L-DOPA Uptake in Astrocytic Endfeet Enwrapping Blood Vessels in Rat Brain.

Astrocyte endfeet surround brain blood vessels and can play a role in the delivery of therapeutic drugs for Parkinson's disease. However, there is no previous evidence of the presence of LAT transporter for L-DOPA in brain astrocytes except in culture. Using systemic L-DOPA administration and a combination of patch clamp, histochemistry and confocal microscopy we found that L-DOPA is accumulated mainly in astrocyte cell bodies, astrocytic endfeet surrounding blood vessels, and pericytes. In brain slices: (1) astrocytes were exposed to ASP(+), a fluorescent monoamine analog of MPP(+); (2) ASP(+) taken up by astrocytes was colocalized with L-DOPA fluorescence in (3) glial somata and in the endfeet attached to blood vessels; (4) these astrocytes have an electrogenic transporter current elicited by ASP(+), but intriguingly not by L-DOPA, suggesting a different pathway for monoamines and L-DOPA via astrocytic membrane. (5) The pattern of monoamine oxidase (MAO type B) allocation in pericytes and astrocytic endfeet was similar to that of L-DOPA accumulation. We conclude that astrocytes control L-DOPA uptake and metabolism and, therefore, may play a key role in regulating brain dopamine level during dopamine-associated diseases. These data also suggest that different transporter mechanisms may exist for monoamines and L-DOPA.

Effects of anti-phencyclidine and anti-(+)-methamphetamine monoclonal antibodies alone and in combination on the discrimination of phencyclidine and (+)-methamphetamine by pigeons.

RATIONALE: Drug-specific monoclonal antibodies against phencyclidine (PCP) and (+)-methamphetamine [(+)-METH] should bind to these drugs to block their discriminative stimulus effects. OBJECTIVES: To determine if mouse monoclonal antibodies against PCP and (+)-METH can block the discriminative stimulus effects of the drugs in pigeons. MATERIALS AND METHODS: Pigeons were trained to discriminate among intramuscular injections of saline, 1 mg/kg PCP, and 2 mg/kg (+)-METH. After responding stabilized, cumulative dose-response curves were obtained for PCP and (+)-METH. Doses of an anti-PCP antibody at 620 mg/kg (anti-PCP mAb6B5) with a K (D) of 1.3 nM for PCP and no measurable affinity for (+)-METH and 1,000 mg/kg doses of anti-(+)-METH antibody (anti-METH mAb6H7) with a K (D) of 41 nM for (+)-METH and no measurable affinity for PCP were subsequently administered, first alone and later in combination after which the dose-response curves were redetermined. RESULTS: When the antibodies were given alone, the anti-PCP antibody blocked the discriminative stimulus effects of PCP, but not those of (+)-METH, and the anti-(+)-METH antibody blocked the discriminative stimulus effects of (+)-METH, but not those of PCP. The anti-PCP antibody shifted the PCP dose-response curve further to the right and for a longer time than the anti-(+)-METH antibody shifted the dose response curve for (+)-METH. When the anti-PCP and anti-(+)-METH antibodies were administered on the same day, the discriminative stimulus effects of both drugs were completely blocked 1 day after antibody administration. CONCLUSIONS: These experiments demonstrate the high specificity of the antibodies for the drugs to which they bind and show that monoclonal antibodies can be combined to antagonize the effects of more than one drug.

Pharmacodynamics of a monoclonal antiphencyclidine Fab with broad selectivity for phencyclidine-like drugs.

