Blockade of muscarinic acetylcholine receptors in the ventral tegmental area disrupts food-related learning in rats

Rationale Stimulation of ventral tegmental area (VTA) muscarinic acetylcholine receptors (mAChRs) is implicated in feeding. Objective To investigate the effects of mAChR blockade in the VTA on food-related learning. Methods In experiment 1, rats (N=12) were placed in chambers containing food and received microinjections of 0 or 5 μg/0.5 μl scopolamine prior to the first four feeding sessions and the alternate dose prior to the tenth feeding session. In experiment 2 (N=9), the effects of daily microinjections of scopolamine on lever pressing under a progressive ratio schedule of food reinforcement were tested. In experiment 3 (N=34), the effects of daily microinjections of scopolamine on lever pressing maintained by conditioned reward were investigated. Results In experiment 1, all rats demonstrated low consumption during session 1. However, pellet consumption for rats initially pretreated with the 0-μg dose rose to and stayed at maximal levels for the remaining sessions, even when pretreated with the 5-μg dose during the tenth session. Pellet consumption for rats initially pretreated with the 5-μg dose remained low, even for the first two sessions following the cessation of scopolamine pretreatment, and gradually rose to maximal levels by the eighth session. In experiment 2, scopolamine significantly decreased break points. In experiment 3, scopolamine failed to significantly decrease responding specifically on the lever producing the conditioned reward. Conclusions Altogether, these data suggest that VTA mAChR stimulation is involved in feeding and food-related learning but may not be involved in responding maintained by conditioned reward.     

Blockade of muscarinic acetylcholine receptors in the ventral tegmental area prevents acquisition of food-rewarded operant responding in rats

Rationale We recently found that muscarinic receptor (mAChR) stimulation in the ventral tegmental area (VTA) is involved in the acquisition of a feeding task.Objective To investigate the involvement of VTA mAChR and nicotinic receptors (nAChR) in the acquisition and performance of a food-rewarded lever-pressing task.Methods In experiment 1 (N=54), rats were trained under a fixed ratio 1 schedule of reinforcement and received bilateral intra-VTA microinjections of scopolamine (0, 2.5 or 5 μg/0.5 μl) or mecamylamine (0, 5 or 10 μg/0.5 μl) before each of the first four sessions. Before session 10, all rats that initially received a dose of either compound now received the vehicle and vice versa. In experiment 2 (N=14), rats were tested with scopolamine or mecamylamine while lever pressing under a progressive ratio schedule of reinforcement.Results In experiment 1, lever pressing by rats initially treated with any mecamylamine dose or the scopolamine vehicle rose to and stayed at maximal levels for the remaining sessions. Responding by rats initially treated with the 2.5- or 5-μg dose of scopolamine remained low, even after the cessation of scopolamine treatment, and gradually rose to maximal levels by the final sessions. Injections of scopolamine 1 to 2 mm dorsal to the VTA had no significant effect on responding. In experiment 2, neither of the compounds significantly affected break points.Conclusions Stimulation of VTA mAChR, but not of nAChR, is necessary for the acquisition of a food-rewarded lever-pressing task and neither is necessary for the performance of the task.     

Long-term clomipramine treatment upregulates forebrain acetylcholine muscarinic receptors, and reduces behavioural sensitivity to scopolamine in mice

We have investigated the effects of long-term treatment with clomipramine, a tricyclic antidepressant, on central muscarinic acetylcholine receptors (mAChR) in mice. Repeated clomipramine administration resulted in an increase in the forebrain receptor density value (Bmax) for [3H]quinuclidinyl benzilate, a muscarinic ligand (P < 0.05), that was dependent on dose per administration (saline or 5, 10, or 20 mg kg(-1) once a day for 7 days) and number of days treated (20 mg kg(-1) for 1, 3, 5, or 7 days). No change in apparent affinity (defined as the reciprocal of the dissociation constant) (KD) occurred. Seven daily treatments with clomipramine (saline or 5, 10, or 20 mg kg(-1)) reduced hyperlocomotion induced by scopolamine (0.5 mg kg(-1), s.c.) dose-dependently, and the effect of 20 mg kg(-1) clomipramine was significant (P < 0.05). These results suggest that an upregulation of mAChR is produced by repeated clomipramine administration, and such a change is responsible for the decreased sensitivity to the muscarinic antagonist scopolamine.     

