Reinforcing aspects of androgens

Are androgens reinforcing? Androgenic-anabolic steroids (AAS) are drugs of abuse. They are taken in large quantities by athletes and others to increase performance, often with negative long-term health consequences. As a result, in 1991, testosterone was declared a controlled substance. Recently, Brower [K.J. Brower, Anabolic steroid abuse and dependence. Curr. Psychiatry Rep. 4 (2002) 377-387.] proposed a two-stage model of AAS dependence. Users initiate steroid use for their anabolic effects on muscle growth. With continued exposure, dependence on the psychoactive effects of AAS develops. However, it is difficult in humans to separate direct psychoactive effects of AAS from the user's psychological dependence on the anabolic effects of AAS. Thus, studies in laboratory animals are useful to explore androgen reinforcement. Testosterone induces a conditioned place preference in rats and mice, and is voluntarily consumed through oral, intravenous, and intracerebroventricular self-administration in hamsters. Active, gonad-intact male and female hamsters will deliver 1 microg/microl testosterone into the lateral ventricles. Indeed, some individuals self-administer testosterone intracerebroventricularly to the point of death. Male rats develop a conditioned place preference to testosterone injections into the nucleus accumbens, an effect blocked by dopamine receptor antagonists. These data suggest that androgen reinforcement is mediated by the brain. Moreover, testosterone appears to act through the mesolimbic dopamine system, a common substrate for drugs of abuse. Nonetheless, androgen reinforcement is not comparable to that of cocaine or heroin. Instead, testosterone resembles other mild reinforcers, such as caffeine, nicotine, or benzodiazepines. The potential for androgen addiction remains to be determined.     

Comparison of 7-nitroindazole with other nitric oxide synthase inhibitors as attenuators of opioid withdrawal

Previously, we demonstrated that two nonselective inhibitors of nitric oxide synthase (NOS),l-N ^G-nitroarginine (l-NNA) andl-N ^G-nitroarginine methyl ester (l-NAME), reduced some signs of morphine withdrawal in rats. The present work extended these studies to include 7-nitroindazole (7-NI), an inhibitor specific for cerebral NOS, andN(5)-(1-iminoethyl)-l-ornithine (l-NIO), a potent inhibitor of endothelial NOS. Behavioral effects of these four NOS inhibitors and clonidine, an ₂-adrenoceptor, agonist, on morphine withdrawal in rats were assessed. Rats received one 75-mg morphine pellet subcutaneously (SC). Three days later, NOS inhibitors were administered IP 1 h before withdrawal was precipitated with naloxone (0.5 mg/kg, SC) and scored. 7-NI,l-NIO,l-NAME andl-NNA produced dose-related decreases in weight loss, diarrhea, wet dog shakes and grooming. 7-NI also reduced mastication, salivation and genital effects. Clonidine produced effects similar to 7-NI. In awake, morphine-naive and morphine-dependent rats not subjected to withdrawal, 7-NI was the only NOS inhibitor that did not increase blood pressure. Because 7-NI attenuated more signs of opioid withdrawal thanl-NNA,l-NAME orl-NIO without causing hypertension, 7-NI appears to warrant further testing as a potential candidate for human use.Abstracts were presented at the annual meetings of the College on Problems of Drug Dependence, West Palm Beach, Fla., 18–23, June 1994; International Narcotics Research Conference, North Falmouth, Cape Cod, Mass., 16–21, July 1994; and a Satellite Symposium to IUPHAR, Montreal, Canada, 22–24, July 1994.     

Potential anti-anxiety, anti-addictive effects of LY 354740, a selective group II glutamate metabotropic receptors agonist in animal models

Despite there being a lot of biochemical data about metabotropic glutamate (mGlu) receptors, our knowledge of the behavioural effects of mGlu receptor agonists/antagonists is still inadequate. LY 354740 is a systemically active agonist of group II mGlu receptors. After peripheral administration, LY 354740 produced anxiolytic-like effects in the conflict drinking test in rats and a four-plate test in mice. It was also found that LY 354740 decreased spontaneous locomotor activity in mice, but did not disturb motor coordination. In behavioural models of depression including the despair test and a tail suspension test, LY 354740 did not produce antidepressant-like effects. LY 354740 inhibited the naloxone-induced symptoms of morphine withdrawal in morphine-dependent mice. The above results indicate that agonists of group II mGlu receptors may play a role in the therapy of anxiety and/or drug-dependence states. The brain sites of action of LY 354740 need to be identified and the mechanism of both the above described effects remains to be elucidated.     