The development of treatment strategies for drug intoxication has been hindered in part by the lack of clinically useful antagonists. Consequently, the major goal of these studies was to determine whether a monoclonal antibody Fab fragment (of IgG) could be used as an effective drug class-selective antagonist and to understand better the dose-response relationships for reversing CNS drug toxicity. Changes in drug-induced locomotor effects in a rat model were used to assess the ability of the antiphencyclidine (anti-PCP) Fab to reverse the behavioral effects of PCP and other potent arylcyclohexylamines. In experiments to determine the pharmacodynamics of Fabinduced antagonism of behavioral effects, the Fab completely reversed all PCP-induced locomotor effects in a Fab dose-dependent manner with a minimal effective dose of 0.18 mole-equivalents of Fab and an ED50 value of about one-third mole-equivalent. The anti-PCP Fab also completely reversed the locomotor effects induced by two other structurally related potent analogs of PCP: 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine. In addition, pharmacological and immunological selectivity was further tested by treatment of the behavioral effects induced by the structurally unrelated locomotor stimulant (+)methamphetamine. The antibody did not effectively reverse the effects of methamphetamine-induced locomotor activity. These results indicate that antibody-based medications can be developed to treat toxicity caused by classes of drugs as well as by individual drugs.

Butorphanol-mediated antinociception in mice: partial agonist effects and mu receptor involvement.

In the present experiments, we characterized the agonist and antagonist effects of butorphanol in mice. In the mouse radiant-heat tail-flick test, the mu agonists morphine and fentanyl and the kappa agonist U50,488H were fully effective as analgesics, whereas butorphanol was partially effective (producing 82% of maximal possible analgesic effect). Naltrexone was approximately equipotent in antagonizing the effects of morphine, fentanyl and butorphanol; in vivo apparent pA2 values for these naltrexone/agonist interactions were 7.5 (unconstrained). Naltrexone was approximately 10 times less potent in antagonizing the effect of U50,488H (average apparent pK(B) = 6.7). The selective mu antagonist beta-funaltrexamine (0.1-1.0 mg/kg) antagonized the effects of butorphanol in a dose-dependent insurmountable manner. Pretreatment with nor-binaltorphimine (32 mg/kg), a kappa-selective antagonist, did not reliably antagonize butorphanol, and naltrindole (20 and 32 mg/kg), a delta-selective antagonist, failed to antagonize the effects of butorphanol. Low doses of butorphanol (1.0, 1.8 or 3.2 mg/kg) caused parallel, rightward shifts in the dose-effect curve for morphine and parallel leftward shifts in the dose-effect curve for U50,488H. Taken together, the results of the present study suggest that butorphanol is a partial agonist in the mouse radiant-heat tail-flick test and that activity at mu receptors accounts for the majority of its antinociceptive effects.

Antiphencyclidine monoclonal Fab fragments reverse phencyclidine-induced behavioral effects and ataxia in rats.

Antiphencyclidine monoclonal antibody binding fragments (anti-PCP Fab) were studied in rats as a possible treatment for phencyclidine (PCP) overdose. Each male Sprague-Dawley rat (n = 4 per group) received an i.v. dose of 1 mg/kg of PCP followed 5 min later (as toxicity maximized) by one of three treatments in a random cross-over design. The treatments were 1 ml of saline, a nonspecific polyclonal human Fab, or a high affinity (Kd = 1.8 nM) anti-PCP monoclonal Fab. The doses of the nonspecific and anti-PCP Fab were 0.3, 1.0 and 3.0 times the mole equivalent (mol-eq) dose of PCP. Changes in locomotor activity and ataxia were the best indicators of PCP-induced behaviors among several time-dependent behavioral changes that were evaluated. PCP administration followed by saline treatment resulted in increases in locomotor activity and ataxia that declined to base line after 35 to 40 min. Anti-PCP Fab at 1.0 and 3.0 times the mol-eq dose of PCP significantly (P < .05) and rapidly reversed PCP-induced behaviors to base-line values. Although the 0.3 mol-eq dose of Fab appeared to slightly decrease the behavioral toxicity, the effects were not statistically different from controls in most cases. No significant effects on PCP-induced behaviors were observed after any dose of the nonspecific Fab. In addition, pharmacological and immunological specificity were tested further by treatment of MK-801 {(+)-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine-}-induced behavioral effects. MK-801 is a PCP-like, noncompetitive N-methyl-D-aspartate receptor antagonist which is structurally unrelated to PCP. The anti-PCP Fab treatment had no effect on MK-801-induced locomotor activity. These data clearly show that anti-PCP Fab is a specific PCP antagonist that can rapidly reverse PCP-induced behavioral toxicity in the rat.

