Interaction between GABA agonists and the cholinergic muscarinic system in rat corpus striatum

Chronic treatment with γ-acetylenic GABA(GAG), a GABA transaminase inhibitor, causes an increase in striatal dopamine receptor binding and function in rat brain suggesting that extrapyramidal side effects may accompany the use of these agents. In the present investigation it was found that chronic administration of THIP, a direct acting GABA receptor agonist, induced a similar increase in dopamine receptor binding. In addition, co-administration of atropine, a cholinergic muscarinic antagonist, was found to completely prevent the GABA-induced dopamine receptor increase. Furthermore, high affinity choline uptake, a measure of cholinergic activity, in striatal synaptosomes is enhanced after the acute administration of either GAG or THIP. Taken together these results support the notion of an interaction between dopaminergic, cholinergic and GABA-ergic neurons in the extrapyramidal system and indicate that co-administration of an anticholinergic agent may be of benefit in preventing the extrapyramidal side effects which may accompany the use of GABAergic agonists.     

Clozapine: selective labeling of sites resembling 5HT6 serotonin receptors may reflect psychoactive profile.

BACKGROUND: Clozapine, the classic atypical neuroleptic, exerts therapeutic actions in schizophrenic patients unresponsive to most neuroleptics. Clozapine interacts with numerous neurotransmitter receptors, and selective actions at novel subtypes of dopamine and serotonin receptors have been proposed to explain clozapine''s unique psychotropic effects. To identify sites with which clozapine preferentially interacts in a therapeutic setting, we have characterized clozapine binding to brain membranes. MATERIALS AND METHODS: [3H]Clozapine binding was examined in rat brain membranes as well as cloned-expressed 5-HT6 serotonin receptors. RESULTS: [3H]Clozapine binds with low nanomolar affinity to two distinct sites. One reflects muscarinic receptors consistent with the drug''s anticholinergic actions. The drug competition profile of the second site most closely resembles 5HT6 serotonin receptors, though serotonin itself displays low affinity. [3H]Clozapine binding levels are similar in all brain regions examined with no concentration in the corpus striatum. CONCLUSIONS: Besides muscarinic receptors, clozapine primarily labels sites with properties resembling 5HT6 serotonin receptors. If this is also the site with which clozapine principally interacts in intact human brain, it may account for the unique beneficial actions of clozapine and other atypical neuroleptics, and provide a molecular target for developing new, safer, and more effective agents.     

Discovery of novel conformationally constrained tropane-based biaryl and arylacetylene ligands as potent and selective norepinephrine transporter inhibitors and potential antidepressants

To further explore the structure-activity relationships of conformationally constrained tropanes, a number of new biaryl and arylacetylene analogs were designed and synthesized. Some of these compounds such as 3a-b, 3d, 3f-h, 5b, and 7g were found to be highly potent and selective or mixed norepinephrine transporter (NET) inhibitors with Ki values of 0.8-9.4 nM. Moreover, all of these compounds display weak to extremely weak muscarinic receptor binding affinity, indicating that as potential antidepressants, they may overcome certain side effects that are of concern with other antidepressants, which are thought to be mediated by their anticholinergic properties.     

Dopamine D3 receptor binding by D3 agonist 7-OH-DPAT (7-hydroxy-dipropylaminotetralin) and antipsychotic drugs measured ex vivo by quantitative autoradiography

Because the dopamine D3 receptor is primarily expressed in regions of the limbic system of brain, it was proposed that it may represent a target for antipsychotic drugs that is free of extrapyramidal side effects. An ex vivo receptor binding technique employing [3H]7-OH-DPAT was used to evaluate in vivo occupancy of dopamine D3 receptors in the rat nucleus accumbens by selective D3 agonist 7-OH-DPAT (7-hydroxy-dipropylaminotetralin) and various antipsychotic drugs. With an ID50 value of 0.07 mg/kg, the selective D3 agonist (+)-7-OH-DPAT had the most potent inhibitory effect on ex vivo binding of [3H]7-OH-DPAT among all drugs tested. Clinical doses of phenothiazine drugs, such as chlorpromazine and levomepromazine, induce binding to D3 receptors in vivo, while atypical antipsychotic drugs, such as clozapine, pimozide, and sulpiride, are very weak in inhibiting ex vivo binding of [3H]7-OH-DPAT, indicating that the role of D3 receptors as targets of antipsychotic drugs free of extrapyramidal side effects may not be important.     