Rapid induction of behavioral and neurochemical tolerance to cocaethylene, a model compound for agonist therapy of cocaine dependence

Rationale: Tolerance to abused drugs may impact on patterns of abuse, and in the case of agonist therapies, may be beneficial in that it reduces the reward value of a given dose of abused drug. Cocaethylene, a psychoactive metabolite resulting from concurrent alcohol and cocaine consumption, was examined because of its use in human research studies of drug reward mechanisms, and its potential as a model compound for an agonist based therapy for cocaine dependence. Objective: Comparisons were made between cocaine and cocaethylene in the acute development of tolerance to the neurochemical and behavioral effects of cocaine. With chronic exposure, tolerance to the behavioral effects of cocaine was examined. Methods: In awake rats with a microdialysis probe in the nucleus accumbens and a jugular catheter, an IV bolus/3-h infusion of cocaine or cocaethylene and a subsequent cocaine challenge was administered while extracellular dopamine and locomotion were monitored. Chronic IV treatment with cocaine, cocaethylene, and a water control was accomplished for 7 days using osmotic minipumps attached to jugular catheters. Animals were then challenged with an IV bolus of cocaine. Results: With acute treatment, the IV bolus of cocaethylene at the beginning of the infusion period resulted in an initial behavioral activation equivalent to that caused by cocaine, after which there was a striking difference in that the cocaethylene group displayed a return to predrug levels of activity, while the cocaine group showed high levels of activity throughout the 3-h period. Both cocaethylene and cocaine resulted in an initial increase in the extracellular concentration of dopamine. However, after that initial increase, levels of dopamine dropped in the cocaethylene group while the cocaine group levels remained elevated. A 1-week infusion of cocaine or cocaethylene resulted in tolerance to the behavioral activating effects of a subsequent cocaine challenge. Conclusions: These results demonstrate a rapid induction of tolerance to the behavioral and neurochemical properties of cocaethylene, resulting in a diminished behavioral response to a cocaine challenge both acutely, and after 7 days. The relevance of these data for the use of cocaethylene as a model compound for an agonist approach to therapy for cocaine dependence is discussed. Received: 22 January 1999 / Final version: 16 April 1999     

Clozapine as a drug of dependence

Rationale: In schizophrenics, clozapine has been reported to induce various withdrawal signs and rapid onset relapse to psychosis on cessation of chronic treatment. Objective: The study was designed to develop an animal model of one aspect of clozapine tolerance and withdrawal using core body temperature measures. Methods: Two groups of 15 female Wistar rats were treated chronically (b.i.d.) with clozapine at 6 or 12 mg/kg per injection for 21 days prior to cessation of drug treatment, withdrawal being studied over 4 consecutive days. Body temperatures were assessed daily throughout the study. Results: Acutely, clozapine induced dose-related hypothermia, to which complete tolerance developed in both groups, the development of tolerance being more rapid in the group treated with 6 mg/kg per injection of clozapine. During withdrawal only the group treated chronically with 12 mg/kg per injection of clozapine showed rapid onset significant hyperthermia. This dissipated progressively over days, and was completely absent after 4 days of withdrawal. Conclusions: Clozapine induced a clear somatic withdrawal sign after chronic treatment. It is suggested that, in future research in both humans and animals, it is important to attempt to differentiate between clozapine withdrawal and clozapine withdrawal-induced relapse to psychosis. It is also important to characterise the clozapine withdrawal syndrome fully in animals; to establish the neurochemical mechanisms involved in such withdrawal; and to determine which novel antipsychotics are most efficacious in inducing clozapine-like withdrawal effects, in suppressing clozapine withdrawal, and in preventing relapse to psychosis in patients being transferred from clozapine to novel atypical antipsychotic drugs. Received: 14 May 1998/Final version: 14 September 1998     

Discriminative stimulus effects of flumazenil in untreated and in diazepam-treated rhesus monkeys