Effect of multiple discrimination reversals on acquisition of a drug discrimination task in rats.

There are many similarities between exteroceptive stimuli and interoceptive stimuli. Nevertheless, it has been suggested that behavior maintained by drug stimuli might be more difficult to reverse than behavior controlled by exteroceptive stimuli. Once a discrimination is established with an exteroceptive stimulus, it can be reversed by switching the reinforcement contingencies, and repeated reversals result in progressively faster relearning of the discrimination. To determine whether faster relearning of successive discrimination reversals also occurs when the discrimination is controlled by an internal drug stimulus, we trained rats to discriminate 3.2mg/kg phencyclidine-(PCP) from saline, in a two-lever food-reinforced operant task. After this discrimination was acquired, the reinforcement contingencies were reversed. A number of such discrimination reversals were performed to determine whether fewer trials would be needed to reach criterion performance with each reversal. Each time the reinforcement contingencies were switched, fewer training sessions were required for the subjects to reach criterion. These results are similar to those observed when a discrimination has been established with exteroceptive stimuli. The present study provides further evidence of the similarity between interoceptive drug stimuli and exteroceptive sensory stimuli.

Effects of food deprivation and satiation on sensitivity to the discriminative-stimulus effects of pentobarbital in pigeons and morphine in rats.

Food deprivation can produce a substantial increase in the self-administration of drugs of abuse, suggesting that food deprivation increases their reinforcing properties. This finding has been replicated with a wide variety of reinforcing drugs. The present experiments examined the effects of food deprivation and satiation on the discriminative stimulus properties of drugs, to determine whether food deprivation affects the discriminative-stimulus effects of drugs in a similar manner. Using pigeons that were trained to discriminate 5mg/kg i.m. pentobarbital from saline, dose-effect curves were determined under both food-deprivation conditions (80% free-feeding body weight) and partial food-satiation conditions (25% and 50% of the amount of full satiation). It was found that generalization curves for both pentobarbital and saline were similar at all levels of food deprivation. In a second set of experiments, rats were trained to discriminate 10mg/kg i.p. morphine from saline, and the discriminative properties of morphine were then tested when the animals were either food-deprived or after a 15min supplemental feeding. The ED(50) value for the food-deprived condition was comparable to that the food-satiated condition (3.6 vs. 4.8mg/kg, respectively). Thus, in both pigeons and rats, there was little evidence that food deprivation increased sensitivity to the discriminative stimulus properties of drugs. Thus, food deprivation must increase drug self-administration by a mechanism other than by increasing the discriminative stimulus properties of self-administered drugs.

[3H]MK-801 binding to well-washed rat brain membranes following cessation of chronic phencyclidine treatment.

Rats were implanted with osmotic minipumps SC that infused either saline or 10 mg/kg/day phencyclidine (PCP) for 10 days, a regimen that produces dependence to PCP. At the end of this 10-day infusion period, the pumps were removed and the rats were sacrificed either immediately or at various time points (12 h, 1, 2, and 7 days) after pump removal. The saturation binding parameters of [3H]MK-801 were then determined in well-washed cortical/hippocampal membranes prepared from these rats. Neither the Bmax nor the Kd of [3H]MK-801 binding in membranes of PCP-treated rats differed from that determined using membranes from saline-treated rats at any time point studied. These results suggest that alterations in PCP receptors do not play a major role in the production of PCP dependence.

Sexual dimorphic effects of chronic phencyclidine in rats.