H1 antihistamines interact with central sigma receptors

Several antihistamines were evaluated for their ability to interact with sigma, muscarinic and histaminic H1 binding sites in rat brain preparations. All of the antihistamines were able to interact with the sigma site, as well as the other two sites. In addition, tripelennamine was found to elicit sigma-like behaviors when administered to rats. This affinity for the sigma site suggests that the compounds may elicit some of their undesirable CNS side effects via this interaction.     

A spin label study of the membrane effect of various psychoactive drugs in human erythrocytes

The effect of psychoactive agents with different clinical actions: three sedative neuroleptics (trifluoperazine, alimemazine tartrate, chlorpromazine), an anticholinergic agent (trihexyphenidyl hydrochloride), two tricyclic antidepressants (imipramine, desipramine) and lithium carbonate on the rotational correlation frequency (V⁺) of the spin label 16NS has been comparatively investigated in whole human erythrocytes. V⁺ was about 40% increased by the three neuroleptics, the anticholinergic agent and the antidepressant molecules at 0.2mM. By contrast, lithium did not induce any significant change in V⁺ at the same concentrations. It can be suggested that the increase in “membrane fluidity”, observed with a wide variety of drugs, is a non specific effect, unrelated to the psychotropic action, that can be ascribed to the amphiphilic properties of the tested drugs.     

Identification of a butyrophenone analog as a potential atypical antipsychotic agent: 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one

The synthesis and exploration of novel butyrophenones have led to the identification of a diazepane analogue of haloperidol, 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one (compound 13) with an interesting multireceptor binding profile. Compound 13 was evaluated for its binding affinities at DA subtype receptors, 5HT subtype receptors, H-1, M-1 receptors and at NET, DAT, and SERT transporters. At each of these receptors, compound 13 was equipotent or better than several of the standards currently in use. In in vivo mouse and rat models to evaluate its efficacy and propensity to elicit catalepsy and hence EPS in humans, compound 13 showed similar efficacy as clozapine and did not produce catalepsy at five times its ED(50) value.     

Comparative neurochemical changes associated with chronic administration of typical and atypical neuroleptics: implications in tardive dyskinesia

An important goal of current neuroleptic research is to develop antipsychotic compounds with the low incidence of extrapyramidal side effects. The therapeutic success and less side-effect of atypical anti-psychotics such as clozapine and risperidone has focused the attention on the role of receptor systems other than dopaminergic system in the pathophysiology of neuroleptics-associated extrapyramidal side effects. The present study compares the effect of chronic administration of typical and atypical antipsychotics on neurochemical profile in rat forebrain. The study was planned to study changes in extracellular levels of norepinephrine, dopamine and serotonin in forebrain region of brain and tried to correlate them with hyperkinetic motor activities (vacuous chewing movements (VCM's), tongue protrusions and facial jerking) in rats, hall mark of chronic extrapyramidal side-effect of neuroleptic therapy tardive dyskinesia. Chronic administration of haloperidol (1 mg/kg) and chlorpromazine (5 mg/kg) resulted in significant increase in orofacial hyperkinetic movements where as clozapine and risperidone showed less significant increase in orofacial hyperkinetic movements as compared to control. There were also significant decrease in the extracellular levels of neurotransmitters dopamine, norepinephrine and serotonin in fore-brain as measured by HPLC/ED after chronic administration of haloperidol and chlorpromazine. Chronic administration of atypical neuroleptics clozapine and risperidone resulted in the decrease in extracellular concentration of dopamine and norepinephrine but the effect was less significant as compared to typical drugs. However, treatment with atypical neuroleptics resulted in 3 fold increase in serotonin levels as compared to forebrain of control rats. Typical and atypical neuroleptics showed varying effects on neurotransmitters, especially serotonin which may account for the difference in their profile of side effects (Tardive dyskinesia).     

The binding of [3H]AF-DX 384 is reduced in the caudate-putamen of subjects with schizophrenia.

