A head-attachable device for injecting nanoliter volumes of drug solutions into brain sites of freely moving rats.

We describe a head-mounted micropump-injection system designed for the infusion of nanoliter volumes of drug solutions into discrete brain regions of the freely moving rats. Using a miniature step motor, the micropump-injection system can be readily constructed from commercially available supplies. In calibrating the micropump-injection system, we found that it will deliver a reliable volume of 50 nl per infusion over a 1-h period, with an infusion given every 1 min. From in vivo testing, we also found that rats readily self-administered up to 100 infusions of D-amphetamine into the nucleus accumbens at regular intervals, suggesting that this system can deliver constant volumes of infusions over time in freely moving rats. It (1) attaches easily to an implanted guide, (2) is compact and durable, (3) weighs only 10 g, and (4) is well tolerated with no apparent discomfort to the animal. This system overcomes some of the weaknesses of currently used intracranial self-administration systems.

Kappa-opioid receptor activation modifies dopamine uptake in the nucleus accumbens and opposes the effects of cocaine.

Coadministration of kappa-opioid receptor agonists (kappa-agonists) with cocaine prevents alterations in dialysate dopamine (DA) concentration in the nucleus accumbens (Acb) that occur during abstinence from repeated cocaine treatment. Quantitative microdialysis was used to determine the mechanism producing these effects. Rats were injected with cocaine (20 mg/kg, i.p.), or saline, and the selective kappa-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DA(ext)) and extraction fraction (E(d)), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increased E(d), whereas repeated U-69593 treatment decreased E(d), relative to controls. Coadministration of both drugs yielded intermediate E(d) values not different from controls. In vitro DA uptake assays confirmed that repeated U-69593 treatment produces a dose-related, region-specific decrease in DA uptake and showed that acute U-69593 administration increases DA uptake in a nor-binaltorphimine reversible manner. Repeated U-69593 also led to a decrease in [(125)I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that kappa-opioid receptor activation modulates DA uptake in the Acb in a manner opposite to that of cocaine: repeated U-69593 administration decreases the basal rate of DA uptake, and acute U-69593 administration transiently increases DA uptake. kappa-agonist treatment also alters DAT function. The action of kappa-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported "cocaine-antagonist" effect of kappa-opioid receptor agonists.

Autoradiographic evidence that prolonged withdrawal from intermittent cocaine reduces mu-opioid receptor expression in limbic regions of the rat brain.

Numerous reports support evidence that dopaminergic mesolimbic pathways interact with opioid systems to influence the reinforcing properties of cocaine. Withdrawal from chronic administration of cocaine in rats causes an upregulation of mesocorticolimbic mu-opioid receptors during early stages, but information about prolonged cocaine abstinence is lacking. We addressed this issue by treating rats with cocaine or saline (control) intermittently (1 mg/kg, i.v., every 12 min for 2 h daily) for 10 days followed by a 10- or 20-day withdrawal period. The animals were then decapitated and the brains removed for quantitative in vitro autoradiographic analysis of 14 brain regions with (125)I-DAMGO. A separate group of animals received two consecutive cycles of the 10-day cocaine/10-day withdrawal regimen. Only the group that participated in the two consecutive cycles showed a significant effect of treatment: downregulation of mu-opiate receptors in limbic cortical layer 3 (17% lower than saline-treated controls, P = 0.03), the core of the nucleus accumbens (16% decrease, P = 0.05), and the nucleus of the diagonal band (18% decrease, P = 0.05). The mu-receptor may manifest, as do other neural markers (e.g., dopamine transporter, dopamine efflux), a biphasic temporal pattern with upregulation during early phases of cocaine withdrawal but a downregulation at later times.

Cocaine reward and MPTP toxicity: alteration by regional variant dopamine transporter overexpression.

