CalDAG-GEFI mediates striatal cholinergic modulation of dendritic excitability, synaptic plasticity and psychomotor behaviors.

CalDAG-GEFI (CDGI) is a protein highly enriched in the striatum, particularly in the principal spiny projection neurons (SPNs). CDGI is strongly down-regulated in two hyperkinetic conditions related to striatal dysfunction: Huntington's disease and levodopa-induced dyskinesia in Parkinson's disease. We demonstrate that genetic deletion of CDGI in mice disrupts dendritic, but not somatic, M1 muscarinic receptors (M1Rs) signaling in indirect pathway SPNs. Loss of CDGI reduced temporal integration of excitatory postsynaptic potentials at dendritic glutamatergic synapses and impaired the induction of activity-dependent long-term potentiation. CDGI deletion selectively increased psychostimulant-induced repetitive behaviors, disrupted sequence learning, and eliminated M1R blockade of cocaine self-administration. These findings place CDGI as a major, but previously unrecognized, mediator of cholinergic signaling in the striatum. The effects of CDGI deletion on the self-administration of drugs of abuse and its marked alterations in hyperkinetic extrapyramidal disorders highlight CDGI's therapeutic potential.

Differential Effects of Nicotine and Nicotine Withdrawal on Fear Conditioning in Male Rats.

BACKGROUND: Tobacco use is prevalent in individuals who are routinely exposed to stress. However, little is known about how nicotine affects responses to trauma. We examined in rats how nicotine exposure affects fear conditioning, a procedure often used to study stress-related psychiatric illness. METHODS: We examined 2 methods of nicotine exposure: self-administration, modeling voluntary use, and experimenter-programmed subcutaneous administration, modeling medicinal administration (nicotine patch). For self-administered nicotine, rats trained to self-administer nicotine i.v. were fear conditioned (via light cue preceding foot-shock) either immediately after a 12-hour self-administration session or 12 hours later during a period with somatic signs of nicotine withdrawal. For experimenter-delivered nicotine, rats were conditioned after 1-21 days of nicotine delivered by programmable (12 hours on) subcutaneous mini-pumps. Tests to evaluate acoustic startle responses to the conditioning environment (context-potentiated startle) and in the presence or absence of the light cue (fear-potentiated startle) occurred after a 10-day period. RESULTS: Rats fear conditioned immediately after nicotine self-administration showed reduced responses to the shock-associated context, whereas those trained during nicotine withdrawal showed exaggerated responses. Experimenter-programmed nicotine produced effects qualitatively similar to those seen with self-administered nicotine. CONCLUSIONS: Self-administration or experimenter-programmed delivery of nicotine immediately before exposure to aversive events can reduce conditioned fear responses. In contrast, exposure to aversive events during nicotine withdrawal exacerbates fear responses. These studies raise the possibility of developing safe and effective methods to deliver nicotine or related drugs to mitigate the effects of stress while also highlighting the importance of preventing withdrawal in nicotine-dependent individuals.

Sex differences in opioid reinforcement under a fentanyl vs. food choice procedure in rats.

Clinical evidence suggest that men are more sensitive than women to the abuse-related effects of mu-opioid agonists. In contrast, preclinical studies suggest the opposite sex difference. The aim of the present study was to clarify this discrepancy using a fentanyl vs. diluted Ensure® choice procedure to assess sex differences in opioid reinforcement. Sex differences in intravenous (IV) fentanyl self-administration were examined under a fixed-ratio (FR5) schedule, a multi-day progressive-ratio (PR) schedule for behavioral economic analysis, and a concurrent (choice) schedule of fentanyl and diluted Ensure® reinforcement in Sprague-Dawley male and female rats. The fentanyl dose-effect function under the FR5 schedule was significantly shifted upward in females compared to males. Similarly, the reinforcing effectiveness of both fentanyl (3.2 and 10 µg/kg per injection, IV) and diluted Ensure® (18 and 56%) were greater in females than in males as assessed using behavioral economic analysis, irrespective of dose or concentration. However, under a fentanyl vs. foodchoice procedure, males chose 3.2 µg/kg per injection fentanyl injections over 18%, but not 56%, diluted Ensure® at a higher percentage compared to females. Overall, these results suggest that the expression of sex differences in opioid reinforcement depends upon the schedule of reinforcement and that preclinical opioid vs. food choice procedures provide a translationally relevant measure (i.e., behavioral allocation) consistent with the direction of sex differences reported in the clinical literature.

