Discriminative stimulus effects of the type-4 phosphodiesterase inhibitor rolipram in rats.

RATIONALE: Rolipram, an inhibitor of cyclic AMP phosphodiesterase (PDE4) produces discriminative stimulus effects in rats. These effects may be related to a wide range of central nervous system effects described previously. OBJECTIVE: The purposes of the present study were to: (i) assess the specificity of the discriminative stimulus effects of rolipram; (ii) examine the role of beta adrenergic receptors; (iii) assess the effects of imipramine and nisoxetine; and (iv) determine whether SKF 38393, a compound which also increases cAMP levels, substitutes for rolipram. METHODS: Rats were trained to discriminate 0.1 mg/kg rolipram from its vehicle in a two-lever task. Following discrimination training, substitution and antagonism tests were carried out. RESULTS: In generalization tests, the PDE4 inhibitors ICI 63,197 and Ro 20-1724 substituted for rolipram in a dose-dependent manner (substitution at 0.3 mg/kg and 3 mg/kg, respectively). The selective inhibitors of PDE1, PDE2, and PDE5/6 did not substitute for rolipram; however, a dose of 10 mg/kg of the PDE3 inhibitor milrinone did substitute. The beta adrenergic agonists clenbuterol and dobutamine at least partially substituted for rolipram (0.1 mg/kg and 18 mg/kg, respectively). By contrast, the D1 dopaminergic agonist SKF 38393 and the monoamine uptake inhibitors imipramine and nisoxetine were ineffective (at doses up to 3, 10, and 10 mg/kg, respectively). CONCLUSIONS: The present results indicate that the discriminative stimulus effects of rolipram are related to the inhibition of the hydrolytic activity of PDE4. Generalized increases in cyclic nucleotides do not appear to be sufficient for producing rolipram-like effects. It appears that a mechanism involving beta adrenergic receptors may contribute to the effects of rolipram, consistent with previous neuropharmacological data. Finally, the discriminative stimulus effects of rolipram appear to be unrelated to its antidepressant-like effect, but may provide a surrogate marker for central nervous system-related side effects of PDE4 inhibitors.

Discriminative stimulus effects of centrally administered isoproterenol in rats: mediation by beta-1 adrenergic receptors.

RATIONALE: Centrally active beta-1 and beta-2 adrenergic agonists produce antidepressant-like effects in several behavioral tests, suggesting that these receptors may be involved in the mediation of the effects of antidepressant drugs. OBJECTIVES: This study aimed to evaluate the ability of intra-cerebral ventricular (ICV) isoproterenol to produce discriminative stimulus effects mediated by beta adrenergic receptors, establishing a reliable model of in vivo activation of central beta adrenergic receptors. METHODS: Rats were trained to discriminate the non-selective beta adrenergic agonist isoproterenol (10 microg ICV) from artificial cerebral spinal fluid (aCSF) using a water-reinforced two-lever operant task [fixed ratio-10 schedule of reinforcement (FR10)]. For substitution and antagonism tests, drugs were administered IP. RESULTS: Following acquisition of the discrimination, ICV isoproterenol produced dose-related increases in drug-appropriate responding (ED50 = 1.14 microg). The beta-1 selective adrenergic agonist dobutamine fully substituted for isoproterenol at a dose of 0.3 mg/kg (ED50 = 0.15 mg/kg). By contrast, the beta-2 selective adrenergic agonist clenbuterol produced 20% isoproterenol-appropriate responding when administered at doses up to 0.1 mg/kg. The beta adrenergic antagonist propranolol fully antagonized the isoproterenol cue at a dose of 0.03 mg/kg (ID50 = 0.013 mg/kg). The beta-1 selective antagonist betaxolol (ID50 = 0.03 mg/kg) more potently antagonized isoproterenol's cue than did the beta-2 selective antagonist ICI 118,551 (ID50 = 0.41 mg/kg). The antidepressant desipramine (1.0 mg/kg) substituted for isoproterenol. CONCLUSIONS: These results demonstrate that the discriminative stimulus effects of isoproterenol are mediated primarily via beta-1 adrenergic receptors. This provides a functional model for activation of central beta-1 adrenergic receptors, permitting further characterization of the role of this receptor subtype in the mechanism of action of antidepressant drugs.