Polygenic factors play important roles in animal models of substance abuse and susceptibility to dopaminergic neurodegeneration. Genetic factors are also likely to contribute to the etiology of human drug abuse disorders, and may alter human vulnerabilities to Parkinsonian neurodegeneration. The dopamine transporter (DAT; SLC6A3) is densely expressed by the dopaminergic midbrain neurons that play central roles in drug reward and is believed to be a primary site of action for cocaine reward. This transporter is necessary for the action of selective dopaminergic neurotoxins, and is uniquely expressed on neurons that are the primary targets of Parkinsonian neurodegeneration. To study possible influences of variant DAT expression on these processes, we have constructed transgenic mice (THDAT) in which tyrosine hydroxylase (TH) promoter sequences drive expression of a rat DAT cDNA variant, increase striatal DAT expression by 20-30%, and provide modest alterations in striatal levels of dopamine and its metabolites. THDAT mice habituate more rapidly to a novel environment than wildtype littermates. These animals display enhanced reward conferred by cocaine, as measured by conditioned place preference. However, locomotor responses to cocaine administration are similar to those of wildtype mice, except at high cocaine doses. THDAT mice display more than 50% greater losses of dopaminergic neurons following a course of MPTP treatment than do wildtype control mice. These results document a model for allelic variation at a gene locus that can exert significant effects in murine models of human substance abuse vulnerability and dopaminergic neurodegeneration.

Regional binding to corticotropin releasing factor receptors in brain of rats exposed to chronic cocaine and cocaine withdrawal.

Cocaine, as does exposure to other physiological stressors, releases brain corticotropin releasing factor (CRF), and this release habituates during the course of repeated cocaine administration in animals. Due to the many signs of anxiety and responses to stress that are produced by cocaine withdrawal in humans, the present study was designed to assess the effects of chronic cocaine and its withdrawal on regional 125I-Tyr-oCRF binding to the CRF1 receptor in brains of male Lewis rats. Cocaine or saline was intravenously infused for 10 days in a regimen that resembled a self-administration paradigm (1 mg/kg every 12 min for 2 h each day). Tissues were harvested either 15 min after or 10 days after the last cocaine infusion, and the brains were sectioned and prepared for CRF1 receptor autoradiography. Compared with findings in saline controls, there was a 31% lower level of CRF binding sites in the basolateral nucleus of the amygdala immediately after the last cocaine infusion, but not 10 days later. Neuroendocrine and non-neuroendocrine mechanisms associated with CRF1 receptors do not appear to contribute to long-term withdrawal effects.

Effects of strychnine-insensitive glycine receptor ligands in rats discriminating dizocilpine or phencyclidine from saline.

Several pharmacologically distinct sites are known to modulate the N-methyl-D-aspartate (NMDA) receptor/ion complex, including a site within the ion channel which binds uncompetitive antagonists like phencyclidine (PCP) or dizocilpine. Glycine acts as a co-agonist for activation of the NMDA receptor complex through a strychnine-insensitive receptor, which is a potential target for novel therapeutic agents (e.g., anticonvulsants, antidepressants). We evaluated the behavioral effects of glycine receptor ligands in rats trained to discriminate either dizocilpine or PCP from saline, to predict whether glycine receptor ligands might induce undesirable PCP-like subjective effects in humans. Dizocilpine ([+]-MK-801), (-)-MK-801 and PCP produced dose-dependent substitution in these rats with potencies in accord with NMDA receptor affinity. Pentobarbital and drugs acting at other sites of the NMDA receptor, including competitive antagonists (NPC 12626 and LY 274614) and the polyamine antagonist, ifenprodil, did not substitute for either dizocilpine or PCP. In contrast to the uncompetitive antagonists like PCP, none of the strychnine-insensitive glycine receptor ligands substituted. Neither the full agonist, glycine; the partial agonists, 1-amino-1-cyclopropanecarboxylic acid, D-cycloserine or (+)-3-amino-1-hydroxypyrrolid-2-one; nor the antagonists, 7-chloro and 5,7-dichlorokynurenic acid, mimicked the discriminative stimulus effects of dizocilpine or PCP. Further, co-administration of 1-amino-1-cyclopropanecarboxylic acid did not significantly enhance the discriminative stimulus effects of dizocilpine. Intracerebroventricular administration of D-serine, a selective agonist of the strychnine-insensitive glycine receptor, neither mimicked nor blocked the discriminative stimulus effects of PCP. These data suggest that functional antagonists of the strychnine-insensitive glycine receptor may be devoid of the subjective side effects characteristic of NMDA channel ligands.

