Time-related facilitation and suppression of drinking by muscarinic anticholinergic drugs in water non-deprived rats

Effects of tertiary anticholinergic drugs, atropine (1.3, 2.5, 5 and 10 mg/kg, s.c.) and scopolamine (0.13, 0.25, 0.5 and 1 mg/kg, s.c.), and a quaternary anticholinergic drug, methylatropine (1.3, 2.5, 5 and 10 mg/kg, s.c.), on the drinking behavior were investigated in water non-deprived rats that were housed in a 12-hr light-dark situation (light period: 6:00-18:00) with a free access to food. Atropine, 5 mg/kg, and scopolamine, 0.13 and 0.25 mg/kg, administered at 12:00, significantly increased the drinking during the 12:00-18:00 period. Furthermore, lower to medium doses of atropine increased the drinking during the 18:00-6:00 period. In contrast, the drinking did not change during the 12:00-18:00 period, but decreased during the 18:00-6:00 period in a dose-dependent manner after administration of methylatropine at 12:00, whereas the drinking during the 18:00-24:00 period decreased in a dose-dependent manner when both the tertiary and quaternary drugs were administered at 18:00. However, the drinking during the 24:00-6:00 period increased in the rats that were administered atropine at the dose of 5 or 10 mg/kg at 18:00, while the drinking still decreased after methylatropine at the same time. The present results suggest that in water non-deprived rats, central muscarinic cholinergic blockade is effective for both increasing and decreasing drinking behavior, depending on the doses, when the drug is administered, and time span between the drug administration and the behavior observation. It is also suggested that peripheral cholinergic blockade monotonously suppresses the drinking behavior.     

The effects of muscarinic cholinergic blockade upon shock-elicited aggression

Graded dosages of atropine sulfate, atropine methyl nitrate, scopolamine hydrochloride, and scopolamine methyl nitrate were administered to rats and shock-elicited fighting frequencies determined. Central cholinergic blockade decreased fighting at appropriate dosages, but peripheral cholinergic blockade had little or no effect upon shock-elicited aggression. These result suggest that shock-elicited aggression is similar to other kinds of agonistic behavior (e.g., isolation-induced fighting and muricide) in that a central cholinergic system is apparently involved in its mediation.     

Role of cholinergic receptors in locomotion induced by scopolamine and oxotremorine-M

Mesopontine cholinergic neurons activate dopamine neurons important for reward-seeking and locomotor activity. The present studies tested whether cholinergic receptor blockade in the ventral tegmental area (VTA) altered locomotion induced by scopolamine (3 mg/kg i.p.) or by oxotremorine-M (0.1 microg bilaterally in the VTA). It was predicted that cholinergic blockers in the VTA would attenuate these cholinergic-induced locomotor increases. Locomotor activity was increased by scopolamine and oxotremorine-M administration in all treatments. When dihydro-beta-erythroidine (DHBE), a nicotinic receptor antagonist, was applied in VTA prior to oxotremorine-M, locomotion was reduced to slightly above saline baseline levels, but atropine, a muscarinic antagonist, had no effect. This suggests that the locomotor effect of oxotremorine-M at this dose was mediated mainly via nicotinic, not muscarinic, receptors. Intra-VTA injections of DHBE, however, did not attenuate scopolamine-induced locomotion indicating that scopolamine-induced locomotion is not mediated mainly via VTA cholinergic receptors. In mutant mice with a deletion in the M5 muscarinic receptor gene, scopolamine-induced locomotion was increased versus wild type mice after scopolamine injection. This suggests that the M5 receptor has an inhibitory effect on scopolamine-induced locomotion.     

Central cholinergic suppression of hyper-reactivity and aggression in septal-lesioned rats

Rats were made hyper-reactive by the mechanical placement of lesions in the septum. The systemic administration of a tertiary anticholinesterase agent (physostigmine) that readily penetrates to brain tissue suppressed the hyper-reactivity in a dose-related manner. This effect was not manifested by a quaternary anticholinesterase drug (neostigmine) that does not readily penetrate to brain tissue. Similarly, the effect of physostigmine was blocked by pretreatment with a tertiary cholinolytic drug (atropine) but not by pretreatment with a quaternary cholinolytic drug (methylatropine). These data, obtained from studies with septal-lesioned rats, are consistent with the proposition that hyper-reactive, aggressive behavior is suppressed by a centrally acting cholinergic system.     