The behavioral effects of phencyclidine (PCP) were studied in male and female Sprague-Dawley rats to determine if chronic infusions would result in sexually dimorphic effects. Rats were trained to make operant responses for food during 30-min response periods that occurred 4 times each day. After attaining stable baseline behaviors, 10 mg of PCP/kg/day was infused s.c. for 10 days. Females were more profoundly affected than males. In the females, response rates were suppressed to 30-71% of control rates during the first 7 days of infusion. In contrast, response rate in male rats never fell below 77% of control during the infusion period. By the eighth infusion day both sexes had become tolerant to these behavioral effects. After stopping infusions there was clear evidence that behavioral dependence had developed; however, the abstinence effects in males and females were similar. Saturation studies of [3H]dizocilpine (MK-801; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) binding to brain membranes were conducted to determine if there were sex-dependent receptor differences. There were no significant differences in Kd +/- S.D. (7.6 +/- 1.5 and 7.1 +/- 0.9 nM for males and females, respectively) or Bmax +/- S.D. (4.1 +/- 0.2 and 4.0 +/- 0.5 pmol/mg protein for males and females, respectively).

Tolerance to and dependence on MK-801 (dizocilpine) in rats.

Rats were trained to respond under a fixed-ratio 30 schedule for food presentation during four daily 0.5-h sessions occurring every 6 h. After stable baseline response was established, osmotic minipumps were implanted that infused vehicle or (+)-5 methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrogen maleate (dizocilpine; MK-801), SC. Behavioral sessions continued to be conducted daily. After 10 days the infusion pumps were removed. Vehicle and 0.10 mg/kg per day MK-801 did not affect behavior during infusions or after cessation of dosing. Dosing with 0.32 and 0.56 mg/kg per day initially suppressed responding, but tolerance developed to these effects. After the infusions were stopped, a dose-dependent disruption of operant behavior occurred. Response rates for the 0.32 and 0.56 mg/kg per day infusion groups were suppressed to 41 and 27% of preinfusion control response rates, respectively, the day after dosing stopped; however, no physical signs of abstinence were observed. Response rates recovered toward control over the next 2-4 days. In a separate experiment, the suppression of response produced by abstinence from 0.32 mg/kg per day of MK-801 (SC) for 10.5 days was reversed by readministration of MK-801 (IP). These results demonstrate that MK-801 produces dependence, as evidenced by the emergence of a behavioral abstinence syndrome after cessation of dosing.

Effects of cocaine self-administration on ethanol, food and water intake in the rat.

Because cocaine and ethanol are frequently abused in combination, the present study was performed to assess the behavioral consequences of concurrent access to both of these drugs. Rats trained to respond for food under a fixed-ratio 40 (FR 40) schedule of reinforcement during four, 30-min periods each day (every 6 h) were given free access to a 5% (w/v) ethanol solution and to water. Once a stable baseline of food, ethanol and water intake was established, intravenous cocaine was made available under a fixed-ratio 1 (FR1) schedule. After cocaine self-administration had been established for 10 days, the ethanol was removed from the chambers for 3 days. After the ethanol was returned to the chambers, saline was substituted for cocaine for 3 days. Following saline substitution, animals were given increased cocaine availability. Before cocaine was made available, rats consumed primarily the ethanol solution, averaging 31.7 ml of the 5% solution (4.2 g/kg ethanol), 10.2 ml of water and 148 food pellets/day. When cocaine (0.2 mg/kg per injection, i.v.) was made available, rats self-administered an average of 40-85 injections per day. Self-administration of cocaine resulted in small decreases in ethanol and food intake, as well as some changes in the pattern of ethanol intake. However, removing the ethanol from the chambers had no effect upon food and cocaine intake. Substitution of saline for the cocaine altered the pattern, but not the amount of ethanol intake. There was a trend toward increased ethanol intake during the study, which may have been related to repeated cycles of cocaine availability.

Discriminative-stimulus control by morphine in the pigeon under a fixed-interval schedule of reinforcement.

