Conformational changes in muscarinic receptors may produce diminished cholinergic neurotransmission and memory deficits in aged rats

Both clinical and laboratory studies suggest that age-related memory deficits may be due, at least in part, to disturbances in muscarinic acetylcholine (mAChR) receptors. In order to further evaluate this premise, the present studies examined the electrophysiological responses rates of hippocampal pyramidal cells to iontophoretically applied ACh in young, middle-age and aged animals. The relationship between age and muscarinic agonist and antagonist binding in the hippocampus was also examined. In addition, possible age-related changes in receptor-effector coupling were assessed by determining calmodulin levels and the activities of phospholipid methyltransferase I and II. Analysis of electrophysiological data showed selective age-related decrements in the ability of ACh to alter burst rate but not simple spike rate. These age-related decreases in the efficacy of ACh to increase burst rate were not paralleled by decreases in mAChR density as assessed by ³H-QNB binding, but they were temporally paralleled by age-related changes in the ability of oxotremorine to inhibit ³H-QNB binding. In the young animals, the resultant Hill coefficients derived from these analyses approached 1, while in the middle and old aged animals, the Hill coefficients deviated significantly from 1, indicating the possible existence of 2 or more receptor states with differential affinity for oxotremorine in the 2 older age groups. When carbamylcholine was used to inhibit ³H-QNB, these complex binding patterns were seen even in the young, since carbamylcholine induces conformational/orientational changes in the mAChR while oxotremorine does not. It is suggested that declines in mnemonic ability that have been reported previously, parallel the age-related conformation/orientational changes observed in the mAChR since these changes result in a receptor that is “neurophysiologically desensitized.”     

Reduction of oxotremorine-induced analgesia after chronic but not acute restraint stress

The analgesic response (tail-flick latency) induced by the muscarinic cholinergic agonist oxotremorine was investigated in DBA/2 mice exposed to acute (a single 2 h session) and chronic (2 h once daily for 10 days) restraint stress. While a single exposure to stress did not influence the antinociceptive effects of the cholinergic agonist, chronic stress induced a clear-cut reduction of the oxotremorine-induced analgesia. The results show an involvement of cholinergic mechanisms in the adaptive modulation of nociception after chronic stressful events.     

Cholinergic effects on fear conditioning II: nicotinic and muscarinic modulations of atropine-induced disruption of the degraded contingency effect

Rationale In a companion study (Carnicella et al., 2005), we showed that the muscarinic antagonist atropine, when administered after extensive training during both conditioning and testing, affected neither cued nor contextual fear memories when both of them did not compete for the control of the overt behaviour. In contrast, atropine altered the degraded contingency effect (DCE), that is, the processes by which contextual fear memory competes with the cued one for the control of the conditioned response. Atropine-induced disruption of the DCE was fully reversed by the administration of the anticholinesterase inhibitor physostigmine, which suggests a direct cholinergic implication.Objective The present series of experiments was conducted in order to define more precisely the involvement of the cholinergic system in such an effect.Methods Oxotremorine (0.0, 0.0075, 0.015, or 0.03 mg/kg), pilocarpine (0.0, 0.3, 1, or 3 mg/kg), xanomeline (0.0, 2.5, 5.0, 10.0 or 20.0 mg/kg) and nicotine (0.0, 0.1, 0.2, or 0.4 mg/kg) were tested for reversal of the atropine-induced alteration of the DCE.Results Oxotremorine and pilocarpine did not reverse the atropine-induced alteration of the DCE. In contrast, xanomeline and nicotine reversed the effect of atropine on the DCE.Conclusion The present series of experiments reveals complex pharmacological interactions within the cholinergic system when cued and contextual fear memories interact. Results are discussed in this connection and with regard to the relation between the properties of cholinergic agonists and their therapeutic values.     

Muscarinic activity of some secondary and tertiary amines and quaternary ammonium salts structurally related to oxotremorine

A number of secondary and tertiary amines as well as quaternary ammonium compounds, obtained by structural modification of the amino group of oxotremorine (1) and its acetamide analogue (14), were investigated for muscarinic and antimuscarinic activity in vivo and in vitro. For the quaternary ammonium analogues, decrease of in vitro muscarinic potency is well correlated with increase of the size of the quaternary ammonium group, as estimated from increments in apparent molal volumes. A similar decrease of muscarinic potency with increasing substitution at the nitrogen atom is generally observed for the secondary and tertiary amines. For the latter the reduction in muscarinic activity appears to be due to loss of efficacy, since the higher homologues are partial agonists or antagonists. There is a highly significant correlation between muscarinic activity in vitro and central tremorogenic activity of the tertiary amines.     