Effects of Acute and Chronic Treatments with Dopamine D2 and D3 Receptor Ligands on Cocaine versus Food Choice in Rats.

Dopamine D3 receptor ligands are potential medications for psychostimulant addiction. Medication assessment may benefit from preclinical studies that evaluate chronic medication effects on choice between an abused drug and an alternative, nondrug reinforcer. This study compared acute and chronic effects of dopamine D2- and D3-preferring ligands on choice between intravenous cocaine and palatable food in rats. Under baseline conditions, cocaine maintained dose-dependent increases in cocaine choice and reciprocal decreases in food choice. Acutely, the D2 agonist R-(-)-norpropylapomorphine (NPA) and antagonist L-741,626 [3-[[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole] produced leftward and rightward shifts in cocaine dose-effect curves, respectively, whereas the partial agonist terguride had no effect. All three drugs dose-dependently decreased food-maintained responding. Chronically, the effects of R-(-)-norpropylapomorphine and L-741,626 on cocaine self-administration showed marked tolerance, whereas suppression of food-reinforced behavior persisted. Acute effects of the D3 ligands were less systematic and most consistent with nonselective decreases in cocaine- and food-maintained responding. Chronically, the D3 agonist PF-592,379 [5-[(2R,5S)-5-methyl-4-propylmorpholin-2-yl]pyridin-2-amine] increased cocaine choice, whereas an intermediate dose of the D3 antagonist PG01037 [N-[(E)-4-[4-(2,3-dichlorophenyl)piperazin-1-yl]but-2-enyl]-4-pyridin-2-ylbenzamide] produced a therapeutically desirable decrease in cocaine choice early in treatment; however, tolerance to this effect developed, and lower and higher doses were ineffective. D3 ligands failed to significantly modify total cocaine intake but caused persistent decreases in food intake. Thus, D2-and D3-preferring ligands showed distinct profiles, consistent with different pharmacological actions. In addition, these results highlight the role of acute versus chronic treatment as a determinant of test drug effects. With the possible exception of the D3 antagonist PG01037, no ligand was promising in terms of cocaine addiction treatment.

Effects of the GLP-1 Agonist Exendin-4 on Intravenous Ethanol Self-Administration in Mice.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) receptor agonists have been shown to decrease ethanol (EtOH) drinking in rodent assays. The GLP-1 system also powerfully modulates food and fluid intake, gastrointestinal functions, and metabolism. To begin to understand the neurobiological mechanisms by which GLP-1 receptor ligands may be able to control EtOH intake, it is important to ascertain whether they can modulate the direct reinforcing effects of EtOH, without the confound of effects on ingestive behaviors generally. METHODS: We trained experimentally naïve, free-fed C57BL/6J mice to self-administer EtOH intravenously. Once stable EtOH intake was acquired, we tested the effect of acute pretreatment with the GLP-1 receptor agonist Exendin-4. Effect of Exendin-4 on operant behavior reinforced by a palatable liquid food was similarly evaluated as a control. RESULTS: Intravenous EtOH functioned as a positive reinforcer in over half the mice tested. In mice that acquired self-administration, EtOH intake was high, indeed, reaching toxic doses; 3.2 µg/kg Exendin-4 decreased intravenous EtOH intake by at least 70%, but had no significant effect on food-maintained operant responding. CONCLUSIONS: This experiment produced 2 main conclusions. First, although technically challenging and yielding only moderate throughput, the intravenous self-administration procedure in mice is feasible, and sensitive to pharmacological manipulations. Second, GLP-1 receptor agonists can powerfully attenuate voluntary EtOH intake by directly modulating the reinforcing effects of EtOH. These findings support the potential usefulness of GLP-1 receptor ligands in the treatment of alcohol use disorder.

Anatabine significantly decreases nicotine self-administration.