The effects of amphetamine and scopolamine on adjunctive drinking and wheel-running in rats

Two groups of rats were exposed to a fixed-interval 90 s schedule of food reinforcement. One group had access to a drinking tube containing water and the second had access to a running wheel. Amphetamine (0.3–10.0 mg/kg) and scopolamine (0.1–3.0 mg/kg) were assessed for their effects on lever-pressing, adjunctive drinking and adjunctive wheel-running. Low to moderate doses of amphetamine increased overall rates of lever-pressing, whereas the highest dose decreased them. Scopolamine decreased overall lever-pressing rates in a dose-dependent manner. Both drugs changed the within-interval pattern of lever-pressing from one of increasing probability through the interval to almost constant probability throughout. Overall rates of adjunctive drinking and adjunctive wheel-running were decreased by amphetamine and scopolamine. Amphetamine failed to alter the within-interval patterns of either drinking or wheel-running in any substantial manner. The effect of scopolamine was to make the probabilities of each adjunctive behaviour more even through the interval. Although the two drugs had different actions, there was little difference in the way drinking and wheel-running were affected by each.     

Effects of psychotropic drugs on FI responding and adjunctive drinking in rats

Effects of d-amphetamine (AM), chlorpromazine (CPZ) and diazepam (DZ) on schedule controlled responding (lever-pressing) and adjunctive drinking under a fixed interval (FI) 1.5 min schedule of food reinforcement in rats were investigated. The drinking was measured with a drinkometer and a lickometer. AM 0.13–1.0 mg/kg SC increased the total responses, and decreased the total amount of drinking and licking counts dose-dependently. A marked response increase in the early portion (0–30 sec component of the FI) and mid portion (30–60 sec component), and decrease of the drinking in the mid portion and terminal portion (60–90 sec component occurred. CPZ 0.25–2.0 mg/kg SC decreased responses, drinking and licking in proportion with the doses. After CPZ, a response decrease in the mid and terminal portions was observed, but not in the early portion. Higher doses of CPZ decreased the drinking and licking in the whole range of the interval. A small dose of DZ (0.25 mg/kg SC) produced a significant response increase. Higher doses of DZ also increased responding, but the change was not significant. The drinking and licking were suppressed by DZ. A dose-related response increase in the mid portion was observed after DZ, but not in the early and terminal portions except after 0.25 mg/kg. Higher doses of DZ (more than 0.5 mg/kg) decreased drinking and licking throughout the whole range of the FI. The present results suggest that the interpellet distribution of responding, drinking and licking, as well as their total values, yield important information when assessing drug effects on FI responding and adjunctive drinking in rats.     

Drinking induced by parenteral injections of pilocarpine.

Parenteral (IP) injections of pilocarpine, in doses from 3.75 - 30 mg/kg, reliably produced drinking in water-satiated rats. This effect was not diminished by pretreatment with either centrally active (scopolaime, atropine) or peripherally active (methyl scopolamine, methyl atropine) cholinergic blocking agents, suggesting that pilocarpine does not induce drinking via a cholingergic meachnism. Repated injections of low doses, but not high doses, of pilocarpine augments drinking over trials.     

Comparative behavioral effects of several anticholinergic agents in rats

Summary The behavioral effects of JB 329 (Ditran) were compared with those of several anticholinergic agents in rats.The tertiary nitrogen-containing compounds (i.e. JB 329, JB 336, atropine, scopolamine) caused an increase in their spontaneous motor activity, decrease in food and water intake and an over-all decrease in the response in the various phases of the multiple schedules of reinforcement (i.e. shock avoidance, fixed-ratio water reinforcement, and time-out). The potency of JB 329 was similar to that of scopolamine and greater than that of JB 336. Atropine was 10 or more times less potent than scopolamine.The quaternary analogues of these drugs showed similar, though very slight, and sometimes reverse effects. The site of action of the tertiary compounds thus appears to be central.Supported by a General Research Support grant and a grant No. UI 00472 from the U. S. Public Health Service.     

