RESUMEN
Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4ß2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4ß2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.
Asunto(s)
Acetilcolina/metabolismo , Neocórtex/metabolismo , Células Piramidales/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/fisiología , Acetilcolina/farmacología , Animales , Agonistas Colinérgicos/farmacología , Potenciales Postsinápticos Excitadores , Ácido Glutámico/metabolismo , Neocórtex/efectos de los fármacos , Vías Nerviosas , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Ratas , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , TálamoRESUMEN
Negative and cognitive symptoms of schizophrenia contribute to an impaired social and professional life for schizophrenic patients, and in most cases, these symptoms are treatment resistant. Therefore, identification of new treatment strategies is sorely needed. Metabotropic glutamate receptors (mGlu) and muscarinic (M) receptors for acetylcholine have been considered promising targets for novel antipsychotics. Among them, mGlu2 and M4 subtypes seem to be of particular importance. In the present study, the effect of mutual activation of mGlu2 and M4 receptors was assessed in MK-801-based animal models of negative and cognitive symptoms of schizophrenia, that is, social interaction and novel object recognition tests. Low sub-effective doses of LY487379 (0.5 mg/kg), a positive allosteric activator of the mGlu2 receptor, and VU152100 (0.25-0.5 mg/kg), a positive allosteric modulator of the M4 receptor, were simultaneously administered in the aforementioned tests. Combined administration of these compounds prevented MK-801-induced disturbances in social interactions and object recognition when acutely administered 30 min before MK-801. Prolonged (7 days) administration of these compounds resulted in the loss of effectiveness in preventing MK-801-induced disruptions in the novel object recognition test but not in the social interaction test. In the next set of experiments, MK-801 (0.3 mg/kg) was administered for seven consecutive days, and the activity of the compounds was investigated on day eight, during which time MK-801 was not administered. In this model, based on prolonged MK-801 administration, the effectiveness of the compounds to treat MK-801-induced disruptions was evident at low doses which were ineffective in preventing the behavioural disturbances induced by an acute MK-801 injection. Combined administration of the compounds did not exert better efficacy than each compound given alone. Pharmacokinetic analysis confirmed a lack of possible drug-drug interactions after combined administration of LY487379 and VU152100. Our data show that modulation of M4 and mGlu2 receptors may potentially be beneficial in the treatment of negative and cognitive symptoms of schizophrenia.
Asunto(s)
Antipsicóticos/uso terapéutico , Benzamidas/uso terapéutico , Memoria a Corto Plazo/efectos de los fármacos , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Conducta Social , Sulfonamidas/uso terapéutico , Animales , Antipsicóticos/administración & dosificación , Antipsicóticos/farmacología , Benzamidas/administración & dosificación , Benzamidas/farmacología , Maleato de Dizocilpina/toxicidad , Quimioterapia Combinada , Agonistas de Aminoácidos Excitadores/administración & dosificación , Agonistas de Aminoácidos Excitadores/farmacología , Agonistas de Aminoácidos Excitadores/uso terapéutico , Antagonistas de Aminoácidos Excitadores/toxicidad , Masculino , Ratones , Pirazoles/administración & dosificación , Pirazoles/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Receptor Muscarínico M4/efectos de los fármacos , Receptores de Glutamato , Esquizofrenia/etiología , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacologíaRESUMEN
Striatal dopamine (DA) and acetylcholine (ACh) regulate motivated behaviors and striatal plasticity. Interactions between these neurotransmitters may be important, through synchronous changes in parent neuron activities and reciprocal presynaptic regulation of release. How DA signaling is regulated by striatal muscarinic receptors (mAChRs) is unresolved; contradictory reports indicate suppression or facilitation, implicating several mAChR subtypes on various neurons. We investigated whether mAChR regulation of DA signaling varies with presynaptic activity and identified the mAChRs responsible in sensorimotor- versus limbic-associated striatum. We detected DA in real time at carbon fiber microelectrodes in mouse striatal slices. Broad-spectrum mAChR agonists [oxotremorine-M, APET (arecaidine propargyl ester tosylate)] decreased DA release evoked by low-frequency stimuli (1-10 Hz, four pulses) but increased the sensitivity of DA release to presynaptic activity, even enhancing release by high frequencies (e.g., >25 Hz for four pulses). These bidirectional effects depended on ACh input to striatal nicotinic receptors (nAChRs) on DA axons but not GABA or glutamate input. In caudate-putamen (CPu), knock-out of M(2)- or M(4)-mAChRs (not M(5)) prevented mAChR control of DA, indicating that M(2)- and M(4)-mAChRs are required. In nucleus accumbens (NAc) core or shell, mAChR function was prevented in M(4)-knock-outs, but not M(2)- or M(5)-knock-outs. These data indicate that striatal mAChRs, by inhibiting ACh release from cholinergic interneurons and thus modifying nAChR activity, offer variable control of DA release probability that promotes how DA release reflects activation of dopaminergic axons. Furthermore, different coupling of striatal M(2)/M(4)-mAChRs to the control of DA release in CPu versus NAc suggests targets to influence DA/ACh function differentially between striatal domains.