Rationale: Long-term use of benzodiazepine agonists can have adverse effects (e.g., development of dependence), thereby limiting their clinical usefulness. Objectives: The goal of the current study was to examine the discriminative stimulus effects of flumazenil in untreated and diazepam-treated monkeys to determine whether this type of procedure could be used to examine benzodiazepine dependence. Methods: Flumazenil (0.32 mg/kg s.c.) was established as a discriminative stimulus in eight monkeys receiving 5.6 mg/kg/day of diazepam (p.o.); four responded under a fixed ratio (FR)5 schedule of stimulus-shock termination (SST) and four responded under a FR5 schedule of food presentation. For comparison, 1.0 mg/kg flumazenil (s.c.) was established as a discriminative stimulus in four untreated monkeys responding under a FR5 schedule of SST. Results: Flumazenil dose-dependently increased responding on the flumazenil-appropriate lever in all monkeys. In diazepam-treated monkeys, Ro 15-4513, ethyl beta-carboline-3-carboxylate and bretazenil substituted for flumazenil with pentylenetetrazole substituting in some monkeys; other drugs failed to substitute for flumazenil. Acute administration of 10.0 mg/kg diazepam (s.c.) shifted the flumazenil dose–effect curve threefold to the right of the control dose–effect curve. Temporary suspension of diazepam treatment produced a time-related increase in flumazenil-lever responding that was reversed by diazepam. In untreated monkeys, midazolam substituted for flumazenil, with other drugs, including those with primary mechanisms of action at non-γ-aminobutyric acid_A receptors, substituting in some monkeys. Ro 15-4513 did not substitute in any untreated monkey. Conclusions: The flumazenil discriminative stimulus appears to be pharmacologically selective in treated monkeys with only negative and low efficacy positive modulators substituting for flumazenil; in contrast, a variety of drugs substitute for flumazenil in untreated monkeys. This apparent difference in selectivity suggests that diazepam treatment modifies the flumazenil discriminative stimulus perhaps due to the development of dependence. Received: 30 November 1998 / Final version: 25 May 1999     

Heroin self-administration in dependent Wistar rats: increased sensitivity to naloxone

  Rationale: Non-dependent and dependent opiate users appear to be driven by two distinct motivational factors: the primary reinforcing properties of the drug, and the negative reinforcing effects associated with relieving the negative affective component of opiate withdrawal in the dependent state. Objective: To investigate the motivational significance of opioid dependence on heroin self-administration (HSA) in rodents. Methods: Rats were trained to self-administer heroin intravenously (0.06 mg/kg per infusion; FR1), and opiate dependence was induced by subcutaneous implantation of two morphine (75 mg base) pellets.Rats in a non-dependent control group received placebo pellets. Three days after pellet implantation, HSA was resumed in daily 3-h sessions until baseline criteria were met and testing was conducted with subcutaneous injections of vehicle or naloxone (0, 0.003, 0.01, 0.03 mg/kg) 115 min into the session. Results: Morphine-dependent rats significantly increased HSA upon 0.01 mg/kg naloxone treatment, but decreased response rates at 0.03 mg/kg. Placebo pellet-implanted rats increased heroin intake at the 0.01 and 0.03 mg/kg doses. In a second experiment, the HSA session was shortened to 1 h and the training dose reduced to 0.03 mg/kg per infusion in new groups of animals. HSA in placebo pellet-implanted rats was increased only following the highest dose of the antagonist, while dependent rats were still affected by naloxone doses of 0.003–0.03 mg/kg. When subjected to a progressive-ratio schedule (experiment 3), breaking point values in dependent animals were 198% above baseline. Conclusions: The present study supports the hypothesis that dependence-induction by morphine-pellet implant in rats resulted in increased sensitivity to very small naloxone doses, as measured by changes in HSA. Taken together, these data suggest that opiate dependence, as measured by changes in sensitivity to naloxone, is a continuum which can contribute to the motivational state of drug-seeking. Received: 5 June 1998 / Final version: 21 December 1998     

Effect of low concentrations of K+ and Cl− on the Na+-dependent neuronal uptake of [3H] dopamine