Nicotine addiction is associated with many lethal disorders (cancer, cardiovascular and pulmonary disease), and more effective medications to aid smoking cessation are urgently needed. Anatabine is 1 of the most abundant minor tobacco alkaloids, but relatively little is known about its interactions with the abuse-related effects of nicotine. The acute effects of anatabine or saline on nicotine- and food-maintained responding were examined in 7 rhesus monkeys (Macaca mulatta). Nicotine (0.01 mg/kg/inj, base) and banana-flavored food pellets (1 g) were available under a second-order schedule (FR 2 [VR 16:S]). Anatabine or saline injections were administered 15 min before the 11:00 a.m. food self-administration session began. Anatabine (0.18-3.2 mg/kg, IM) dose-dependently reduced nicotine self-administration (0.01 mg/kg/inj) (p = .036-0.0003). Food-maintained responding was decreased only at the highest dose of anatabine (3.2 mg/kg; p = .003). Each monkey returned to baseline levels of nicotine self-administration after anatabine treatment, and there was no evidence of catheter malfunction. Next, the effects of anatabine and saline on the nicotine dose-effect curve (0.001-0.1 mg/kg/inj) were evaluated. Anatabine (0.32 and 1.0 mg/kg, IM) decreased the peak of the nicotine dose-effect curve (p < .001 - p < .0001), with no significant effect on food-maintained responding. The abuse liability of anatabine also was examined, and monkeys did not self-administer anatabine (0.0032-0.32 mg/kg/inj) above saline levels. These findings are consistent with anatabine's effects on nicotine self-administration in rats (Caine et al., 2014). These data suggest that anatabine could be an effective agonist medication for treatment of nicotine addiction.

Nicotine-like behavioral effects of the minor tobacco alkaloids nornicotine, anabasine, and anatabine in male rodents.

Tobacco use is associated with lethal diseases in an estimated 440,000 persons in the United States each year (Centers for Disease Control and Prevention, 2005). Successful smoking quit-rates are estimated at 5%-8%, even though a quarter of those attempts included use of smoking-cessation aids (Messer et al., 2008; Henningfield et al., 2009). Current projections are that 16% of the U.S. population-35 million people-will still smoke in 2025, thus more effective smoking-cessation aids are urgently needed (Pollock et al., 2009). The minor tobacco alkaloids may be promising candidates, but further research is necessary (Hoffman & Evans, 2013). Accordingly, we systematically evaluated the minor tobacco alkaloids nornicotine, anabasine, and anatabine using assays of behavioral tolerability, nicotine withdrawal, nicotine discrimination, and nicotine self-administration in male rodents. At doses that were well tolerated, all 3 minor alkaloids dose-dependently engendered robust substitution for a nicotine discriminative stimulus in mice (0.32 mg/kg, IP), and anabasine attenuated nicotine withdrawal. When the ED50 dose of each alkaloid was administered in combination with nicotine, the discriminative stimulus effects of nicotine were not enhanced by any of the alkaloids, and anatabine blunted nicotine's effects. In drug self-administration studies, only nornicotine was self-administered by rats that self-administered nicotine intravenously; anabasine and anatabine had no reinforcing effects. Moreover, prior administration of each of the minor tobacco alkaloids dose-dependently decreased nicotine self-administration. Collectively these results suggest that the minor tobacco alkaloids may substitute for the subjective effects of nicotine and attenuate withdrawal and craving without the abuse liability of nicotine.

Acute and chronic effects of the M1/M4-preferring muscarinic agonist xanomeline on cocaine vs. food choice in rats.

RATIONALE: We previously showed that the M1/M4-preferring muscarinic agonist xanomeline can acutely attenuate or eliminate cocaine self-administration in mice. OBJECTIVE: Medications used to treat addictions will arguably be administered in (sub)chronic or repeated regimens. Tests of acute effects often fail to predict chronic effects, highlighting the need for chronic testing of candidate medications. METHODS: Rats were trained to lever press under a concurrent FR5 FR5 schedule of intravenous cocaine and food reinforcement. Once baseline behavior stabilized, the effects of 7 days once-daily injections of xanomeline were evaluated. RESULTS: Xanomeline pretreatment dose-dependently (1.8-10 mg/kg/day) shifted the dose-effect curve for cocaine rightward (up to 5.6-fold increase in A 50), with reallocation of behavior to the food-reinforced lever. There was no indication of tolerance, rather effects grew over days. The suppression of cocaine choice appeared surmountable at high cocaine doses, and xanomeline treatment did not significantly decrease total-session cocaine or food intake. CONCLUSIONS: In terms of xanomeline's potential for promoting abstinence from cocaine in humans, the findings were mixed. Xanomeline did produce reallocation of behavior from cocaine to food with a robust increase in food reinforcers earned at some cocaine/xanomeline dose combinations. However, effects appeared surmountable, and food-maintained behavior was also decreased at some xanomeline/cocaine dose combinations, suggesting clinical usefulness may be limited. These data nevertheless support the notion that chronic muscarinic receptor stimulation can reduce cocaine self-administration. Future studies should show whether ligands with higher selectivity for M1 or M1/M4 subtypes would be less limited by undesired effects and can achieve higher efficacy.