Asunto(s)
Acetilcolina/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Interneuronas/metabolismo , Receptores Muscarínicos/metabolismo , Transmisión Sináptica/fisiología , Animales , Ganglios Basales/citología , Ganglios Basales/efectos de los fármacos , Ganglios Basales/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Electrofisiología , Interneuronas/citología , Interneuronas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Muscarínicos/farmacología , Neostriado/citología , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Núcleo Accumbens/citología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/genética , Receptor Muscarínico M4/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/genética , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Despite the discovery of a diverse range of novel agonists and allosteric modulators of the M(4) muscarinic acetylcholine (ACh) receptor (mAChR), little is known about how such ligands activate the receptor. We used site-directed mutagenesis of conserved residues in transmembrane 3 (TMIII), a key region involved in G protein-coupled receptor activation, to probe the binding and function of prototypical orthosteric mAChR agonists, allosteric modulators, and "atypical" agonists. We found that most mutations did not affect the binding of the allosteric modulators, with the exception of W108(3.28)A and L109(3.29)A (which may contribute directly to the interface between allosteric and orthosteric sites) and mutation D112(3.32)N (which may cause a global disruption of a hydrogen bond network). Although numerous mutations affected signaling, we did not identify amino acids that were important for the functional activity of any one class of agonist (orthosteric, allosteric, or atypical) to the exclusion of any others, suggesting that TMIII is key for the transmission of stimulus irrespective of the agonist. We also identified two key residues, Trp108(3.28) and Asp112(3.32), that are essential for the transmission of binding cooperativity between 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine- 2-carboxylic acid cyclopropylamide (LY2033298) and ACh. Finally, we found that LY2033298 was able to rescue functionally impaired signaling of ACh at the majority of mutants tested in a manner that was inversely correlated with the ACh signaling efficacy, indicating that a key part of the mechanism of the positive cooperativity mediated by LY2033298 on the endogenous agonist involves a global drive of the receptor toward an active conformation.