Noradrenergic lesions differentially alter the expression of two subtypes of low Km cAMP-sensitive phosphodiesterase type 4 (PDE4A and PDE4B) in rat brain.

This study examined the effects of selective, central noradrenergic dennervation with 6-hydroxydopamine (6-OHDA) on the expression of type 4 phosphodiesterases (PDE4). Twenty-one days following i.c.v. injection of 6-OHDA (200 microg) hypothalamus, neostriatum, and cerebellum were dissected. Infusion of 6-OHDA reduced norepinephrine (NE) content in all the brain areas examined (to 17%, 76% and 16% of sham-operated controls in hypothalamus, striatum, and cerebellum, respectively). 6-OHDA injections also reduced dopamine levels in hypothalamus (53%) and neostriatum (68%). Administration of desipramine (20 mg/kg, i.p.) 30 min prior to 6-OHDA injection protected neostriatal and cerebellar noradrenergic neurons NE levels (110-122% of the control levels). Desipramine partially attenuated the 6-OHDA-mediated decrease in NE content of hypothalamus, but had little or no effect on either striatal or hypothalamic dopamine (DA) levels. Western blot analysis using a PDE4A-selective antibody revealed three major bands (109 kDa PDE4A5, 102 kDa PDE4AX and 76 kDa PDE4A1) in hypothalamus and striatum. Infusion of 6-OHDA decreased the expression of PDE4A5 and PDE4AX but not of PDE4A1 in hypothalamus, as determined by quantitative Western blotting. Pretreatment of rats with desipramine attenuated the 6-OHDA-induced down-regulation of PDE4A5 and PDE4AX bands in hypothalamus. The PDE4B selective antibody K118 labels 5 major bands in all the brain regions studied. One hundred kDa PDE4B3, 86 kDa PDE4B2 and a 78 kDa PDE4B band was identified using recombinant proteins. Treatment of rats with 6-OHDA resulted in a 52% decrease in the PDE4B3 and 58% decrease in 78 kDa PDE4B variant in hypothalamus; administration of desipramine attenuated the 6-OHDA-induced down-regulation of both PDE4B variants. Neither 6-OHDA nor desipramine altered striatal PDE4A or PDE4B isozymes. In contrast, cerebellar PDE4B3 variant is up-regulated by 6-OHDA treatment and were partially normalized to control values by desipramine pretreatment. These data demonstrate that PDE4 subtypes are differentially regulated by presynaptic noradrenergic activity and may play an important role in the maintaining homeostasis of noradrenergic signal transduction in rat brain.

Effects of antidepressants in rats trained to discriminate the beta-2 adrenergic agonist clenbuterol.

The ability of indirectly acting agonists such as norepinephrine uptake inhibitors, serotonin reuptake inhibitors, and atypical antidepressants to substitute for clenbuterol, a beta-2 adrenergic agonist, was examined in rats trained to discriminate 0.03 mg/kg clenbuterol and saline using a fixed-ratio 10 (FR 10) schedule with water reinforcement. The beta-2 selective adrenergic agonist clenbuterol produced an orderly dose-response relationship, and its discriminative stimulus effects were antagonized by the beta-adrenergic antagonist propranolol. It was found that the effects of tricyclic antidepressants and selective norepinephrine uptake inhibitors did not generalize to the discriminative stimulus effects of clenbuterol, with the exception of high doses of protriptyline. Moreover, compounds from other drug classes, including fluoxetine and phenelzine, did not substitute for clenbuterol. Atypical antidepressants, such as trazodone, rolipram, and bupropion also did not engender drug-appropriate responding. Prenalterol and dobutamine, both purported to be beta-1 adrenergic receptor agonists, partially substituted for clenbuterol, but at relatively high doses. The present results show that the antidepressants tested do not share discriminative stimulus effects with clenbuterol, a beta-2 adrenergic agonist.