Cocaine versus food choice procedure in rats: environmental manipulations and effects of amphetamine.

We have adapted a nonhuman primate model of cocaine versus food choice to the rat species. To evaluate the procedure, we tested cocaine versus food choice under a variety of environmental manipulations as well as pharmacological pretreatments. Complete cocaine-choice dose-effect curves (0-1.0 mg/kg/infusion) were obtained for each condition under concurrent fixed ratio schedules of reinforcement. Percentage of responding emitted on the cocaine-reinforced lever was not affected significantly by removal of cocaine-associated visual or auditory cues, but it was decreased after removal of response-contingent or response-independent cocaine infusions. Cocaine choice was sensitive to the magnitude and fixed ratio requirement of both the cocaine and food reinforcers. We also tested the effects of acute (0.32, 0.56, 1.0, 1.8 mg/kg) and chronic (0.1, 0.32 mg/kg/hr) d-amphetamine treatment on cocaine choice. Acute and chronic d-amphetamine had opposite effects, with acute increasing and chronic decreasing cocaine choice, similar to observations in humans and in nonhuman primates. The results suggest feasibility and utility of the choice procedure in rats and support its comparability to similar procedures used in humans and monkeys.

Cocaine self-administration in dopamine D3 receptor knockout mice.

The dopamine D3 receptor has received attention over the last two decades as a target for medications development for substance abuse disorders. Results have remained mixed. Despite emergence of more D3-selective ligands, possible attribution of observed effects to D2 receptors remains a concern. Knockout mice may help shed light on mechanisms. Here we evaluated the effect of constitutive D3 receptor inactivation ("knockout") on the reinforcing effects of cocaine. We tested D3 wild-type (WT), heterozygous (D3⁺/⁻), and knockout (D3⁻/⁻), mice in acquisition and maintenance of intravenous self-administration across a broad range of cocaine doses, using a fixed ratio (FR) 1 and a progressive ratio (PR) schedule of reinforcement, along with parallel food-reinforced studies. Generally, D3⁻/⁻ mice showed cocaine self-administration comparable to WT controls across assays. Moderate and nonsignificant trends toward lesser reinforcing effects of a low cocaine dose (0.32 mg/kg) were apparent in acquisition and PR studies, consistent with the idea that the D3 receptor may play a subtle role in the reinforcing effects of low cocaine doses under low FR conditions. However, those effects with cocaine self-administration were more subtle than the lower responding of D3 knockout mice observed with food-maintained behavior. In addition, the D3 antagonist PG01037 failed to affect cocaine self-administration under an FR 1 schedule in WT mice. The present data do not support a necessary role for the D3 receptor in the direct reinforcing effects of cocaine.

Lack of abuse potential in a highly selective dopamine D3 agonist, PF-592,379, in drug self-administration and drug discrimination in rats.

Dopamine D3-preferring agonists are commonly used to treat Parkinson's disease and restless leg syndrome; however, laboratory animal studies suggest that they may possess a moderate abuse potential. These studies aimed to compare the highly selective, full D3 agonist PF-592,379 to the less selective D3 agonist 7-OH-DPAT, and the indirect dopamine agonist cocaine in drug self-administration and discrimination assays. Although rats readily acquired high rates of fixed ratio (FR)1 responding for cocaine, experimentally naive rats failed to acquire responding when 7-OH-DPAT or PF-592,379 was made available during an 18-session acquisition period. Cocaine also maintained dose-dependent levels of responding when available under a FR5 or a progressive ratio (PR) schedule of reinforcement. Although 7-OH-DPAT maintained modest levels of responding when substituted under a FR5, it failed to maintain significant levels of PR responding. PF-592,379 maintained saline-like rates of responding when substituted under FR5 or PR schedules of reinforcement. Similar behavioral profiles were observed in cocaine discrimination assays, with 7-OH-DPAT partially substituting for cocaine, and PF-592,379 producing saline-like effects over a wide range of doses. Together, the results of these studies predict that highly selective D3 agonists, such as PF-592,379, will have low abuse potential in humans.