Clinical studies of cholinergic pharmacotherapy, together with the putative role of the muscarinic receptor system in the neurophysiology of human behavior, support a possible muscarinic cholinergic involvement in schizophrenia. The present study has measured the density of [3H]AF-DX 384 labelled receptors (muscarinic M2 and M4) in the caudate-putamen, obtained at autopsy, from 19 subjects who had schizophrenia, and 20 subjects who did not have schizophrenia. [3H]AF-DX 384 binding was reduced in caudate-putamen from schizophrenic subjects (104 +/- 10.3 vs 145 +/- 901 fmol mg(-1) TE; mean +/- s.e.; p = 0.007). Preliminary analysis of patient drug data as well as rat studies suggest that the reduced [3H]AF-DX 384 binding in caudate-putamen of schizophrenic subjects is not wholly due to antipsychotic drug treatment, or anticholinergic medication for the treatment of extrapyramidal effects. These data suggest that the muscarinic cholinergic system may be involved in the pathology of schizophrenia.     

Differential Effect of Subchronic Treatment with Various Neuroleptic Agents on Serotonin2 Receptors in Rat Cerebral Cortex

In addition to antidepressant drugs, some neuroleptic (NL) drugs reduce serotonin2 (5-HT2) receptor binding sites after chronic administration. The present study was undertaken to characterize further this property of NL drugs. Scatchard analysis of [3H]spiperone binding in rat cerebral cortex revealed that 21-day treatment with chlorpromazine (CPZ), cis-flupenthixol, and thioridazine reduced 5-HT2 radioligand binding density by 60, 27, and 18%, respectively. The more selective dopamine-D2 antagonists haloperidol and sulpiride were totally ineffective in this regard. No reduction in 5-HT2 ligand binding sites occurred after 1 day of treatment with CPZ but 3-days of treatment was effective and this reduction persisted, although diminished, for at least 72 h after the last injection. cis-Flupenthixol and d-butaclamol were also effective after 3 days of treatment but trans-flupenthixol and l-butaclamol were not, indicating stereo-specificity of the response mechanism. Female rats showed the same response to CPZ as did male rats. Central 5,7-dihydroxytryptamine-induced lesions of 5-HT neurons demonstrated that intact 5-HT neurons were not required for the reduction of 5-HT2 receptor ligand binding by CPZ. Since CPZ has high affinity for many receptors, including alpha 1, histamine1, and muscarinic receptors, the role of these effects in producing 5-HT2 receptor down-regulation was considered by studying the effects of prazosin, atropine, and pyrilamine administration on 5-HT2 radioligand binding. Results indicate that no one of these actions appears to account for the down-regulation of 5-HT2 receptors by CPZ. Several of these effects, in combination, or some unique mechanism, may be involved.     

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.     

Multiple actions of phencyclidine: discriminant structure-activity relationships from molecular conformations and assay conditions

A major approach to the elucidation of the mechanisms by which phencyclidine (PCP) elicits the varied and complex responses observed in biological systems consists of comparisons of the pharmacological profiles of PCP derivatives with those of drugs for which the mechanism of action is better understood. Such studies depend on the definition of discriminant structure-activity relationships and on comparisons of rank orders of potency for the actions of PCP derivatives. Using the muscarinic cholinergic system in brain and the potassium ion channels in cardiac muscle as targets for PCP derivatives, we review the elucidation of the structural determinants for PCP recognition at muscarinic receptors, and the dramatic effect of assay conditions on the rank order of potency for the action of PCP derivatives at intracellular sites. The structural and physicochemical considerations illustrated here must be considered basic to the establishment of any assay aimed at the elucidation of the mechanism of action of PCP.     

Antipsychotic-Like Effect of the Muscarinic Acetylcholine Receptor Agonist BuTAC in Non-Human Primates

Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic receptors have been suggested as possible future targets for the treatment of schizophrenia and drug abuse. The muscarinic ligand (5R,6R)-6-(3-butylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (BuTAC) exhibits high affinity for muscarinic receptors with no or substantially less affinity for a large number of other receptors and binding sites, including the dopamine receptors and the dopamine transporter. In the present study, we wanted to examine the possible antipsychotic-like effects of BuTAC in primates. To this end, we investigated the effects of BuTAC on d-amphetamine-induced behaviour in antipsychotic-naive Cebus paella monkeys. Possible adverse events of BuTAC, were evaluated in the same monkeys as well as in monkeys sensitized to antipsychotic-induced extrapyramidal side effects. The present data suggests that, the muscarinic receptor ligand BuTAC exhibits antipsychotic-like behaviour in primates. The behavioural data of BuTAC as well as the new biochemical data further substantiate the rationale for the use of muscarinic M₁/M₂/M₄-preferring receptor agonists as novel pharmacological tools in the treatment of schizophrenia.     