Central cholinergic blockade of the conditioned avoidance response in rats

Summary The effects of arecoline, Tremorine and chlorpromazine were studied in conditioned rats in shuttlebox avoidance and lever-press avoidance procedures. Conditioned avoidance response (CAR) blockade produced by subtremor doses of arecoline and Tremorine was felt to result from central cholinergic stimulation since the effects of both drugs were prevented by the tertiary anticholinergic, atropine, but not by the quaternary agent, methantheline. In both avoidance procedures the observed CAR blockade produced by these cholinergics suggested not reduction in fear or anxiety drive, popularly ascribed to chlorpromazine, but rather heightened anxiety and indecision about appropriate performance for avoiding the impending danger of shock. The behavioral pattern of the trained animals under the influence of these cholinergic drugs was, in fact, remarkably similar to behavior of the unconditioned rats during the earlier stages of avoidance training. The possible implications of these findings are discussed.A portion of this paper was presented at the AAAS meetings held in Cleveland, Ohio, December, 1963. Supported in part by USPHS Grant MH 07767-01 and an equipment grant from the Smith, Kline, and French Foundation.Generously supplied by Abbott Laboratories, Chicago, Illinois.     

Recognition of muscarinic cholinergic receptors in human SK-N-SH neuroblastoma cells by quaternary and tertiary ligands is dependent upon temperature, cell integrity, and the presence of agonists

The recognition of muscarinic cholinergic receptors (mAChRs) in human SK-N-SH neuroblastoma cells by hydrophilic (quaternary) and lipophilic (tertiary) ligands has been examined. When quiescent cells were incubated at 37 degrees, the same maximum number of mAChRs was revealed by antagonists that possessed either a quaternary nitrogen, e.g., N-methylscopolamine (NMS) and N-methylquinuclidinyl benzilate, or a tertiary nitrogen, e.g., scopolamine and quinuclidinyl benzilate (QNB). If cells were incubated at 0 degree, the quaternary [3H]NMS labeled 15-20% fewer sites than the tertiary [3H]scopolamine; but upon warming to 37 degrees, these inaccessible sites also became labeled. This suggests that mAChRs are present at both cell surface and sequestered sites in this cell, and that an equilibrium exists between the two cellular compartments. In competition studies at 37 degrees, NMS detected a population of [3H]QNB-binding sites which exhibited a very low affinity for the quaternary antagonist. However, the sites were not evident when mAChRs were labeled with [3H]scopolamine, suggesting that factors other than the lipophilic nature of the probe are involved. Although mAChRs were equally accessible to charged and uncharged antagonists at 37 degrees, the quaternary agonist carbamoylcholine competed for the sites labeled by quaternary antagonists with a 10- to 29-fold higher affinity than those labeled by tertiary antagonists, whereas the tertiary agonist OXO-2 displaced all sites with an equal affinity. However, carbamoylcholine competed equally well for [3H]scopolamine-and [3H]NMS-binding sites in either hypotonic cell lysates at 37 degrees or in intact cells maintained at 0 degree. These results suggest that, at 37 degrees, agonists induce the sequestration of cell surface receptors into a lipophilic environment in which receptors become inaccessible to quaternary, but not tertiary, ligands. Addition of NMS inhibited the stimulation of phosphoinositide hydrolysis elicited by either carbamoylcholine or OXO-2. The Ki values were similar for both agonists. It is concluded that mAChRs in SK-N-SH cells cycle between cell surface and sequestered sites. At 37 degrees, this cycling is rapid and all receptors have access to the cell surface compartment, whereas at 0 degree, receptor translocation is prevented and a population of sequestered mAChRs is detected. When cells are exposed to an agonist at 37 degrees, the equilibrium shifts such that more mAChRs are found in a sequestered cell compartment that is inaccessible to quaternary ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of muscarinic receptors mediating second messenger responses in the rabbit aorta