Cocaine withdrawal reduces dopamine transporter binding in the shell of the nucleus accumbens.

We have previously shown that withdrawal from repeated, intermittent infusions of cocaine in Lewis rats results in a long-lasting reduction in dopamine transporter levels in the nucleus accumbens. The reduction is dose-dependent, requires multiple injections as well as about a 10-day withdrawal period. In this investigation, we show that the decrease (34%) occurs in the shell rather than in the core of the nucleus accumbens, and that a second cycle of cocaine administration and withdrawal has no additional effect. Also, there were no changes in transporter binding in the caudate putamen, the olfactory tubercle or the ventral tegmental area. These results indicate that the limbic portions of the nucleus accumbens are involved in neurochemical adaptations during withdrawal from cocaine.

Withdrawal of repeated intravenous infusions of cocaine persistently reduces binding to dopamine transporters in the nucleus accumbens of Lewis rats.

Male, Lewis rats were administered cocaine or saline i.v. in an intermittent fashion for 5, 10 or 20 days and killed at various times afterwards. Dopamine transporter binding was then measured in dorsal striatum and nucleus accumbens. Transporter binding was not changed in dorsal striatum under any conditions tested. In the nucleus accumbens, however, binding was decreased in animals given cocaine (10 mg/kg total) for 10 days and withdrawn for 10, 30 or 60 days, but not in animals withdrawn for 0, 1, 3 and 6 days. There were no changes in animals given cocaine for 5 days and withdrawn for 10, or in animals given drug for 20 days and withdrawn for 1 day. Animals given only 1/10 of the cocaine dose had no changes in nucleus accumbens after 10 days of administration and 10 days of withdrawal. Scatchard analysis in control animals indicated that there were significant differences in both Kd and Bmax when comparing nucleus accumbens with dorsal striatum. Within the nucleus accumbens, decreases in binding after a cessation of cocaine administration were associated with a change in Bmax and not in Kd. These data indicate that long-lasting changes in the mesolimbic dopaminergic system can occur during the withdrawal period, and may contribute to behavioral effects during this period.

Effects of caramiphen and phencyclidine alone and in combination on behavior in the rat.

Because dextromethorphan (DM) has been shown to inhibit the locomotor stimulant effects of phencyclidine (PCP), this study explored further the possible interaction between drugs acting on DM and PCP receptor sites. Caramiphen, an antitussive that binds with high affinity to the DM site, was injected (IP) alone (15-120 mg/kg) or at two doses (15 or 60 mg/kg) 15 min before a challenge dose of PCP (1.25-20 mg/kg). Caramiphen alone dose-dependently increased ataxia, increased stereotypy, and had no effect on locomotor activity. PCP alone dose-dependently increased ataxia, stereotypy, and locomotor activity, the latter showing an inverted U-shaped function. At both pretreatment doses, caramiphen enhanced locomotor activity and stereotypy when combined with low PCP doses but decreased these behaviors at high PCP doses. Caramiphen produced a dose-dependent additive effect on ataxia when combined with all PCP doses. It was concluded that, although caramiphen, like DM, inhibited the locomotor stimulant effects of selected doses of PCP, that interaction appeared to be due to other behaviors (e.g., ataxia/stereotypy) elicited by caramiphen combined with high doses of PCP. This study underscored the importance of using full dose ranges of PCP when attempting to antagonize its behavioral effects with other drugs.

Housing conditions influence acquisition of sufentanil aerosol self-administration in rats.