The stimulus control by morphine under a fixed-interval (FI) 180-s schedule of reinforcement was examined in five pigeons trained to discriminate 5.0mg/kg of i.m. morphine from saline. After training, dose-response relationships were determined for morphine, d-amphetamine, pentobarbital, fentanyl, and MK-801. At low doses of morphine, responding of individual subjects was predominantly on the saline-appropriate key. At intermediate doses responding occurred on both keys throughout the interval. Following doses of 5.6mg/kg and higher, responding on both keys was observed in the early portions of the interval with responding shifting to the morphine-appropriate key as the interval progressed. Thus, a graded dose-effect curve for morphine was obtained under the FI schedule. The fentanyl dose-effect curve was similar to that obtained with morphine, but the other test drugs were not generalized. Five other pigeons were trained to discriminate 5.0mg/kg of morphine from saline under a fixed-ratio (FR) 100 schedule of reinforcement. During testing with morphine, responses were confined to the saline-appropriate key at 0.3 and 1.0mg/kg of morphine, but at higher doses responses were confined to the drug key. In contrast to the results obtained using the FI schedule, a quantal relationship for responding on the two keys was observed when a FR schedule was employed.

Phencyclidine dependence: the relationship of dose and serum concentrations to operant behavioral effects.

The dependence-producing properties of 10 days of chronic i.v. infusions of phencyclidine (PCP) and the relationship between PCP serum concentrations and behavioral effects were studied in Sprague-Dawley rats. For dependence studies, rats were trained to respond for food under a fixed-ratio 30 schedule during half-hour response periods every 6 hr. After training, implantation of jugular catheters, and restabilization of behavior, the rats were infused with PCP.HCl at 3.2, 5.6, 10.0 or 17.8 mg/kg/day (n = 5 or 6 per dose). The two higher doses initially decreased response rates, but tolerance developed within 4 to 5 days. When PCP infusions were terminated, dose-dependent decreases in session response rate occurred in the three highest dose groups (P less than .05). Mild, overt signs of abstinence were observed only in the highest dose group. Response rates returned to base line within 2 to 3 days after stopping PCP infusions. PCP serum concentrations in rats infused with 10 mg of PCP.HCl/kg/day for 10 days were stable from hour 24 to day 10 (mean steady-state concentration (+/- S.D.) = 97 (+/- 20) ng of PCP/ml; n = 4). The average terminal elimination half-life after stopping infusions on day 10 was 4.6 hr. Comparison of the average response rates with the average serum concentrations showed that during the first 24 hr of infusions, the rate of responding for food decreased as PCP concentrations increased; however, once the animals became tolerant to PCP there was no relationship. In contrast, during the first 24 hr after stopping infusions, response rates decreased as serum concentrations decreased.

Effects of terminating chronic phencyclidine on schedule-controlled behavior in rats.

Six rats were trained to respond under a multiple fixed-ratio 30, fixed-interval 3-min schedule for food presentation. Acute administration of phencyclidine (0.1-3.2 mg/kg, IP) produced decreases in fixed-ratio response rates at doses above 0.3 mg/kg, but fixed-interval response rates were only decreased at the highest dose. However, the pattern of fixed-interval responding (as evidenced by quarter-life values) was affected at doses above 0.3 mg/kg. Osmotic minipumps were implanted, SC, which infused saline (2 rats) or phencyclidine (4 rats, 10.0 mg/kg/day) for 10 days, and then removed. Daily behavioral sessions were conducted during infusions and for 10 days afterwards. The effects of phencyclidine infusions on fixed-ratio responding were variable. Fixed-interval response rate and quarter-life values were only modestly affected during drug infusion. All three parameters were markedly affected upon cessation of chronic phencyclidine dosing, but there did not appear to be differential effects between the schedule components. No effects on responding were observed during or after saline infusions.

Phencyclidine pharmacokinetics and concentration-response relationships in the pigeon.