Establishing morphine and U-50,488H as discriminative stimuli in a three-choice assay with pigeons.

Pigeons were trained in a 3-choice assay to discriminate among injections of 5.6 mg/kg U-50,488H, 5.6 mg/kg morphine, and vehicle solution. In dose-response tests, subjects rarely responded on the U-50,488H-appropriate key when morphine was administered or on the morphine-appropriate key when they received U-50,488H. A high dose of naltrexone (1.0 mg/kg) completely blocked the morphine cue but failed to block completely the U-50,488H cue. In generalization tests, d-amphetamine primarily engendered saline-appropriate responding. Ethylketazocine produced mixed results, in that moderate doses produced responding on both the morphine- and U-50,488H-appropriate keys, but 3.2 mg/kg engendered primarily morphine-appropriate responding. These results demonstrate the feasibility, but not necessarily the value, of 3-choice discrimination procedures involving mu and kappa agonists and vehicle.

Behavioral contingencies modulate tolerance to discriminative stimulus effects of morphine.

Experiments examined how learning processes modulate tolerance to discriminative stimulus effects of morphine. Rats were trained to discriminate saline and 3.2 mg/kg morphine, and the doses of morphine required to mimic the training dose were determined before, during and after repeated treatment with saline or high doses of morphine (10 mg/kg, b.i.d.). In one set of experiments, training was either suspended or continued with saline and the original training dose during a 2-week treatment regimen. When training was suspended, high-dose morphine treatment increased the dose of morphine required for stimulus effects approximately 3-fold. Tolerance persisted 2 days after treatment ended, but disappeared within 7 days. In contrast, continued training with saline and 3.2 mg/kg morphine during high-dose treatment both attenuated development of tolerance and transferred control to lower doses. Transfer of control to lower doses appeared conditional upon recent termination of high-dose treatment, as it disappeared within 7 days. Treatment with saline did not change the doses of morphine required for stimulus effects under either training condition. A final experiment examined whether high-dose treatment could transfer control to higher doses of morphine. The treatment dose of 10 mg/kg morphine itself was used as the training dose during a 2-week treatment regimen. The dose of morphine required for stimulus effects increased 2- to 4-fold during treatment, but quickly returned to control values when treatment ended. These results extend previous findings that conditioning and pharmacodynamic processes jointly regulate development of tolerance to discriminative effects of morphine.

Effects of (+)-fenfluramine on 3,4-methylenedioxymethamphetamine (MDMA) discrimination in rats.

This study examined the effects of a presumed neurotoxic dose regimen of (+)-fenfluramine on the discrimination of MDMA and (+)-amphetamine in male Sprague-Dawley rats trained to discriminate 1.5 mg/kg MDMA from saline in a two-choice operant task. Substitution tests were conducted with saline, several doses of MDMA (0.19-1.5 mg/kg), and (+)-amphetamine (0.125-1.0 mg/kg) prior to and again following the administration of (+)-fenfluramine (4.0 mg/kg twice a day for 4 days; n = 11) or a similar pattern of saline injections (n = 10). During pretreatment substitution tests, lower doses of MDMA elicited drug-appropriate responding in a dose-dependent manner, although none of these doses substituted for the training dose. Likewise, no dose of (+)-amphetamine substituted for the training drug during pretreatment substitution tests. The discrimination of MDMA was disrupted in some animals following (+)-fenfluramine treatment, but with subsequent training, discrimination criteria were met. In posttreatment substitution tests, the lowest dose of MDMA produced significantly higher drug-appropriate responding in (+)-fenfluramine treated animals but not in saline-treated animals. The amount of drug-appropriate responding during posttreatment substitution tests with (+)-amphetamine varied little from pretreatment substitution tests in saline-treated animals, but was greater at all doses in (+)-fenfluramine-treated animals; the highest dose of (+)-amphetamine substituted for MDMA subsequent to (+)-fenfluramine treatment. These results support previous findings that the long-lasting serotonergic effects of fenfluramine may have functional consequences that can be detected using a drug discrimination procedure. Specifically, serotonin depletion may unmask or strengthen the stimulant-like effects of MDMA.