Asunto(s)
Proteínas de la Membrana/fisiología , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M4/efectos de los fármacos , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ensayo de Unión Radioligante , Receptor Muscarínico M4/química , Receptor Muscarínico M4/genéticaRESUMEN
Alzheimer's disease (AD) is a profoundly debilitating neurodegenerative disorder characterized most notably by progressive cognitive decline, but also agitation and behavioral disturbances that are extremely disruptive to patient and caregiver. Current pharmacological treatments for these symptoms have limited efficacy and significant side effects. We have recently reported the discovery of Compound 24, an M4 positive allosteric modulator (PAM) that is potent, highly selective, and devoid of cholinergic-like side effects in rats. In order to further evaluate the translatability of the effects of compound 24 in primates, here we describe the effect of Compound 24 on three behavioral and cognition assays in rhesus monkeys, the stimulant induced motor activity (SIMA) assay, the object retrieval detour task (ORD), and the visuo-spatial paired-associates learning (vsPAL) task. As far as we know, this is the first such characterization of an M4 PAM in non-human primate. Compound 24 and the clinical standard olanzapine attenuated amphetamine induced hyperactivity to a similar degree. In addition, Compound 24 demonstrated procognitive effects in scopolamine-impaired ORD and vsPAL, and these effects were of similar magnitude to donepezil. These findings suggest that M4 PAMs may be beneficial to diseases such as Alzheimer's disease and schizophrenia, which are marked by behavioral disturbances as well as deficits in cognitive function.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/psicología , Conducta Animal/efectos de los fármacos , Colinérgicos/farmacología , Trastornos del Conocimiento/tratamiento farmacológico , Receptor Muscarínico M4/efectos de los fármacos , Esquizofrenia/tratamiento farmacológico , Psicología del Esquizofrénico , Anfetamina/antagonistas & inhibidores , Anfetamina/farmacología , Animales , Aprendizaje por Asociación/efectos de los fármacos , Estimulantes del Sistema Nervioso Central , Colinérgicos/farmacocinética , Trastornos del Conocimiento/psicología , Hipercinesia/inducido químicamente , Hipercinesia/prevención & control , Macaca mulatta , Masculino , Actividad Motora/efectos de los fármacos , Olanzapina/farmacología , Orientación/efectos de los fármacosRESUMEN
The central cholinergic system regulates both the circadian clock and sleep-wake cycle and may participate in the feedback control of vigilance states on neural excitability in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigate the mechanisms for cholinergic modulation of SCN neuron excitability. Cell-attached recordings indicate that the nonspecific cholinergic agonist carbachol (CCh) inhibited 55% and excited 21% SCN neurons, leaving 24% nonresponsive. Similar response proportions were produced by two muscarinic receptor [muscarinic acetylcholine receptor (mAChR)] agonists, muscarine and McN-A-343 (M1/4 agonist), but not by two nicotinic receptor (nAChR) agonists, nicotine and choline (alpha7-nAChR agonist), which, however, produced similar response proportions. Whole cell and perforated-patch recordings indicate that CCh inhibition of firing was mediated by membrane hyperpolarization due to activation of background K(+) currents, which were sensitive to submillimolar concentrations of Ba(2+) and to millimolar concentrations of TEA. RT-PCR analysis demonstrated the presence of mRNA for M1 to M5 mAChRs in SCN. The CCh-induced hyperpolarization and activation of background K(+) currents were blocked by M4 antagonists and to a lesser degree by M1 antagonists but were insensitive to the antagonists for M2 or M3, suggesting the involvement of M4 and M1 mAChRs in mediating CCh inhibition of firing. CCh enhancement of firing was mediated by membrane depolarization, as a result of postsynaptic inhibition of background K(+) currents. The multiple actions of cholinergic modulation via multiple receptors and ion channels may allow acetylcholine to finely control SCN neuron excitability in different physiological settings.
Asunto(s)
Neuronas/fisiología , Sistema Nervioso Parasimpático/fisiología , Núcleo Supraquiasmático/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Carbacol/farmacología , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M4/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estimulación Química , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D(2) dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D(2) dopamine receptor modulation of calcium (Ca(2+)) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M(4) muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4-an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.
