine receptor antagonists and the session continued. The agonist concentrations at the time of each self-administration (satiety thresholds) were calculated during the session. The antagonists accelerated self-administration of both agonists with a concomitant increase in the calculated satiety thresholds. The maximum agonist concentration ratio was proportional to the dose of antagonist. The time courses of the changes in agonist concentration ratio were independent of the agonist and of the dose of antagonist. Schild analysis of the maximum agonist concentration ratio as a function of the antagonist dose allowed apparent pA₂ (or K(dose)) to be measured. Antagonist K(dose) values should provide a quantitative basis for receptor identification in behavioral pharmacology. The assay system may also measure the pharmacokinetics of antagonist elimination from the brain. Agonist self-administration represents a sensitive in vivo pharmacological assay system that provides information useful for pharmacokinetic/pharmacodynamic modeling of antagonist effects.

The composition, structural properties and binding of very-low-density and low-density lipoproteins to the LDL receptor in normo- and hypertriglyceridemia: relation to the apolipoprotein E phenotype.

Abstract The composition, apolipoprotein structure and lipoprotein binding to the LDL receptor were studied for very-low-density (VLDL) and low-density lipoprotein (LDL) particles isolated from subjects with apoE phenotype E3/3 (E3), E2/2 or E2/3 (E2+) and E3/4 or E4/4 (E4+) and a wide range of plasma triglyceride (TG) contents. The data combined for all three phenotype groups can be summarized as follows. (i) A decrease in accessibility of VLDL tryptophan residues to I- anions with a decrease in tryptophan surface density, concomitant with an increase in VLDL dimensions, reflects the increased efficiency of protein-protein interactions. (ii) A gradual increase in the quenching constant for LDL apoB fluorescence with an increase in TG/cholesterol (Chol) ratio reflects the ‘freezing’ effect of Chol molecules on apoB dynamics. (iii) Different mechanisms specific for a particular lipoprotein from E3/3 or E2/3 subjects are responsible for apoE-mediated VLDL binding and apoB-mediated LDL binding to the LDL receptor in a solid-phase binding assay. (iv) The ‘spacing’ effect of apoC-III molecules on apoE-mediated VLDL binding results in a decrease in the number of binding sites. (v) The maximum of the dependence of the LDL binding affinity constant on relative tryptophan density corresponds to LDL intermediate size. VLDL particles from hypertriglyceridemic E2/3 heterozygotic individuals had remnant-like properties (increased cholesterol, apoE and decreased apoC-III content) while their binding efficiency was unchanged. Based on the affinity constant value and LDL-Chol content, increased competition between VLDL and LDL for the binding to the LDL receptor upon increase in plasma TG is suggested, and LDL from hypertriglyceridemic E3/3 homozygotic individuals is the most efficient competitor.

Satiety threshold during maintained cocaine self-administration in outbred mice.

Male Swiss Webster mice maintained cocaine self-administration in a regular and dose-dependent manner. These characteristics made it possible to apply the satiety threshold model of drug self-administration developed recently for cocaine self-administration in rats. Non-linear regression analysis revealed that cocaine satiety threshold was 1.3 ± 0.6 mg/kg and the functional half-life of the cocaine was 8.1 ± 2.2 min. Whether the self-administration of cocaine was maintained by lever presses or nose pokes did not influence the inter-injection intervals. The results are consistent with the pharmacological model of maintained cocaine self-administration. The ability to determine addiction-relevant phenotypes (the satiety threshold and functional half-life of cocaine) in inbred strains of mice may help to identify the genetic determinants of cocaine self-administration behavior.

The Affinity of D2-Like Dopamine Receptor Antagonists Determines the Time to Maximal Effect on Cocaine Self-Administration

Differences in the time to maximal effect (Tmax) of a series of dopamine receptor antagonists on the self-administration of cocaine are not consistent with their lipophilicity (octanol-water partition coefficients at pH 7.4) and expected rapid entry into the brain after intravenous injection. It was hypothesized that the Tmax reflects the time required for maximal occupancy of receptors, which would occur as equilibrium was approached. If so, the Tmax should be related to the affinity for the relevant receptor population. This hypothesis was tested using a series of nine antagonists having a 2500-fold range of Ki or Kd values for D2-like dopamine receptors. Rats self-administered cocaine at regular intervals and then were injected intravenously with a dose of antagonist, and the self-administration of cocaine was continued for 6 to 10 h. The level of cocaine at the time of every self-administration (satiety threshold) was calculated throughout the session. The satiety threshold was stable before the injection of antagonist and then increased approximately 3-fold over the baseline value at doses of antagonists selected to produce this approximately equivalent maximal magnitude of effect (maximum increase in the equiactive cocaine concentration, satiety threshold; Cmax). Despite the similar Cmax, the mean Tmax varied between 5 and 157 min across this series of antagonists. Furthermore, there was a strong and significant correlation between the in vivo Tmax values for each antagonist and the affinity for D2-like dopamine receptors measured in vitro. It is concluded that the cocaine self-administration paradigm offers a reliable and predictive bioassay for measuring the affinity of a competitive antagonist for D2-like dopamine receptors.

The effects of a repeated dose of a recombinant humanized anti-cocaine monoclonal antibody on cocaine self-administration in rats

BACKGROUND Immunotherapy has shown potential as a treatment for cocaine abuse. The humanized recombinant anti-cocaine monoclonal antibody (mAb) with the preclinical designation h2E2 has been shown to decrease cocaine concentrations in the brain in rats, but its effects on cocaine self-administration behavior have never been tested. METHODS The amount of cocaine needed to reinstate self-administration behavior (priming threshold) was calculated and the inter-injection intervals at unit doses of 0.3μmol/kg and 3μmol/kg during maintained self-administration were measured over a five-week baseline period. Rats trained to self-administer cocaine were infused with two doses of h2E2 (120mg/kg i.v.) 35days apart. Priming threshold and inter-injection intervals were measured for 35days after both injections. RESULTS After both injections of h2E2, priming thresholds were significantly increased (3-fold) compared to expected baseline and then gradually declined over 35days. A significant decrease (15-33%) in inter-injection intervals during maintained self-administration was also observed following both h2E2 infusions at the lower dose, and after the first injection at the higher dose. No significant decreases in body weight were observed after either injection, indicating a lack of toxicity following a second injection. CONCLUSIONS These data predict that the safety and effectiveness of h2E2 will be maintained after multiple treatments of this potential immunotherapy for cocaine abuse.