The rebound excitation triggered by anticholinergic drugs from ovine pyloric antrum, small bowel and gallbladder.

The effect of anticholinergic drugs on gastrointestinal motility is complex and incompletely recognized. Accordingly, in 6 adult sheep bipolar electrodes and strain gage force transducers were surgically attached to the antral, small intestinal and gallbladder wall at the serosal side. During chronic experiments the myoelectric and mechanical recordings were performed in fasted and non-fasted animals before and after various doses of hexamethonium, atropine and pirenzepine given intravenously. Hexamethonium administration triggered rebound excitation after an inhibitory period almost in all the recording sites. Administration of atropine and pirenzepine evoked these secondary contractions mostly in the small intestine and gallbladder. No rebounds were observed when the anticholinergic drugs were given during feeding. In fasted animals, rebound excitation arrived later but more frequently than in non-fasted animals. The excitatory changes were dose-dependent. In the gallbladder, these values were lower than in the small intestine. The frequency of the recurrent pattern was dependent upon the dose of the anticholinergic drug used. It is concluded that nicotinic receptors are more important than muscarinic receptors in the initiation of the rebound excitation in pyloric antrum while in the small bowel and gallbladder the role of both cholinergic receptors is similar. The anticholinergic drugs should be used with caution in all these clinical situations, where the enhancement of gastrointestinal motility must be avoided.     

The neuropharmacology of emesis: the role of receptors in neuromodulation of nausea and vomiting

The basic pharmacological mechanisms involved in mediating nausea and vomiting are still poorly understood. Several classes of drugs have been identified that alleviate the symptoms of nausea and vomiting, either prophylactically or acutely. None of these is completely effective in all cases. They include antihistamines, dopamine antagonists, steroids, cannabinoids, benzodiazepines, serotonin antagonists, and anticholinergics. This paper examines the evidence that links each of these classes of drugs with the distribution of specific neurotransmitter receptor sites on which they may be acting. Studies on the central nervous system distribution of binding sites for one of these classes of drugs, the anticholinergics, are described. Binding sites for the muscarinic cholinergic radioligand [3H]quinuclidinylbenzilate occur in different concentrations throughout the dorsal vagal complex of the rabbit medulla oblongata. The distribution of such sites in this nonvomiting experimental animal is markedly different from that in the cat, an animal that has been used for many physiological and pharmacological studies of emesis. A previous study has suggested that muscarinic binding sites may occur presynaptically on vagal afferent terminals that synapse in the dorsal vagal complex of the cat; this appears not to be the case in the rabbit. Possible implications of these findings for the identification of the site of action of anticholinergic, antiemetic drugs are discussed.     

Changes in extracellular dopamine in the rat globus pallidus induced by typical and atypical antipsychotic drugs

Typical antipsychotic drugs with a high extrapyramidal motor side-effects liability markedly increase extracellular dopamine in the caudate-putamen, while atypical antipsychotic drugs with a low incidence of extrapyramidal motor side-effects have less pronounced stimulating actions on striatal dopamine. Therefore, it has been suggested that the extrapyramidal motor side-effects liability of antipsychotic drugs (APD) is correlated with their ability to increase extracellular dopamine in the caudate-putamen. The globus pallidus (GP) is another basal ganglia structure probably mediating extrapyramidal motor side-effects of typical antipsychotic drugs. Therefore, the present study sought to determine whether extracellular dopamine in the globus pallidus might be a further indicator to differentiate neurochemical actions of typical and atypical antipsychotic drugs. Using in vivo microdialysis we compared effects on pallidal dopamine induced by typical and atypical antipsychotic drugs in rats. Experiment I demonstrated that systemic administration of haloperidol (1 mg/kg; i.p.) and clozapine (20 mg/kg; i.p.) induced a significant pallidal dopamine release to about 160 and 180% of baseline, respectively. Experiment II revealed that reverse microdialysis of raclopride and clozapine using a cumulative dosing regimen did not stimulate extracellular dopamine in the globus pallidus if low (1microM) or intermediate (10 and 100 microM) concentrations were used. Only at a high concentration (1,000 microM), raclopride and clozapine induced a significant pallidal dopamine release to about 130 and 300% of baseline values, respectively. Thus, effects of typical and atypical antipsychotic drugs on pallidal dopamine were similar and thus, may not be related to their differential extrapyramidal motor side-effects liability. Furthermore, the finding that reverse microdialysis of raclopride over a wide range of concentrations did not stimulate pallidal dopamine concentrations tentatively suggests that pallidal dopamine release under basal conditions is not regulated by D2 autoreceptors.     