Contribution of both M1 and M4 receptors to muscarinic agonist-mediated attenuation of the cocaine discriminative stimulus in mice.

RATIONALE: We previously showed that muscarinic agonists with M(1) and/or M(4) receptor affinities attenuated cocaine discrimination and self-administration in wild-type mice but not in M(1)/M(4) double-knockout mice. OBJECTIVE: This study aims to elucidate the respective contributions of M(1) and M(4) receptors to this effect. METHODS: Knockout mice lacking either the M(1) subtype (M (1) (-/-) ) or the M(4) subtype (M (4) (-/-) ) and wild-type mice were trained to discriminate 10 mg/kg cocaine from saline. Muscarinic ligands were tested for modulation of cocaine discrimination: xanomeline (M(1)/M(4)-preferring agonist), VU0357017 (M(1)-selective partial agonist), 77-LH-28-1 (M(1) agonist), and BQCA (M(1)-selective positive allosteric modulator). RESULTS: Xanomeline produced rightward shifts in the cocaine dose-effect curve in all three genotypes, but most robustly in wild-type mice. VU0357017 produced rightward shifts in the cocaine dose-effect curve in wild-type and M (4) (-/-) mice, but not in M (1) (-/-) mice. Response rates were suppressed by xanomeline in wild-type and M (1) (-/-) but not in M (4) (-/-) mice and were unaltered by VU0357017. 77-LH-28-1 and BQCA also showed evidence of attenuating cocaine's discriminative stimulus, but at doses that suppressed responding or had other undesirable effects. Intriguingly, both VU0357017 and 77-LH-28-1 exhibited U-shaped dose-effect functions in attenuating cocaine discrimination. None of the drugs substituted for the cocaine stimulus. CONCLUSIONS: Attenuation of the cocaine stimulus by VU0357017 depended upon M(1) receptors, and full effects of xanomeline depended upon both M(1) and M(4) receptors. Therefore M(1)-selective agonists and mixed M(1)/M(4) agonists may be promising leads for developing medications that block cocaine's effects.

Psychomotor stimulant effects of cocaine in rats and 15 mouse strains.

Relative to intravenous drug self-administration, locomotor activity is easier to measure with high throughput, particularly in mice. Therefore its potential to predict differences in self-administration between genotypes (e.g., targeted mutations, recombinant inbred strains) is appealing, but such predictive value is unverified. The main goal of this study was to evaluate the utility of the locomotor assay for accurately predicting differences in cocaine self-administration. A second goal was to evaluate any correlation between activity in a novel environment, and cocaine-induced hyperactivity, between strains. We evaluated locomotor activity in male and female Sprague-Dawley rats and 15 mouse strains (129S1/SvImJ, 129S6/SvEvTac, 129X1/SvJ, A/J, BALB/cByJ, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, SJL/J, SPRET/EiJ, and outbred Swiss Webster and CD-1/ICR), as well as cocaine self-administration in BALB substrains. All but BALB/cJ mice showed locomotor habituation and significant cocaine-induced hyperactivity. BALB/cJ mice also failed to self-administer cocaine. BALB/cByJ mice showed modest locomotor habituation, cocaine-induced locomotion, and cocaine self-administration. As previously reported, female rats showed greater cocaine-induced locomotion than males, but this was only observed in one of 15 mouse strains (FVB/NJ), and the reverse was observed in two strains (129X1/SvJ, BALB/cByJ). The intriguing phenotype of the BALB/cJ strain may indicate some correlation between all-or-none locomotion in a novel environment, and stimulant and reinforcing effects of cocaine. However, neither novelty- nor cocaine-induced activity offered a clear prediction of relative reinforcing effects among strains. Additionally, these results should aid in selecting mouse strains for future studies in which relative locomotor responsiveness to psychostimulants is a necessary consideration.

Psychomotor stimulation by dopamine D1-like but not D2-like agonists in most mouse strains.