Tolerance to the discriminative stimulus properties of d-amphetamine

Male rats (F-344) responding for milk on a VI 20 sec schedule of reinforcement were trained to discriminate which of two levers to press on the basis of whether they had been injected with d-amphetamine (0.50, 1.00 or 1.50 mg/kg) or saline 15 min prior to daily training sessions. Dose-response functions determined for each of the three (n = 6) training-dose groups indicated that ED₅₀ values were directly correlated with training dose. Two days following chronic amphetamine injections (a total of 78 mg/kg over 4 days) rats were tested for tolerance at a dose which normally produced about 80% drug-lever responding. Rats in all three groups showed tolerance to the cue properties of amphetamine. In the 0.50 and 1.00 mg/kg groups, complete tolerance was shown as evidenced by the fact that the drug lever responding did not differ from that which was appropriate following saline injections.     

Effects of azaprophen, scopolamine and trihexyphenidyl on schedule-controlled behavior, before and after chronic physostigmine

The effects of the muscarinic acetylcholine receptor antagonists, azaprophen (0.3-10.0 mg/kg), scopolamine (0.01-3.0 mg/kg) and trihexyphenidyl (0.3-10.0 mg/kg) were examined in rats using a VI 18 s schedule of food reinforcement, before and after chronic physostigmine administration. All three compounds produced dose-dependent decreases [corrected] in the rate of responding. Scopolamine was more potent than trihexyphenidyl which was equipotent to azaprophen. All three compounds antagonized the response rate-decreasing effects of physostigmine in a dose-dependent fashion. Following 43 consecutive daily administrations of physostigmine (0.4 mg/kg), partial tolerance developed to its response rate-decreasing effects. When the three antagonists were again examined (alone and in combination with physostigmine), their effects were generally unchanged. These results further characterize the behavioral effects of azaprophen, scopolamine and trihexyphenidyl. These results also suggest that tolerance to physostigmine's effects can be mediated through behavioral rather than pharmacological mechanisms.     

Effects of acute and chronic administration of phenobarbital and d-amphetamine on schedule-controlled behavior

The effects of acute and chronic administration of phenobarbital and d-amphetamine were determined in rats responding under a multiple fixed-interval five minute fixed-ratio 30 (mult FI 5 FR 30) schedule of food presentation. After determining the acute effects of each drug, the drugs were injected daily with one group of rats receiving the drugs before each behavioral session while another group received the drugs immediately after each daily session. After four to seven consecutive injections, tolerance developed to the effects of phenobarbital on the average rates of responding under FI and FR schedule components only if the drug was administered before each session. Tolerance was more pronounced for responding during the terminal portions of the FI component than for responding during either the initial portions of the FI or the FR component. Evidence for a selective tolerance to the effects of the drug on responding during the final segments of the FI was also obtained in rats responding under an FI 5 schedule. In contrast, injections of d-amphetamine for seven to eight consecutive days failed to produce any tolerance to the effects of the drug on responding under mult FI 5 FR 30, FI 5, or FR 30 schedules. These results indicate that the development of tolerance to the effects of phenobarbital depended both upon the temporal relationship of the drug effects to the behavioral testing and upon the schedules controlling behavior. These findings are discussed in terms of theories of behavioral tolerance.     

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [³⁵S]GTPγS binding, using the anti-Gα_q antibody sc-393. Increased specific [³⁵S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gα_q, selectively coupled with the M₁ mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M₁ mAChR agonists in the present study were generally lower than previously reported in M₁-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [³⁵S]GTPγS binding to Gα_q was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M₁-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the G_q-coupled M₁ mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.     

Mechanism of tolerance development to 2,5-dimethoxy-4-iodoamphetamine in rats: down-regulation of the 5-HT2A, but not 5-HT2C, receptor