In vivo apparent pA2 analysis for naltrexone antagonism of discriminative stimulus and analgesic effects of opiate agonists in rats.

Six opiate agonists were characterized by in vivo apparent pA2 analysis with respect to their discriminative stimulus, rate-decreasing and analgesic effects, by using the antagonist naltrexone. In drug discrimination experiments, rats were trained to discriminate 3.2 mg/kg of morphine from saline under a fixed-ratio 15 schedule of food reinforcement. In analgesia experiments, rat's tails were immersed into 55 degrees C water and latency for tail withdrawal was measured. Naltrexone (0.01-1.0 mg/kg) antagonized discriminative stimulus effects of all agonists, rate-decreasing effects of etorphine, morphine, fentanyl, buprenorphine and GPA 1657 [(1)-B-2'-hydroxy-2,9-dimethyl-5-phenyl-6,7-benzomorphan] and analgesic effects of etorphine, morphine, buprenorphine and GPA 1657. Analgesic effects of fentanyl and nalbuphine were not tested. Naltrexone apparent pA2 values across the three behavioral measures were etorphine (7.2-7.4 mol/kg), fentanyl (7.3-7.4 mol/kg), morphine (7.5-8.4 mol/kg), GPA 1657 (7.0-7.3 mol/kg), buprenorphine (7.5-7.7 mol/kg) and nalbuphine (7.7 mol/kg). Apparent pA2 values averaged 7.5 mol/kg and slopes of the naltrexone Schild regressions were not different from unity, suggesting that the measured behavioral effects of these agonists are mediated by mu opioid receptors. Nalbuphine also was used as an antagonist in the tail-withdrawal assay. The apparent pA2 values for nalbuphine were etorphine (4.9 mol/kg), morphine (5.9 mol/kg), GPA 1657 (5.7 mol/kg) and buprenorphine (5.5 mol/kg). Slopes of the Schild regressions differed, suggesting that nalbuphine's modest analgesic effects may have prevented proper conditions for an accurate Schild analysis.

The effects of d-amphetamine and diazepam on schedule-induced defecation in rats.

The effects of d-amphetamine (0.32 to 5.6 mg/kg) and diazepam (0.10 to 5.6 mg/kg) on defecation were examined in two groups of rats. One group was exposed to a fixed-time 60-s (FT 60-s) schedule of food delivery and the other group was exposed to massed-food sessions. During vehicle control sessions, rats exposed to the FT 60-s schedule excreted a significantly higher number of fecal boli than rats exposed to massed-food sessions. d-Amphetamine, at doses above 0.56 mg/kg, significantly reduced defecation (boli produced) in both groups, although the magnitude of the drug's effect was larger in the group exposed to the FT 60-s schedule. For both groups, diazepam only produced a significant decrease in defecation at the highest dose (5.6 mg/kg). These results appear to be inconsistent with interpretations of adjunctive behavior that emphasize arousal or emotion as mechanisms.

Effects of cocaine on fixed-ratio responding of rats: modulation by required response force.

The effects of acute cocaine administrations (5.6-32 mg/kg) were determined in rats responding under a multiple fixed-ratio 15 (FR 15) FR 15 schedule of food delivery. The minimum response effort required in one schedule component was 25 g, whereas in the other component it was 200 g. Cocaine produced generally dose-dependent decreases in rate of responding and increases in preratio pause time under each component. There was, however, a significant interaction between force and drug dose, and drug effects were larger in the component requiring 200 g for lever operation. Although a number of other parameters have been shown previously to modulate the effects of cocaine on schedule-controlled responding, the present data constitute the first demonstration that minimum response effort does so.