Asunto(s)
Acetilcolina/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Interneuronas/metabolismo , Proteínas RGS/metabolismo , Receptor Muscarínico M4/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Autorreceptores/efectos de los fármacos , Autorreceptores/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Interneuronas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Agonistas Muscarínicos/farmacología , Oxotremorina/farmacología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , Proteínas RGS/genética , Ratas , Receptor Muscarínico M4/efectos de los fármacos , Receptores de Dopamina D2/efectos de los fármacos , Receptores de Dopamina D2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Analgesic properties of orthosteric agonists of the muscarinic M4 receptor subtype have been documented in literature reports, with evidence from pharmacological and in vivo receptor knock out (KO) studies. Constitutive M4 receptor KO mice demonstrated an increased response in the formalin pain model, supporting this hypothesis. Two novel positive allosteric modulators (PAM) of the M4 receptor, Compounds 1 and 2, were characterized in rodent models of acute nociception. Results indicated decreased time spent on nociceptive behaviors in the mouse formalin model, and efficacy in the mouse tail flick assay. The analgesic-like effects of Compounds 1 and 2 were shown to be on target, as the compounds lacked any activity in constitutive M4 KO mice, while retaining activity in wild type control littermates. The analgesic-like effects of Compounds 1 and 2 were significantly diminished in KO mice that have selective deletion of the M4 receptor in neurons that co-express the dopaminergic D1 receptor subtype, suggesting a centrally-mediated effect on nociception. The opioid antagonist naloxone did not diminish the effect of Compound 1, indicating the effects of Compound 1 are not secondarily linked to opioid pathways. Compound 1 was evaluated in the rat, where it demonstrated analgesic-like effects in tail flick and a subpopulation of spinal nociceptive sensitive neurons, suggesting some involvement of spinal mechanisms of nociceptive modulation. These studies indicate that M4 PAMs may be a tractable target for pain management assuming an appropriate safety profile, and it appears likely that both spinal and supraspinal pathways may mediate the antinociceptive-like effects.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Nocicepción/efectos de los fármacos , Receptor Muscarínico M4/agonistas , Regulación Alostérica/fisiología , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Colinérgicos/farmacología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas de Narcóticos/farmacología , Nocicepción/fisiología , Dolor/metabolismo , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismoRESUMEN
Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS where they control a variety of neuronal functions. Due to their roles in a number of CNS processes, mAChRs have long been a target of the drug discovery industry; however, the only mAChR ligands approved for use in the clinic are non-selective antagonists for the treatment of Parkinson's disease. This article briefly reviews recent progress made in mAChR drug discovery for Alzheimer's disease (AD), schizophrenia and Parkinson's disease, with particular emphasis on novel target validation, as well as highlighting novel indications such as drug addiction.
Asunto(s)
Fármacos del Sistema Nervioso Central/farmacología , Sistema Nervioso Central/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Antiparkinsonianos/farmacología , Antipsicóticos/farmacología , Sistema Nervioso Central/metabolismo , Fármacos del Sistema Nervioso Central/uso terapéutico , Humanos , Agonistas Muscarínicos/uso terapéutico , Antagonistas Muscarínicos/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismo , Receptores Muscarínicos/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Trastornos Relacionados con Sustancias/metabolismoRESUMEN
The five muscarinic acetylcholine receptors (M(1)-M(5)) are differentially expressed in the brain. M(2) and M(4) are coupled to inhibition of stimulated adenylyl cyclase, while M(1), M(3) and M(5) are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2-affinity order M(1) > or = M(4) >> others-and MT3-highly selective M(4) antagonist-did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M(2), M(4) would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M(4) receptor would be responsible for this regulation.
Asunto(s)
Hipocampo/enzimología , Antagonistas Muscarínicos/farmacología , Neostriado/enzimología , Receptor Muscarínico M4/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/fisiología , Animales , Colforsina/farmacología , AMP Cíclico/biosíntesis , Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Masculino , Agonistas Muscarínicos/farmacología , Neostriado/efectos de los fármacos , Oxotremorina/farmacología , Ratas , Ratas Wistar , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidoresRESUMEN
Herein we describe the discovery and optimization of a new series of 2,3-disubstituted and 2,3,6-trisubstituted muscarinic acetylcholine receptorâ 4 (M4 ) positive allosteric modulators (PAMs). Iterative libraries enabled rapid exploration of one-dimensional structure-activity relationships (SAR) and identification of potency-enhancing heterocycle and N-alkyl pyrazole substituents. Further optimization led to identification of the potent, receptor-subtype-selective, brain-penetrant tool compound 24 (7-[3-[1-[(1-fluorocyclopentyl)methyl]pyrazol-4-yl]-6-methyl-2-pyridyl]-3-methoxycinnoline). It is efficacious in preclinical assays that are predictive of antipsychotic effects, producing dose-dependent reversal of amphetamine-induced hyperlocomotion in rats and mice, but not in M4 knockout mice. Cholinergic-related adverse effects observed in rats treated with 24 at unbound plasma concentrations more than 3-fold higher than an efficacious dose in the hyperlocomotion assay were fewer and less severe than those observed in rats treated with the nonselective M4 agonist xanomeline, suggesting a receptor-subtype-selective PAM has the potential for an improved safety profile.