Phencyclidine (PCP) pharmacokinetics and drug discrimination were examined in pigeons (n = 6 in both groups) after intramuscular doses of 1.48 mg/kg. PCP absorption was rapid with maximum measured plasma concentrations ranging from 559 to 1450 ng/ml at 10-30 min after dosing, which corresponded to the time of maximum PCP stimulus effects in the drug discrimination studies. The terminal elimination half-life was 0.88 hr (harmonic mean). Average values for the volume of distribution and total body clearance were 1.6 l/kg and 18.2 ml/min/kg, respectively. In the behavioral studies, pigeons discriminated PCP-like effects from about 2 min to 2 hr after dosing. An average value for response on the PCP-appropriate key and for PCP concentration at each time point from 2 min to 2 hr was calculated from the individual subject data. Least-squares linear regression analysis of these data showed a highly significant relationship between the ability to discriminate PCP and log PCP concentration (y = 103x - 219, r2 = .810, p less than 0.005). This analysis suggests PCP concentration is a good predictor of behavioral efficacy.

Alterations in rat brain [3H]-TCP binding following chronic phencyclidine administration.

Rats were chronically infused with phencyclidine (PCP, 13.3 mg PCP.HCl/kg/day) or saline, s.c., for 10 days using osmotic minipumps (n = 5 for each group). Twenty-four hours after the cessation of dosing, the rats were sacrificed, and brains were removed for analysis of PCP receptor binding. Saturation studies of the binding of [3H]-TCP to brain homogenates revealed statistically significant increases in the maximum binding capacity (Bmax) and decreases in the affinity for [3H]-TCP in the PCP-treated group.

A comparison of interoceptive and exteroceptive discrimination in the pigeon.

In pigeons performing a conditional discrimination under a second-order, color-tracking procedure, stimulus control of responding was established using a blinking versus a nonblinking light as exteroceptive stimuli (light-discrimination group). Another group performing under the same second-order schedule of reinforcement was trained to discriminate the interoceptive stimuli produced by an IM injection of 1.5 mg/kg phencyclidine (PCP) versus saline (drug-discrimination group). In the drug-discrimination group, administration of PCP or pentobarbital resulted in dose-dependent increases in PCP-appropriate responding, while, in general, d-amphetamine did not result in appreciable drug-appropriate responding. In the light-discrimination group, all three drugs over the same dose ranges resulted in decreased discriminative control over responding. In both groups, doses of PCP and pentobarbital which resulted in intermediate (30 to 70%) levels of stimulus-appropriate responding were associated with responding at a single key position rather than tracking a key color. In contrast, intermediate responding after d-amphetamine administration was not associated with position responding in either group. These results emphasize the similarity between discriminative control maintained by interoceptive drug stimuli and exteroceptive visual stimuli.

Interaction of the discriminative stimulus effects of phencyclidine with those of (+)-N-allylnormetazocine, pentobarbital and d-amphetamine.

Pigeons trained to discriminate 1.0 mg/kg phencyclidine from saline were used to study the interaction between the stimulus effects of phencyclidine and those of (+)-N-allylnormetazocine [(+) NANM], pentobarbital and d-amphetamine using a cumulative-dosing procedure. Both (+) NANM and pentobarbital enhanced the discriminative stimulus effects of phencyclidine. The enhancement of the phencyclidine stimulus by pentobarbital was predicted by adding the effects of the individual drugs, but the enhancement of the phencyclidine stimulus by (+) NANM was sometimes more than would have been expected from adding the effects of the individual drugs. d-Amphetamine did not enhance the discriminative stimulus effects of phencyclidine, but neither did it interfere with these effects. Combinations of (+) NANM or pentobarbital with phencyclidine also enhanced the rate-decreasing effects of phencyclidine, but to a lesser extent than they enhanced the discriminative stimulus effects of phencyclidine. d-Amphetamine only slightly enhanced the rate-decreasing effects of phencyclidine.