Effects of d-amphetamine and ethosuximide on responding under delayed-matching-to-sample procedures with differential and nondifferential outcomes.

Pigeons were exposed to delayed-matching-to-sample (DMTS) procedures in which food or a flash of the feeder light followed correct responses. When these consequences were correlated with a particular stimulus (e.g., food followed matching responses to red and a flash of the feeder light followed matching responses to green), accuracy was higher (i.e., stimulus control was greater) than when discriminative stimuli and consequences were not correlated. Although stimulus control in the absence of drug appeared to be weaker under the uncorrelated procedure, neither d-amphetamine (0.5-3.0 mg/kg) in Experiment 1 nor ethosuximide (40-160 mg/kg) in Experiment 2 disrupted accuracy to a greater extent under that procedure. These results, like those of a prior investigation, suggest that drug effects are similar under DMTS procedures regardless of whether correlated or uncorrelated outcomes are arranged.

Onset of tolerance to discriminative stimulus effects of morphine.

Experiments assessed the onset of tolerance to discriminative stimulus effects of morphine in rats treated repeatedly with twice daily doses of 10 mg/kg morphine. Saline and 3.2 mg/kg morphine were established as discriminative stimuli for food-reinforced fixed-ratio performances in several groups of rats, and initial ED50 values were determined for stimulus and rate-altering effects of morphine. To assess onset of tolerance, training was halted and 10 mg/kg doses of morphine were administered repeatedly at 12-h intervals. In separate experiments, ED50 values were redetermined after various treatment periods. One treatment with 10 mg/kg morphine did not alter the ED50 for stimulus effects of morphine, whereas treatment for one or three days increased the ED50 by approximately 2-fold. Comparisons with published data showed even greater tolerance when treatment lasted one or two weeks. Tolerance to stimulus effects of morphine generally was accompanied by tolerance to its rate-decreasing effects. Repeated treatment with morphine also produced cross-tolerance to morphine-like stimulus effects of methadone and buprenorphine. As with morphine itself, greater tolerance developed with longer treatment. These results suggest that tolerance to discriminative stimulus effects of morphine develops gradually, with magnitude of tolerance increasing as a function of treatment duration.

Tolerance to morphine-like stimulus effects of mu opioid agonists.

Experiments assessed the ability of repeated treatment with morphine to produce tolerance to morphine-like discriminative stimulus effects of buprenorphine, etorphine, methadone, morphine, and nalbuphine. Before treatment, each compound evoked full morphine-like stimulus effects in rats trained to discriminate saline and 3.2 mg/kg morphine. During treatment, training was halted and a dose of 10 mg/kg morphine administered every 12 h for 14-18 days. Repeated treatment with morphine increased the ED50 for stimulus control by etorphine, methadone or buprenorphine 2- to 4-fold and that for morphine 4.5-fold. Repeated treatment produced an insurmountable tolerance to the morphine-like stimulus effects of nalbuphine, so that a dose 150-fold higher than the initial ED50 evoked only 40% generalization. Treatment with a lower dose of morphine (10 mg/kg every 24 h) produced a short-lived surmountable tolerance to stimulus effects of nalbuphine. For etorphine, methadone and morphine, tolerance to morphine-like stimulus effects was accompanied by tolerance to rate suppressing effects. After treatment ended, the ED50 for stimulus control by etorphine, methadone or nalbuphine returned to initial values within 3 days; that for morphine, within 5 days; that for buprenorphine, within 10 days. These results demonstrate that repeated treatment with morphine produces cross-tolerance to compounds that exert morphine-like stimulus effects. Additionally, the results suggest that differences among these compounds in agonist efficacy may be revealed as differences in the degree of tolerance produced by morphine treatment.