At weaning, rats were housed either individually or in pairs and as adults were trained to poke their nose in and out of a port that dispensed a 2-s exposure of sufentanil aerosol (50-micrograms/ml solution). During the acquisition phase, which consisted of five nightly sessions lasting 14-16 h, individually caged rats responded for more sufentanil aerosol than did pair-caged animals when the fixed ratio (FR) requirement was gradually increased from FR 1 to FR 5 over the five sessions. During the maintenance phase, which consisted of daytime 2-h sessions at an FR 5 schedule of reinforcement, there were no differences between individually and pair-caged animals responding for sufentanil or for water vapor. Both groups responded significantly more for sufentanil than for water vapor. Based upon present evidence, it is suggested that environmental and biologic determinants may change psychomotor behavior in a way that could influence the rate by which animals acquire drug-seeking behavior.

Cocaine tolerance and cross-tolerance.

Pharmacological mechanisms by which tolerance develops to the behavioral effects of cocaine were assessed by examining cross-tolerance to specific drugs. Daily experimental sessions were conducted in which rats were trained to press a key under a fixed-ratio 30-response schedule of food reinforcement (each 30th response produced food). Each of the drugs studied decreased rates of responding before initiating daily (10 mg/kg, i.p.) treatment with cocaine. Treatment with cocaine produced a small, significant shift to the right in the cocaine dose-effect curve; the ED50 values changed from 13.3 to 21.7 mg/kg. Cross-tolerance was not conferred to the indirect agonist, d-amphetamine, the direct agonist apomorphine, the D1-selective agonists SKF 38393 or fenoldopam, or the D2-selective agonists quinpirole or (-)-NPA. Cross-tolerance was conferred to the close structural analog of cocaine, WIN 35,428, but not to another dopamine uptake inhibitor, GBR 12909. Tolerant rats showed no change in specific binding of [3H]SCH 23390 to D1 receptors, [3H]spiperone to D2 receptors, [3H]GBR 12935 to dopamine uptake sites in striatum, [3H]paroxetine to serotonin uptake sites or [3H]mazindol to norepinephrine uptake sites in cortex or hippocampus. In addition, there were no changes in transmitter levels indicative of neurotoxicity. Serum levels of cocaine were not appreciably different in groups of cocaine- and saline-treated rats. The present results suggest that the modest tolerance that can develop to the behavioral effects of cocaine does not confer significant functional or metabolic changes in the effects of drugs acting on dopaminergic systems. Importantly, the tolerance produced by repeated administration of cocaine does not produce a cross-tolerance to GBR 12909, suggesting differences in mechanism among different structural forms of dopamine uptake inhibitors.

Carbamazepine produces nonspecific effects on cocaine self-administration in rats.

Anecdotal evidence in humans suggest that carbamazepine suppresses cocaine-induced rush and craving. Such claims are unsupported in controlled trials using a placebo control. In the present study, rats were trained to self-administer i.v. cocaine in daily 2-hr sessions in which every tenth lever press delivered 1 mg/kg cocaine. After responding was stable, they were injected before each session with the vehicle for 2 days followed by carbamazepine for 2 days. At a 7 mg/kg dose, carbamazepine was without effect, whereas 15 mg/kg suppressed responding for cocaine only on the second (day 4) day of carbamazepine treatment. With 4 consecutive days of treatment, carbamazepine (15 mg/kg) reduced cocaine-maintained responding slightly, but significantly. In another group of animals trained to lever-press for food reinforcement, carbamazepine (15 mg/kg) also significantly decreased the rate of responding, suggesting that the suppression of responding was not specific to cocaine-reinforced behavior.

Withdrawal of repeated cocaine decreases autoradiographic [3H]mazindol-labelling of dopamine transporter in rat nucleus accumbens.

The in vitro autoradiographic distribution of desipramine-insensitive specific [3H]mazindol binding sites (labelling the dopamine transporter) was determined in brain sections from rats receiving repeated i.v. infusions of saline or cocaine (1 mg/kg, every 12 min for 2 h/day), for 10 days. Brains were removed either within 15 min of or 10 days after the last treatment. A marked dorsal-to-ventral gradient in [3H]mazindol binding appeared in the striatum with the dorsal caudate putamen showing the greatest binding and the medial shell of the nucleus accumbens the least. Cocaine-associated changes in [3H]mazindol-labelled dopamine uptake sites occurred only in the nucleus accumbens (57 and 66% decrease in the lateral core and medial shell, respectively), of animals 10 days after the last treatment. Down-regulation of the dopamine transporter in the nucleus accumbens by withdrawal of chronic cocaine may be one of the mechanisms involved in cocaine's long-term abstinence effects.