The second messenger response of cultured smooth muscle and endothelial cells of the rabbit aorta to acetylcholine (ACh) was investigated. ACh induced a concentration-dependent accumulation of inositol-monophosphate (InsP1) in the smooth muscle cells and a concentration-dependent reduction in basal production of adenosine 3',5'-cyclic monophosphate (cAMP) in the endothelial cells. Atropine, scopolamine and nitric oxide (NO) inhibited the ACh-induced accumulation of InsP1. The IC50 values were 55 +/- 4.3 nM, 81.2 +/- 6.5 microM and 13.3 +/- 3.5 microM, respectively. On the other hand, the inhibition of reduction in basal production of cAMP was inhibited by scopolamine (IC50 = 55.3 +/- 4.3 nM) and atropine (IC50 = 63.2 +/- 5.2 microM). Pirenzepine inhibited both the ACh-induced accumulation of InsP1 (IC50 = 1.5 +/- 0.01 microM) and reduction of basal production of cAMP (IC50 = 812 +/- 33.5 nM). However, unlike scopolamine or atropine, the M1-selective ligand was not selective to either the endothelial or smooth muscle receptors. These results demonstrate for the first time the second messenger response of the action of ACh on the endothelial muscarinic receptors and the inhibition of InsP1 formation by NO. In addition, the results also support our earlier findings that the muscarinic receptors in the endothelium and smooth muscle of the rabbit aorta can be differentiated by atropine and scopolamine, namely, the endothelial receptors have high affinity for scopolamine but extremely low affinity for atropine whilst the reverse holds true for the smooth muscle receptors.     

Atropine,scopolamine and hippocampal lesion effects on alternation performance of rats

Interactions between effects of hippocampal lesions and cholinergic blocking agents were examined using two behavioral tasks, delayed spatial alternation and go-no go temporal alternation. Atrophine and scopolamine were administered to control rats and to rats with lesions in three areas of the hippocampal formation. Large hippocampal lesions disrupted performance on both tasks as did administration of atrophine or scopolamine to control rats and to rats with lesions in restricted areas of the hippocampus. However, the effects of atropine and scopolamine on performance of rats with large lesions involving all regions of the hippocampal formation depended upon the alternation task used. Atropine and scopolamine disrupted delayed spatial alternation performance of all groups tested, but atropine failed to significantly disrupt go-no go alternation performance of the group with large unrestricted hippocampal lesions. Both the size of the lesion and the behavioral task were found to be important determinants of the effects of cholinergic blockers on the performance of rats with hippocampal lesions.     

Central cholinergic interactions in somato-vegetative reflexes

In cats lightly anaesthetized with urethane (600 mg/kg, i.p.), electrical stimulation of the sciatic nerve elicited frequency-dependent pressor reflexes and contractions of the nictitating membrane. Administration of oxotremorine (0.2 mg/kg, i.v.) or physostigmine (0.5 mg/kg, i.p.) resulted in depression of the pressor reflexes. At the same time, physostigmine enhanced the reflex contractions of the nictitating membrane, while oxotremorine induced sustained contraction of the latter. All these effects were antagonized by the tertiary amine scopolamine, but not by the quaternary atropine methylbromi.The results point to a role of central cholinergic mechanisms in the integration of somato-vegetative reflexes, and give evidence that the sympathetic driving of different effectors is not uniformly organized by the central nervous system.     

Changes in sensitivity to operant effects of dopaminergic and cholinergic agents following morphine withdrawal in rats

Rats trained to bar-press on either a FI 15 sec or FR 30 schedule for water reinforcement were administered various doses of appomorphine, haloperidol, pilocarpine and scopolamine both before and 1--4 months after a 3-day period of continuous morphine administration. All drugs monotonically depressed response rates on both schedules with increasing dose. Chronic morphrine administration produced a persistent increase in sensitivity to apomorphine and pilocarpine and a persistent decrease in sensitivity to haloperidol and scopolamine. The results suggest that both dopaminergic and cholinergic supersensitivity are likely to be involved in protracted abstinence phenomena.     