Many neurological and psychiatric disorders are treated with dopamine modulators. Studies in mice may reveal genetic factors underlying those disorders or responsiveness to various treatments, and species and strain differences both complicate the use of mice and provide valuable tools. We evaluated psychomotor effects of the dopamine D1-like agonist R-6-Br-APB and the dopamine D2-like agonist quinelorane using a locomotor activity procedure in 15 mouse strains (inbred 129S1/SvImJ, 129S6/SvEvTac, 129X1/SvJ, A/J, BALB/cByJ, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, SJL/J, SPRET/EiJ, outbred Swiss Webster, and CD-1) and Sprague-Dawley rats, using groups of both females and males. Both D1 and D2 stimulation produced hyperactivity in the rats, and surprisingly, only two mouse strains were similar in that regard (C3H/HeJ, SPRET/EiJ). In contrast, the majority of mouse strains exhibited hyperactivity only with D1 stimulation, whereas D2 stimulation had no effect or decreased activity. BALB substrains, A/J and FVB/NJ mice showed only decreased activity after either D1 or D2 stimulation. CAST/EiJ mice exhibited hyperactivity exclusively with D2 stimulation. Sex differences were observed but no systematic trend emerged: For example, of the five strains in which a main factor of sex was identified for the stimulant effects of the D1 agonist, responsiveness was greatest in females in three of those strains and in males in two of those strains. These results should aid in the selection of mouse strains for future studies in which D1 or D2 responsiveness is a necessary consideration in the experimental design.

False positive in the intravenous drug self-administration test in C57BL/6J mice.

The objective of this study was to examine C57BL/6J (B6) mice during extinction conditions, after food training, and for rates and patterns of operant behavior that seems similar to behavior maintained by intravenous cocaine injections. The rationale was to evaluate the potential for false positives in the intravenous self-administration test using protocols common in studies of knockout mice backcrossed to B6. An additional aim was to assess the influence of food-associated and drug-associated cues and mouse strain. Mice were allowed to acquire lever pressing reinforced by sweetened condensed milk under a fixed ratio 1 then fixed ratio 2 schedule of reinforcement accompanied by a flashing light. A catheter base was then implanted for simulation of intravenous self-administration conditions. Mice were allowed to lever press with cues remaining the same as during food training but without further scheduled consequences (i.e. no drug or food reinforcers delivered). All mice sustained lever pressing for several weeks, and over half met commonly used criteria for 'self-administration behavior.' Thus, B6 mice showed perseveration of a previously reinforced behavior that closely resembled rates and patterns of drug self-administration. This effect in B6 mice was greater than with A/J mice, and the lack of extinction was even more robust in the presence of cocaine-associated cues than with food-associated cues. We suggest that a necessary criterion for positive results in the intravenous drug self-administration test include an increase in responding when cocaine is made available after extinction with saline self-administration.

Monoamine transporters: vulnerable and vital doorkeepers.

Transporters of dopamine, serotonin, and norepinephrine have been empirically used as medication targets for several mental illnesses in the last decades. These protein-targeted medications are effective only for subpopulations of patients with transporter-related brain disorders. Since the cDNA clonings in early 1990s, molecular studies of these transporters have revealed a wealth of information about the transporters' structure-activity relationship (SAR), neuropharmacology, cell biology, biochemistry, pharmacogenetics, and the diseases related to the human genes encoding these transporters among related regulators. Such new information creates a unique opportunity to develop transporter-specific medications based on SAR, mRNA, DNA, and perhaps transporter trafficking regulation for a number of highly relevant diseases including substance abuse, depression, schizophrenia, and Parkinson's disease.

Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation.

Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus. More importantly, we tested the hypothesis that muscarinic receptor agonists with varied receptor subtype selectivity can blunt cocaine's discriminative stimulus and reinforcing effects; we hypothesized a critical role for the M(1) and/or M(4) receptor subtypes in this modulation. Mice were trained to discriminate cocaine from saline, or to self-administer intravenous cocaine chronically. The nonselective muscarinic antagonists scopolamine and methylscopolamine, the nonselective muscarinic agonists oxotremorine and pilocarpine, the M(1)/M(4)-preferring agonist xanomeline, the putative M(1)-selective agonist (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride (McN-A-343), and the novel M(1)-selective agonist 1-(1-2-methylbenzyl)-1,4-bipiperidin-4-yl)-1H benzo[d]imidazol-2(3H)-one (TBPB) were tested as substitution and/or pretreatment to cocaine. Both muscarinic antagonists partially substituted for cocaine and enhanced its discriminative stimulus. Conversely, muscarinic agonists blunted cocaine discrimination and abolished cocaine self-administration with varying effects on food-maintained behavior. Specifically, increasing selectivity for the M(1) subtype (oxotremorine < xanomeline < TBPB) conferred lesser nonspecific rate-suppressing effects, with no rate suppression for TBPB. In mutant mice lacking M(1) and M(4) receptors, xanomeline failed to diminish cocaine discrimination while rate-decreasing effects were intact. Our data suggest that central M(1) receptor activation attenuates cocaine's abuse-related effects, whereas non-M(1)/M(4) receptors probably contribute to undesirable effects of muscarinic stimulation. These data provide the first demonstration of anticocaine effects of systemically applied, M(1) receptor agonists and suggest the possibility of a new approach to pharmacotherapy for cocaine addiction.

Modulation of prepulse inhibition through both M(1) and M (4) muscarinic receptors in mice.

RATIONALE: Muscarinic cholinergic M(1) and M(4) receptors may participate in schizophrenia's etiology and have been proposed as targets for antipsychotic medications. OBJECTIVE: Here, we investigated the involvement of these receptors in behavioral measures pertinent to schizophrenia using knockout mice lacking M(1) receptors (M(1)-/-), M(4) receptors (M(4)-/-), or both (M(1)-/-M(4)-/-). METHODS: We measured prepulse inhibition (PPI) of startle without drugs and after treatment with scopolamine (0.32-1.8 mg/kg), xanomeline (3.2 mg/kg), oxotremorine (0.032-0.1 mg/kg), clozapine (1.0-5.6 mg/kg), or haloperidol (0.32-3.2 mg/kg). RESULTS: In female (but not male) mice, combined deletion of both M(1) and M(4) receptors decreased PPI relative to wild-type mice, while knockout of either receptor alone had no significant effect. Scopolamine disrupted PPI in wild-type and M(4)-/- mice, but not in female M(1)-/-M(4)-/- or female M(1)-/- mice. When administered before scopolamine, xanomeline restored PPI in wild-type mice and M(1)-/- mice, but not in M(4)-/- mice. In contrast, pretreatment with oxotremorine increased PPI regardless of genotype. Effects of clozapine and haloperidol on PPI were not hindered by either mutation. CONCLUSIONS: Deletion of both M(1) and M(4) receptors can disrupt PPI, suggesting that (at least partially redundant) M(1) and M(4) receptor-dependent functions are involved in sensorimotor gating mechanisms. PPI-disrupting effects of muscarinic antagonists appeared dependent upon M(1) receptor blockade. Our data also suggest that xanomeline exerts antipsychotic-like effects mainly through M(4) receptor stimulation, while stimulation of non-M(1)/M(4) subtypes may also have antipsychotic potential. Finally, our results do not support a role of M(1)/M(4) receptors in mediating antipsychotic-like effects of clozapine.

Lack of cocaine self-administration in mice expressing a cocaine-insensitive dopamine transporter.

Cocaine addiction is a worldwide public health problem for which there are no established treatments. The dopamine transporter (DAT) is suspected as the primary target mediating cocaine's abuse-related effects based on numerous pharmacological studies. However, in a previous study, DAT knockout mice were reported to self-administer cocaine, generating much debate regarding the importance of the DAT in cocaine's abuse-related effects. Here, we show that mice expressing a "knockin" of a cocaine-insensitive but functional DAT did not self-administer cocaine intravenously despite normal food-maintained responding and normal intravenous self-administration of amphetamine and a direct dopamine agonist. Our results have three implications. First, they imply a crucial role for high-affinity DAT binding of cocaine in mediating its reinforcing effects, reconciling mouse genetic engineering approaches with data from classic pharmacological studies. Second, they demonstrate the usefulness of knockin strategies that modify specific amino acid sequences within a protein. Third, they show that it is possible to alter the DAT protein sequence in such a way as to selectively target its interaction with cocaine, while sparing other behaviors dependent on DAT function. Thus, molecular engineering technology could advance the development of highly specialized compounds such as a dopamine-sparing "cocaine antagonist."