Chronic cocaine administration and withdrawal of cocaine modify neurotensin binding in rat brain.

Neurotensin (NT) is a peptide colocalized with dopamine (DA) within some mesocorticolimbic DA neurons that are affected by cocaine. We assessed whether chronic treatment with cocaine and withdrawal from cocaine would alter NT binding within these and other areas in the brain. Rats were given infusions repeatedly of isotonic saline or cocaine (1 mg/kg i.v. every 12 min for 2 hr over 10 days) and then were killed within 15 min of the last treatment session ("cocaine" or "saline") or 10 days later ("withdrawal"). Brains were processed for NT receptor autoradiography. Cocaine affected NT binding in the mesocortical regions differently from other areas. Within the mesocorticolimbic system, NT binding in the parabrachial pigmented nucleus of the ventral tegmental area (VTA) was 67% lower in cocaine-treated rats killed immediately after or 10 days after their final infusion than in rats given saline. In contrast to the perikaryal region, significantly more NT binding occurred postsynaptically in the terminal areas of the VTA (prefrontal cortex [PFC] and substantia nigra, pars compacta) 10 days after withdrawal of cocaine than in the saline controls. NT binding in the nucleus accumbens was unaffected by cocaine or its withdrawal. Cocaine also decreased NT binding in non-mesocorticolimbic areas, including the dorsal hypothalamic area and the zona incerta, but binding returned toward control levels 10 days after withdrawal from cocaine. These data suggest that in central areas poor in DA uptake sites such as the PFC, NT may be a critical element in the inactivation of DA. Chronic cocaine treatment and its withdrawal appear to uncouple the normal NT-DA interaction at both the cell bodies and terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of phencyclidine-like behavior in rats by dextrorphan but not dextromethorphan.

The behavioral effects of dextromethorphan (DM), dextrorphan (DO) and phencyclidine (PCP) were compared in rats. DO (15-120 mg/kg) was similar to PCP (1.25-20 mg/kg) in inducing dose-dependent locomotor hyperactivity, stereotypy and ataxia. DM (15-120 mg/kg) induced moderate hyperactivity only at the higher doses about 45 min after treatment. DM and DO modified the locomotor facilitation induced by 10 mg/kg PCP in opposite directions. Pretreatment with DO facilitated, whereas DM dose-dependently inhibited PCP-elicited hyperactivity. Although the metabolism of DM in rats is unknown, the recently reported abuse of DM in humans may occur by its conversion to DO in the organism, i.e., to a metabolite which produces PCP-like effects.

Separate neural mechanisms mediate sufentanil-induced pupillary responses in the cat.

The pharmacologic characteristics of a highly selective mu receptor agonist, sufentanil, were studied on the cat's pupillary responses (size, light reflex and fluctuations) measured with an infrared video pupillometer. The pupillary effects of sufentanil were also compared with those of morphine and clonidine, known mydriatics in the cat. Sufentanil (0.3-10 micrograms/kg i.v.) dose-dependently increased pupillary size and decreased light reflex and fluctuations. Naltrexone (10 micrograms/kg i.v.) pretreatment shifted the dose-response curve to the right by a factor of 26 for pupillary size, 9.5 for light reflex and 7.2 for fluctuations (nonvalid bioassay). Equivalent mydriatic doses of sufentanil (1 micrograms/kg), morphine (0.5 mg/kg) and clonidine (10 micrograms/kg) produced divergent effects on the light reflex and fluctuations. At these doses, morphine was more effective than sufentanil in inhibiting fluctuations. Clonidine was a more potent inhibitor of fluctuations but significantly enhanced the light reflex. Sufentanil (compared with morphine in a previous study) was 298 times more potent than morphine as a mydriatic, 100 times more potent in inhibiting the light reflex, and only slightly more potent in inhibiting fluctuations. These results indicate that separate neural mechanisms control the three pupillary components and that mu opioid receptors are more involved in mediating opiate-induced mydriasis than in inhibiting the light reflex and fluctuations in the cat.