Muscarinic cholinergic and histamine H1 receptor binding of phenothiazine drug metabolites

In vitro binding affinities of chlorpromazine, fluphenazine, levomepromazine, perphenazine and some of their metabolites for dopamine D2 receptors, alpha 1- and alpha 2 adrenoceptors in rat brain were previously reported from our laboratories. The present study reports the in vitro binding affinities of the same compounds for muscarinic cholinergic receptors and for histamine H1 receptors in rat brain, using 3H-quinuclidinyl benzilate and 3H-mepyramine as radioligands. Chlorpromazine, levomepromazine, and their metabolites had 5-30 times higher binding affinities for muscarinic cholinergic receptors than fluphenazine, perphenazine and their metabolites. Levomepromazine was the most potent and fluphenazine the least potent of the four drugs in histamine H1 receptor binding. 7-Hydroxy levomepromazine, 3-hydroxy levomepromazine and 7-hydroxy fluphenazine had only 10% of the potency of the parent drug in histamine H1 receptor binding, while the 7-hydroxy-metabolites of chlorpromazine and perphenazine had about 75% of the potency of the parent drug in this binding system. Their histamine H1 receptor binding affinities indicate that metabolites may contribute to the sedative effects of chlorpromazine and levomepromazine.     

Typical and Atypical Neuroleptics Induce Alteration in Blood-Brain Barrier and Brain 59FeCl3 Uptake

Abstract: Long-term neuroleptic medication of schizophrenic patients induces extrapyramidal motor side effects, of which tardive dyskinesia (TD) is the most severe. The etiology of TD is still obscure. Recently, it was suggested that abnormal iron metabolism may play a crucial role in neuroleptic-induced dopamine D₂ receptor super-sensitivity. The apparent relationship between neuroleptics and iron is further supported by the increase of iron in the basal ganglia of patients with TD. We now report on the ability of neuroleptics to alter the blood-brain barrier in the rat and to potentiate the normally limited iron transport into the brain. Thus, chronic treatment of rats with chlorpromazine and haloperidol facilitated ⁵⁹Fe³⁺ uptake into brain cells. In contrast, clozapine, an atypical antipsychotic neuroleptic with little extrapyramidal motor side effects, caused iron sedimentation in brain blood vessels with no sign of detectable iron in the cells. Moreover, chronic treatment with chlorpromazine and haloperidol caused a 43% and 24% reduction, respectively, in liver nonheme iron, whereas clozapine induced an 81% increase. The apparent different potentials of chlorpromazine, haloperidol, and clozapine to increase iron transport into the brain from its peripheral stores may be linked to the severity of extrapyramidal motor side effects they induce and to the pathophysiology of TD.     

Neuroleptics-induced changes of tyrosine hydroxylase activity in rat striatum in vitro and in vivo.

The potency of haloperidol and chlorpromazine, but not clozapine, for increasing homovanillic acid and activating tyrosine hydroxylase in the striatum was significantly weakened after the repeated administration in rats. These findings suggest that clozapine could supply enough dopamine to surmount the blockade of dopamine receptors in the striatum even after the repeated administration. This property of clozapine seems to be the cause of low incidence of extrapyramidal side effects in clinical use.     

The effects of muscarinic receptor blockers on the turnover rate of acetylcholine in various regions of the rat brain.

The actions of antimuscarinic agents (benztropine, trihexyphenidyl, and scopolamine) on the dynamics of acetylcholine (ACh) in central cholinergic neurons were examined in various rat brain areas. It was found that the pattern of changes in ACh turnover (TRACh) elicited by these drugs exhibited marked regional variations. After administration of the anticholinergic drugs, the TRACh in hippocampus and thalamus was increased, in cortex it was decreased, and in striatum it was unchanged. ACh concentration in the cortex and striatum was decreased while in hippocampus and thalamus ACh levels were unaltered. Further analysis of the cholinergic septo-hippocampal pathway using lesions of the fimbria-fornix and local drug injections into the septum argue against an in vivo action of these drugs on presynaptic or cell body muscarinic autoreceptors. Moreover, the data suggest that muscarinic receptor blockers cause an increased TRACh only in those areas where a feedback loop is operative, possibly by inhibiting a neuronal feedback loop involving at least one noncholinergic interneuron.