  Rationale: Defining the mechanism of tolerance development to hallucinogenic drugs will help to explain their mechanism of action. Objectives: The present study was conducted to determine first, if tolerance develops to the discriminative stimulus (DS) properties of the hallucinogen, 2,5 dimethoxy-4-iodo-amphetamine (DOI) and second, the mechanism mediating tolerance. Methods: Rats were trained to discriminate 0.75 mg/kg DOI from saline on a concurrent VI-30-min schedule of reinforcement with a 15-min time-out for incorrect responses. To evaluate tolerance development, rats were assigned to one of four groups and treated with either chronic saline or chronic DOI. Prior to chronic treatment, two groups were tested for choice behavior following vehicle administration while the remaining two groups were tested following the administration of 0.375 mg/kg DOI. One group from each pre-test condition was injected with either saline or DOI (1 mg/kg) for 8 days. Twenty-four hours after the last chronic injection the pre-test treatments were replicated. Using receptor autoradiography, the density of 5-HT_2A and 5-HT_2C receptors was measured in independent groups of rats that had received identical treatment conditions. Results: Animals receiving chronic DOI showed a 60% decrease in DOI lever responding (from 100% to 40%) when tested on 0.375 mg/kg DOI, while animals receiving chronic saline showed no change in percent choice (100%) on the DOI lever. Significant changes in binding were observed in 5-HT_2A receptors but not 5-HT_2C receptors. The results of tests with antagonists were consistent with the changes in binding. Conclusions: These results suggest that behavioral tolerance to DOI reflects neuroadaptive changes in 5-HT_2A receptors. Received: 17 July 1998 / Final version: 19 January 1999     

Serotonergic modulation of anticholinergic effects on cognition and behavior in elderly humans

Cholinergic neurotransmission is thought to be modulated by serotonin as documented in animal and human studies. We examined the effects of the muscarinic antagonist scopolamine (0.4 mg IV) given alone or together with the serotonin mixed agonist/antagonistm-chlorophenylpiperazine (m-CPP, 0.08 mg/kg IV), and the selective 5-HT₃ receptor antagonist ondansetron (0.15 mg/kg IV). Ten normal elderly volunteers each received five separate pharmacologic challenges (placebo, ondansetron, scopolamine, scopolamine + ondansetron, and scopolamine + m-CPP). Cognitive, behavioral, and physiologic variables were analyzed using repeated measures analysis of variance. The acute effects of scopolamine in certain cognitive, behavioral, and physiological measures were significantly exaggerated by the addition of m-CPP. Scopolamine's cognitive effects were unaffected by ondansetron at the dose tested, nor did ondansetron given alone affect basal cognitive performance. This pilot study suggests that the serotonin mixed agonist/antagonist m-CPP may influence cholinergic neurotransmission. The changes associated with the combination of scopolamine and m-CPP do not appear to be secondary to simple pharmacokinetic alterations and suggest a complex interaction between the cholinergic and serotonergic systems centrally.     

Barbiturate dependence and drug preference

Rats were administered chronic multiple injections of amphetamine (AMPH) using dosage regimens which produce tolerance to the AMPH facilitation of self-stimulation responding, or reverse tolerance (sensitization) to the locomotor stimulant and stereotypy-producing effects of the drug. Subsequently rats were challenged with AMPH at behaviorally relevant doses and times and striatal and mesolimbic dopamine (DA) dynamics were assessed using the conversion of ³H-tyrosine to ³H-DA, and endogenous levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as indices of dopaminergic function. Acute administration of AMPH produced dose and time related changes in all indices of DA function in both the striatal and mesolimbic brain regions. Co-administration of haloperidol during chronic AMPH pretreatment prevented the appearance of most of the behavioral changes induced by chronic AMPH, suggesting an important role for DA systems. However, following chronic AMPH treatment, no additional biochemical changes in striatal or mesolimbic DA metabolism could be detected which would parallel the development of tolerance to AMPH facilitation of self-stimulation behavior or reverse tolerance to AMPH as reflected in enhanced post-stereotypy locomotor activity or a suggested increased intensity of stereotypy. Challenge with AMPH after chronic AMPH pretreatment did accelerate the changes in striatal but not mesolimbic DA metabolism, correlating with the more rapid onset of stereotypy induced by chronic AMPH. Thus, while DA systems appear to be a critical link, not only in the acute effects of AMPH, but also in the development of tolerance and reverse tolerance, most of the behavioral differences between acutely and chronically treated animals are not reflected by comparable differences in DA synthesis and metabolism.     

Tolerance to p-chloroamphetamines effects on Sidman avoidance performance and catecholamine metabolism.