The specific uptake of [³H] dopamine (DA) was studied using a crude synaptosomal fraction obtained from rat striatum. In a medium containing a 10 mM NaHC03/NaH2PO4 buffer and no added K⁺ ions, addition of NaCl elicited an increase in DA uptake for Na⁺ concentrations from 10 to 60 mM, and then a decrease of uptake for Na⁺ concentrations up to 130 mM. These data confirm that rather low NaCl concentrations produce a maximal DA uptake. This biphasic curve of uptake resulted from significant changes in the V _max of the DA uptake. Except for 10 mM Na⁺, this curve was not significantly modified when 9 mM NaHCO₃/NaH₂PO₄ were replaced by 9 mM NaCl. This result indicates that the Cl^– dependence of the DA uptake is mainly secondary to the Na⁺ dependence. Addition of KCl up to 3 mM did not modify the ascending part of the NaCl-dependent uptake curve. In contrast, the reduction in uptake produced by high Na⁺ concentrations was prevented in a concentration-dependent manner by KCl; this effect resulted from a decrease in the K_m and an increase in the V _max for the uptake.Measurements of membrane potential, with the help of the fluorescent probe 3,3-diethylthiadicarbocyanine iodide [DiSC₂(5)] and purified synaptosomes prepared from rat striatum and cerebral cortex, revealed that addition of 3 mM KCl to a medium containing a high Na⁺ concentration and no K⁺ ions produced a marked and stable decrease in the fluorescence level. This decrease which corresponds to an increase in membrane polarization was blocked by 0.1 mM ouabain. These data suggest that low K⁺ concentrations are likely to prevent the decrease in uptake elicited by high Na⁺ concentrations by restoration, via a Na⁺/K⁺ ATPase-mediated mechanism, of the membrane potential and/or a transmembrane electrochemical Na⁺ gradient more favourable to DA uptake.     

Withdrawal from oral cocaine in rats: ultrasonic vocalizations and tactile startle

While anxiety appears to characterize humans who administer high doses of cocaine or experience withdrawal from cocaine, it is difficult to capture this aspect of cocaine effects in animals. The present study investigated if acute or protracted withdrawal from prolonged low-dose cocaine that is self-administered via the oral route could be detected in tactile startle and vocal distress responses of rats. Adult, male Long-Evans rats had access to cocaine solution (0.1 mg/ml) either for 24 or 4 h/day using the two-bottle choice technique. The amount of solution consumed from each bottle was measured daily for 30 or 60 days. On days 1, 3, 7, 14, 21, 28 of withdrawal, startle and ultrasonic vocal responses (USV, 15–35 kHz) were measured in response to 18 airpuff stimuli (20 psi). Rats drank an average of 5–20 mg/kg per day of the cocaine solution. On average, about half of the daily liquid was consumed from the cocaine solution-containing bottle. USVs were emitted at significantly increased rates on day 3 of withdrawal from 30 or 60 days of cocaine drinking. Startle reactions were slightly, but non-significantly increased on day 1 of withdrawal. Comparable to withdrawal from ethanol, morphine, and diazepam treatments, withdrawal from oral self-administration of low to moderate doses of cocaine increases the rate of ultrasonic vocalizations while increasing minimally the amplitude of startle responses to low-intensity tactile stimuli. Nevertheless, no correlation between the total amount of cocaine self-administered or the duration of treatment with the intensity of the withdrawal manifestations could be detected.     

The cocaine-insensitive component of non-exocytotic efflux of noradrenaline from adrenergic axons in the isolated rat tail artery

Summary The working hypothesis was that the cocaine-insensitive component of non-exocytotic efflux of noradrenaline represents diffusion of the unprotonated amine across the axonal membrane. It was tested by examination of the effect of changing axoplasmic pH —and thus the fraction of extravesicular noradrenaline in the unprotonated form — on the overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from rat tail arteries. The catechols were assayed by liquid chromatography with amperometric detection. To dissipate the H⁺ gradient across the axonal membrane, the tissues were incubated in media of different pH, in which Na⁺ was completely replaced with K⁺ and which were HCO₃ ^– - (and Ca²⁺-)free. Exposure of the tissues to these media produced substantial, but reversible increases in the overflow of noradrenaline. Subsequently, the overflows of both noradrenaline and the glycol kept rising, but their ratio did not change.Cocaine (0.1 mmol/1) lowered the (noradrenaline overflow: glycol overflow) ratio significantly. The ratio observed in its presence increased steeply with decreasing external and, presumably, axoplasmic pH. Addition of valinomycin and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mol/1 each) to the cocaine-containing media more than doubled the overflows without altering significantly the ratio. Under identical conditions, the overflow of noradrenaline from preparations with inactive neuronal monoamine oxidase did not decrease with decreasing pH.Since, in the presence of cocaine, the overflow ratio increased — rather than decreased — with decreasing pH, and because the overflow or noradrenaline from preparations with inactive monoamine oxidase did not decline with pH, the cocaine-insensitive component of noradrenaline efflux does not seem proportional to the axoplasmic concentration of the unprotonated amine. The data can, however, be accounted for by postulating that the component reflects the concentration of the protonated form of noradrenaline.