Discriminative stimulus effects of inhaled cocaine in squirrel monkeys.

Squirrel monkeys (N = 4) were trained with food reinforcement to press one of two levers after administration of IV cocaine (0.3 or 1.0 mg/kg) or the other lever after saline. After training, IV cocaine (0.03-3.0 mg/kg) produced dose-related increases in the percentage of responses on the cocaine lever (ED50 = 0.15 mg/kg). Cocaine delivered IM also produced dose-related increases in cocaine-appropriate responding (ED50 = 0.32 mg/kg), but was approximately half as potent as IV cocaine. Similar relative potency relations were obtained for decreases in response rates produced by cocaine. Prior to some sessions subjects were placed in a Plexiglas chamber and exposed for 60 s to cocaine vapor created with an ultrasonic nebulizer. Exposure to vapor from cocaine solutions (1.0-30.0 mg/ml) produced concentration-dependent increases in cocaine-appropriate responding and decreases in response rates. Exposure to vapor from a 30 mg/ml concentration produced virtually exclusive cocaine-appropriate responding. Concentration-effect curves for inhaled cocaine were similar to dose-effect curves obtained when cocaine was administered by the other routes. The time course of the minimally effective concentration of inhaled cocaine was compared to that of the minimally effective doses of systemically administered cocaine. Inhaled cocaine had a duration of action longer than IV cocaine. The results indicate that inhaled cocaine vapor has effects qualitatively similar to those of IV cocaine, and may have a duration of action longer than that of an IV cocaine dose producing a similar degree of drug-appropriate responding.

Ibogaine fails to reduce naloxone-precipitated withdrawal in the morphine-dependent rat.

Because of anecdotal reports in which ibogaine eliminates opioid withdrawal symptoms in humans, we studied this phenomenon in the rat model. Ibogaine (5, 10, 20 and 40 mg kg-1, s.c.) was administered 15 min before naloxone (0.5 mg kg-1, s.c.) in morphine dependent rats (3 days after the s.c. implantation of a 75 mg morphine pellet). Of the 12 withdrawal signs scored, the only significant changes observed after ibogaine (compared with vehicle control) was a decrease in grooming (10 mg kg-1) and an increase in teeth chatter (5 mg kg-1). In spite of ibogaine's apparent interaction with several neurotransmitter receptor systems, it does not alleviate opioid withdrawal in this animal model at non-tremorigenic (5 and 10 mg kg-1) or tremorigenic (20 and 40 mg kg-1) doses.

Multiple, but not acute, infusions of cocaine alter the release of prolactin in male rats.

Hypothalamic dopamine tonically inhibits the release of prolactin (PRL) from the anterior pituitary gland. Cocaine, in turn, alters dopaminergic transmission. We compared the effects of acute and repeated injections of cocaine on the release of PRL in male rats to assess whether cocaine could affect dopaminergically mediated hormonal responses. We found that the concentration of PRL in plasma was not affected by single i.v. injections of 1, 3 or 10 mg/kg of cocaine. However, in rats infused repeatedly with 1 mg/kg of cocaine for 5 s every 12 min for 2 h over 10 days, the pre-infusion concentrations of PRL increased in a time-dependent manner whereas cocaine uniformly decreased post-infusion levels of PRL. Repeated administration of cocaine may produce long-term changes in either the tuberoinfundibular dopaminergic neurons or the adenohypophysial dopamine D2-receptors, or both. Changes in the peripheral concentration of PRL after multiple injections of cocaine and during cocaine withdrawal may reflect dopaminergic activity in the hypothalamus. In contrast, single injections of cocaine increased adrenocorticotropin (ACTH) in a dose-dependent manner whereas repeated infusions did not increase peripheral concentrations of ACTH or corticosterone. It seems that repeated injections of cocaine do not result in persistent changes in the hypothalamo-pituitary-adrenal axis.