Rats trained to bar-press to avoid shock in a non-discriminated, Sidman avoidance paradigm were injected intraperitoneally with either saline or 5 mg/kg of PCA twice daily for 7.5 days. When tested 48 hr later, the administration of PCA increased response rates in saline-pretreated but not PCA-pretreated rats, indicating the development of physiological tolerance. Complete recovery from tolerance was observed within 2 weeks after the termination of chronic drug treatment. The rates of disappearance of PCA from the brains of tolerant and non-tolerant rats were similar, suggesting that tolerance was not due to an altered disposition of the drug. The effects of acute and chronic PCA administration on the conversion of [³H]-tyrosine to [³H]-norepinephrine and [³H]-dopamine and in vivo tyrosine hydroxylase activity were measured in whole brains 15 min after PCA administration. The acute administration of PCA increased the formation of both catecholamines and increased in vivo tyrosine hydroxylase activity. However, neither of these effects was observed in tolerant rats challenged with PCA. These data suggest that tolerance to the facilitatory effect of PCA is associated with tolerance to the ability of the drug to increase the rate of synthesis of catecholamines.     

Effects of chronic phencyclidine on fixed-ratio responding: No relation to neurotransmitter receptor binding in rat cerebral cortex

The effects of chronic phencyclidine (3.2 mg/kg for 25 days) on responding maintained under a fixed-ratio 30 schedule of food presentation were studied in rats. Initially phencyclidine produced large decreases in the overall rate of responding. This decrease was due primarily to long pauses in responding and secondarily to a decrease in local rates of responding. Although tolerance developed to the rate-decreasing effects of phencyclidine in each subject, the extent and pattern of its development differed among the subjects. After the chronic drug regimen, the rats were sacrificed. Ligand binding to muscarinic cholinergic, opiate, adrenergic, and serotonergic receptors in cortex was then compared to that in rats which received saline with operant training, phencyclidine alone, or saline alone. Neither operant behavior alone, phencyclidine alone, nor the interaction of phencyclidine and operant behavior was found to alter binding to these receptors. The results indicate that behavioral tolerance develops to phencyclidine, but it is not accompanied by changes in binding to the receptors studied.     

Tolerance to the effects of daily nicotine on rat bar pressing behavior for water reinforcement

The effects of daily nicotine tartrate given IP in a dose of 0.25 mg/kg twice a day for 15 days was studied on bar pressing behavior for water reinforcement in the rat. A modified FR_{1 5} schedule was used. Nicotine injections caused an initial disruption of bar pressing behavior with rapid tolerance development. Antidiuretic hormone (5–10 units/kg, SC) administration also suppressed water drinking behavior after an initial latency in contrast to the effects of nicotine. It is concluded that tolerance occurs to daily nicotine administration.     

Antagonism of oxotremorine-induced behavioral suppression by antimuscarinic drugs

The effects of benztropine, scopolamine, atropine, methylatropine, amitriptyline, phencyclidine, and meperidine were determined alone and in conjunction with a behaviourally suppressing dose (0.05 mg/kg IM) of oxotremorine in pigeons responding under a multiple fixed-ratio 30 fixed-interval 5-min schedule of grain presentation. Benztropine, scopolamine, atropine, and amitriptyline antagonized the behavioral suppressing effects of oxotremorine at doses that alone decreased responding. In contrast, methylatropine, phencyclidine, and meperidine did not antagonize the effects of oxotremorine. These results suggest that benztropine, scopolamine, atropine, and amitriptyline decrease responding due to an action at central muscarinic receptors, whereas the behavioral effects of methylatropine, phenyclidine, and meperidine are due to actions other than at central muscarinic receptors.     

Amphetamine and the reward system: Evidence for tolerance and post-drug depression

Existing reports of tolerance to the behavioral effects of d-amphetamine are most parsimoniously interpreted as reflecting behavioral adaptation to the disruptive effects of the drug rather than physiological tolerance. The present study shows that physiological tolerance does develop to the facilitation of self-stimulation behavior which the drug produces. Rats were trained to bar-press for electrical stimulation of the medial forebrain bundle and tested for facilitation of responding following the administration of 0.25 or 0.50 mg/kg d-amphetamine. Testing was terminated for 4 days during which increasing doses (1.0–12.0 mg/kg) of the drug were given. 16 h after the last injection, the test doses (0.25 or 0.50 mg/kg) no longer produced facilitation of self-stimulation. In addition, testing on the following day with no further drug administration showed a depression of responding indicating depression of the sensitivity of the reward system of the brain.     

Task-dependent development of tolerance to scopolamine

Rats were chronically treated with once daily injections of either 0.5 mg/kg scopolamine hydrochloride or isotonic saline for 21 days. When spontaneous locomotor activity or acquisition of active avoidance in a two-way shuttle box were measured at 48 hours after the cessation of chronic treatment, no differences were observed between the two chronically treated groups. Tolerance to scopolamine's locomotor stimulatory effects was evident as the increase in locomotor activity following acute treatment was smaller in the group which had been chronically treated with scopolamine. On the other hand, acutely administered scopolamine facilitated the acquisition of active avoidance responding to an equal degree in both chronically treated groups. The reasons which may account for this task-dependent tolerance development to scopolamine are discussed.     