Effect of oxotremorine on homovanillic acid concentration in the striatum of the rat

The effects of oxotremorine and other cholinomimetics on the concentration of homovanillic acid (HVA) in the striatum was studied in rats. Oxotremorine, arecoline, nicotine, and physostigmine increased the concentration of striatal HVA. The increase in striatal HVA induced by oxotremorine was counteracted by atropine but it was unaffected by atropine methylbromide, mecamylamine or hemicholinium-3. These results suggest that oxotremorine increases the concentration of HVA in the striatum of the rat via a direct action on muscarinic receptors in dopamine neurons.     

Amantadine antagonism of oxotremorine effects

Summary Administration of oxotremorine to mice produced centrally-mediated effects, such as catalepsy and tremor, and peripheral muscarinic actions, such as diarrhoea and lachrymation. Pretreatment with amantadine (25–200 mg/kg) prevented these oxotremorine-induced effects in mice. Catalepsy was most susceptible and tremor most resistant to the administration of amantadine. The possible mechanisms involved are discussed.Our results validate the use of the oxotremorine model in the search for novel antiparkinsonian drugs.     

Genetical differences in sensitivity to tremorine and oxotremorine in mice

Male mice from 14 strains were injected i.p. with tremorine (3.0 mg/kg) or oxotremorine (0.15 or 0.1 mg/kg). Large inter-strain differences in the degree and duration of the subsequent hypothermia were noted. 2 strains, BALB/c and Simpson, were particularly sensitive to the hypothermic effect of oxotremorine. The offspring from a cross between BALB/c and Simpson were less sensitive than the parental strains, suggesting genetic complementation. A set of 7 recombinant inbred (RI) lines derived from strains C57BL and BALB/c were tested with oxotremorine. 5 RI lines resembled strain C57BL in their response and 2 RI lines resembled strain BALB/c. It was concluded that strains C57BL and BALB/c differ at a gene which has a major effect on the response to oxotremorine.     

Behavioral and physiological effects associated with changes in muscarinic receptors following administration of an irreversible cholinergic agonist (BM 123)

Previous work in our laboratory has shown that the aziridinium ion of BM 123 (N-[4(2-chloroethylmethylamino)-2-butynyl]-2 pyrrolidone) is a potent and selective muscarinic agonist and binds irreversibly to muscarinic receptors (mAChR). The present series of experiments was designed to study the effects of BM 123 on behavioral and physiological variables known to be sensitive to manipulations of the cholinergic neurotransmitter system. BM 123 was injected into the tail vein of Sprague-Dawley rats, reducing mAChR to approximately 10% of normal as judged by [³H](–)QNB binding. Oxotremorine was injected IV for purposes of comparison. Behavioral and physiological variables were measured daily for 26 days. Physiological variables (e.g., tremor, chromodacryorrhea, salivation, and temperature) showed effects in less than 5 min after injection and returned to their pretreatment baselines within minutes. Nociceptive thresholds, dependent on sensory-perceptual processes, showed peak changes of approximately +230% and returned to normal within hours. Motoric responses, i.e., drinking and general activity, recovered in 3–4 days. Learned responses and those requiring temporal discrimination took 8–11 days to recover and were the only responses paralleling the return of the mAChRs to their normal levels. Changes elicited by oxotremorine recovered more rapidly than those elicited by BM 123. The results suggest that the different variables measured are dependent on different densities of functional receptors. Implications for a theoretical model are discussed.     

Attenuation of cholinergic analgesia by nifedipine

Nociception was tested in mice receiving oxotremorine or physostigmine either after the dihydropyridine calcium channel blocker nifedipine or the non-calcium antagonist vasodilator hydralazine. Nifedipine did not change the reaction time to thermal stimulation (tail-flick test), but attenuated the prolonging action on tail-flick latencies exerted by the two cholinomimetic agents. Hydralazine had no effect alone nor modified the action of cholinomimetics. The results suggest that attenuation of cholinergic analgesia by nifedipine might be related to not yet defined neuronal changes produced by calcium channel blockade, but changes in the pharmacokinetics of oxotremorine and physostigmine cannot be ruled out.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.