Modulation of the aversive qualities of shock through a central inhibitory cholinergic system in the rat

Evidence has been supplied which suggests that a central inhibitory cholinergic (i.e., muscarinic) system may be involved in modulating the aversive qualities of electric shock in the rat. Central cholinergic stimulation via the administration of pilocarpine or arecoline elevated the threshold for grid shock, while central acting anticholinergics (i.e., scopolamine and atropine) produced decrements in the threshold. Peripheral acting anticholinergics (e.g., methyl scopolamine, methyl atropine) were less potent than central acting drugs given in equivalent doses, while peripheral cholinergic stimulants (i.e., neostigmine, carbachol) were inactive. In addition, only the central acting stimulant pilocarpine, and not carbachol, was able to block the decrements noted in response to scopolamine hydrobromide administration. Finally, only arecoline, and not nicotine, was able to elevate the aversive threshold indicating that muscarinic receptor sites are probably involved in mediating the effects of central cholinergic stimulants.     

A cholinergic link in the centrally mediated actions of angiotensin II

This study was designed to determine the role of brain cholinergic mechanisms in the centrally mediated blood pressure and drinking responses to angiotensin II (ANG II). Atropine (10 mg/kg, IV), but not methyl atropine (8 mg/kg, IV), significantly inhibited drinking and BP responses to ANG II intracerebroventricularly (ICV). BP responses to IV ANG II were not inhibited. Both atropine (20 mg/kg, IP) and methyl atropine (20 mg/kg, IP) inhibited cellular-dehydration thirst induced by injection of hypertonic saline. Hemicholinium (80 μg/kg, ICV) had similar effects to atropine on BP and drinking responses to ICV ANG II. Phentolamine (0.5 mg/kg, IV) had a small but significant inhibitory effect on the drinking but not the BP response to ANG II ICV. It is concluded that brain cholinergic mechanisms contribute both to the drinking and pressor response to ICV ANG II, and to the drinking response to cellular-dehydration thirst. There may also be a peripheral cholinergic component to cellular dehydration but not ICV ANG II-induced thirst.     

Cholinergic muscarinic effects on insulin release in mice

Insulin secretion and blood glucose homeostasis were studied in mice following administration of cholinergic agonists, antagonists and other possible modifiers of cholinergic insulin secretory mechanisms. It was observed that administration to mice of the cholinergic agonists acetylcholine, carbachol and pilocarpine resulted in an increase in plasma immuno-reactive insulin levels accompanied by a significant fall in blood glucose levels. Nicotine had no effect. Carbachol was found to enhance insulin release in a dose-dependent manner. Muscarinic blockade by atropine or methylatropine totally suppressed carbachol-induced insulin secretion. No blocking effect was accomplished by beta-adrenoceptor blockade. Glucose-induced insulin secretion was not affected by atropine in normal non-fasted mice. In mice fasted for 24 h, however, the insulin response to glucose was impaired by atropine suggesting that the nutritional state is important for cholinergic modulation of glucose-induced insulin response. Pretreatment of animals with the glycogenolytic hydrolase, acid amyloglucosidase, enhanced tolbutamide-induced insulin release but did not influence insulin secretion stimulated by carbachol. Pretreatment with the monoamine oxidase inhibitor, pargyline, plus L-dopa, leading to an intracellular accumulation of dopamine in the insulin cells, totally suppressed carbachol-induced insulin release. It is suggested that, in mice, cholinergic stimulation promotes insulin secretion through activation of muscarinic receptors on the insulin cell. Blockade of these receptors does not influence glucose-stimulated insulin release in the non-fasting state, but may impair the insulin response to glucose after fasting. Cholinergic stimulation of insulin release is inhibited after L-dopa-induced accumulation of dopamine in the insulin cells. In contrast to tolbutamide-induced insulin release cholinergic insulin release is not dependent on acid amyloglucosidase activity.     