Mianserin as a discriminative stimulus in rats: asymmetrical cross-generalization with scopolamine

The present study was conducted to determine if the tetracyclic antidepressant mianserin could be established as a discrminative stimulus in rats. One group of rats was trained to discriminate mianserin (4.0 mg/kg, IP) from saline in a two-lever drug discrimination procedure, and a second group of rats was trained to discriminate the muscarinic cholinergic antagonist scopolamine (0.25 mg/kg, IP) from saline. Generalization testing with the training drugs yielded an ED₅₀ of 0.502 mg/kg for the mianserin-trained rats and an ED₅₀ of 0.048 mg/kg for the scopolamine-trained rats. Asymmetrical cross-generalization between mianserin and scopolamine was observed, because scopolamine produced mianserin-appropriate responding, but mianserin did not produce scopolamine-appropriate responding. This study is the first demonstration that rats can be trained to discriminate mianserin from saline and that antagonism of muscarinic cholinergic receptors is sufficient to produce mianserin-appropriate responding.     

Chronic amphetamine: Tolerance and reverse tolerance reflect different behavioral actions of the drug

Chronic administration of amphetamine (AMPH) has been reported to produce tolerance to the drug's behavioral effects in some paradigms (self-stimulation, discriminative stimulus, self-administration) and an enhanced effect or reverse tolerance when other behaviors are monitored (locomotor activity, stereotypy). The present study investigated whether the two phenomena are, in fact, related to the particular behavior monitored or reflect the marked differences in the injection regimens (1X vs. 3X daily injections) used to produce the phenomena. The effects of chronic AMPH administered once or three times daily on AMPH facilitation of self-stimulation responding and on the locomotor stimulant and stereotypy-producing effects of the drug were assessed. Regardless of the injection regimen used, chronic AMPH resulted in an enhancement of the locomotor stimulant effects of the drug as well as a more rapid onset and greater intensity of the stereotypy produced. In the self-stimulation paradigm, only the 3X daily regimen significantly reduced the effectiveness of a challenge dose of AMPH (tolerance), although the 1X regimen produced effects that were qualitatively similar but quantitatively less. Perhaps behavioral tasks in which tolerance develops reflect the mood-altering properties of the drug in humans whereas a process similar to reverse tolerance may underlie the increased susceptibility to psychoses elicited by the drug with repeated use.     

Interactions between morphine, scopolamine and nicotine: Schedule-controlled responding in rats

Functional interactions between drugs acting on either opioid or cholinergic systems have been demonstrated for both neurochemical and behavioral measures. This study used schedule-controlled responding and isobolographic analyses to examine interactions between the µ opioid receptor agonist morphine and the muscarinic acetylcholine receptor antagonist scopolamine as well as the nicotinic acetylcholine receptor agonist nicotine. In 8 rats responding under a fixed ratio 5 schedule of food presentation, morphine (3.2-10 mg/kg), scopolamine (0.032-1.0 mg/kg), and nicotine (0.1-1 mg/kg) each dose-dependently decreased responding. Acute injection of scopolamine shifted the morphine dose-response curved leftward and downward and acute injection of morphine shifted the scopolamine and nicotine dose-response curves leftward and downward. The interaction between morphine and nicotine was additive; however, the interaction between morphine and scopolamine was infra-additive or supra-additive, depending on whether scopolamine or morphine was administered first. These results provide quantitative evidence regarding potentially important interactions between drugs acting on either opioid or cholinergic systems, although these interactions are modest and appear to depend on the specific conditions of drug administration.     

Pharmacological and behavioral components of tolerance to LSD and mescaline in rats

A fixed-ratio schedule of water reinforcement (FR-10) was used to examine the relative contributions of pharmacological and behavioral mechanisms in the development of tolerance to the disruptive effects of LSD and mescaline in the rat. Rats treated daily with LSD or mescaline before operant testing developed tolerance to the impairment of responding, while rats treated daily after each session did not display tolerance when the drugs were administered before testing. These results indicate that behavioral compensatory mechanisms may be involved in the development of tolerance to the disruptive effects of LSD and mescaline on fixed-ratio (FR-10) performance.