Differential effect of selective beta 1 and nonselective beta-adrenoceptor blockade on epinephrine and atropine response in normal humans.

Sympathetic stimulation with epinephrine (EPI) combined with parasympathetic blockade with atropine was studied in 10 healthy volunteers premedicated with placebo or three different beta-adrenoceptor blockers: atenolol (62.5 micrograms/kg, beta 1-selective), propranolol (62.5 micrograms/kg, nonselective), and pindolol (7.5 micrograms/kg, nonselective with intrinsic sympathomimetic activity, ISA). EPI infusion (0.06 microgram/kg/min) after placebo increased heart rate (HR) and systolic blood pressure (SBP) and decreased diastolic BP (DBP). Pretreatment with atenolol reduced the HR increase, and caused similar changes in BP. In contrast, pretreatment with propranolol and pindolol decreased HR and increased BP. Combined EPI and atropine (15 micrograms/kg) after placebo increased HR by 40% without causing BP changes. Similar HR changes were observed after administration of all beta-adrenoceptor blockers, but whereas a marked pressor response was observed after propranolol and pindolol a blunted response was observed after atenolol. Propranolol and pindolol reduced myocardial oxygen demand estimated by the HR x BP product after EPI, but this response was abolished by atropine. Serum potassium decreased from 3.9 +/- 0.2 to 3.2 +/- 0.3 mM after EPi and atropine. This effect was less after atenolol, and potassium increased after premedication with propranolol and pindolol. Our results show that nonselective beta-adrenoceptor blockade has a favorable effect on potassium homeostasis and oxygen demand parameters during EPI infusion but causes a marked pressor response, contrary to a beta 1-selective agent, during combined sympathetic stimulation and parasympathetic blockade. They also highlight the importance of the vasodilator cholinergic system as a defense mechanism in such situations.     

The nature of the scheduled reinforcer and adjunctive drinking in nondeprived rhesus monkeys

Adjunctive drinking was generated in three free-feeding rhesus monkeys by the contingent and intermittent delivery of flavored pellets. The amount of drinking generated was greater when pellet availability was restricted under fixed-interval schedules compared to a massed-reinforcer control condition. The volume of water consumed depended upon the fixed-interval of pellet delivery (FI 180 sec to FI 1800 sec). Peak amounts of water consumed ranged from 532 ml to 650 ml during the 2 hr sessions and the schedule which generated the most drinking was either FI 420 or FI 600 sec, across monkeys. Variables which did not appear to influence the amount of drinking generated within the session were the amount of water consumed outside the session, the rates of responding maintained by pellet delivery and the pattern of responding for pellet delivery. However, when either cocaine or diazepam was the scheduled reinforcer, these same free-feeding monkeys did not engage in adjunctive drinking. The ability of cocaine and diazepam to generate adjunctive drinking was determined first by gradually decreasing the frequency of pellet delivery while keeping drug delivery constant using a second-order schedule of pellet delivery [FR n(FI 300 sec: drug delivery) with n ranging from 1 to 6]. Second, a range of drug doses was tested under a FI 300 sec schedule (cocaine: 0.01-0.3 mg/kg/injection; diazepam: 0.01-0.56 mg/kg/injection). These results suggest that there may be some restriction on the generation of adjunctive drinking depending upon the nature of the scheduled reinforcer.     

Differential cholinergic influences on the immobility response in various strains of domestic fowl

A series of five experiments examined the effects of two anticholinergic drugs, atropine and scopolamine, on the duration of tonic immobility (TI) and susceptibility to the TI response in both Production Red and White Leghorn chickens (Gallus gallus), in an attempt to resolve previous contradictory findings about the effects of cholinergic manipulations on tonic immobility. These two anticholinergic drugs significantly reduced the duration of TI and, therefore, supported the conclusion that cholinergic systems are involved with the immobility response. However, the effects of these drugs on TI differed depending on the age, strain, local population, and handling experience of the individual birds.