Asunto(s)
Descubrimiento de Drogas , Piridinas/química , Piridinas/farmacología , Receptor Muscarínico M4/efectos de los fármacos , Regulación Alostérica , Animales , Humanos , Ratas , Receptor Muscarínico M4/metabolismo , Relación Estructura-ActividadRESUMEN
We remember our lives as sequences of events, but it is unclear how these memories are controlled during retrieval. In rats, the medial prefrontal cortex (mPFC) is positioned to influence sequence memory through extensive top-down inputs to regions heavily interconnected with the hippocampus, notably the nucleus reuniens of the thalamus (RE) and perirhinal cortex (PER). Here, we used an hM4Di synaptic-silencing approach to test our hypothesis that specific mPFCâRE and mPFCâPER projections regulate sequence memory retrieval. First, we found non-overlapping populations of mPFC cells project to RE and PER. Second, suppressing mPFC activity impaired sequence memory. Third, inhibiting mPFCâRE and mPFCâPER pathways effectively abolished sequence memory. Finally, a sequential lag analysis showed that the mPFCâRE pathway contributes to a working memory retrieval strategy, whereas the mPFCâPER pathway supports a temporal context memory retrieval strategy. These findings demonstrate that mPFCâRE and mPFCâPER pathways serve as top-down mechanisms that control distinct sequence memory retrieval strategies.
Asunto(s)
Memoria a Corto Plazo/fisiología , Vías Nerviosas/fisiología , Corteza Prefrontal/fisiología , Receptor Muscarínico M4/metabolismo , Animales , Clozapina/análogos & derivados , Clozapina/farmacología , Antagonistas del GABA/farmacología , Hipocampo/fisiología , Memoria a Corto Plazo/efectos de los fármacos , Núcleos Talámicos de la Línea Media/efectos de los fármacos , Núcleos Talámicos de la Línea Media/fisiología , Corteza Perirrinal/efectos de los fármacos , Corteza Perirrinal/fisiología , Corteza Prefrontal/citología , Corteza Prefrontal/efectos de los fármacos , Ratas , Ratas Long-Evans , Receptor Muscarínico M4/efectos de los fármacos , Antagonistas de la Serotonina/farmacologíaRESUMEN
Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Reflujo Gastroesofágico/fisiopatología , Receptores Muscarínicos/fisiología , Sistema Respiratorio/irrigación sanguínea , Animales , Atropina/farmacología , Diaminas/farmacología , Femenino , Bloqueadores Ganglionares/farmacología , Cobayas , Hexametonio/farmacología , Técnicas In Vitro , Masculino , Antagonistas Muscarínicos/farmacología , Antagonistas Nicotínicos/farmacología , Parasimpatolíticos/farmacología , Piperidinas/farmacología , Pirenzepina/farmacología , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M4/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Flujo Sanguíneo Regional/fisiologíaRESUMEN
RATIONALE: Nonselective muscarinic acetylcholine antagonists have been used for several years as antiparkinsonian drugs. However, there are at least five subtypes of muscarinic receptor (M1-5). Neostriatal M4 receptors have been implicated in aspects of motor function, and it has been suggested that M4 antagonists could be used as treatments for parkinsonism. OBJECTIVE: Currently, there is a lack of highly selective M4 antagonists that readily penetrate the blood brain barrier. Thus, the present studies focused upon the effects of tropicamide, a muscarinic acetylcholine receptor antagonist with moderate binding selectivity for the M4 receptor subtype. MATERIALS AND METHODS: Tremulous jaw movements were used as a model of parkinsonian tremor in these studies, and the effects of tropicamide were compared with those of the nonselective muscarinic antagonist atropine. RESULTS: Tropicamide suppressed the tremulous jaw movements induced by the muscarinic agonist pilocarpine and the dopamine antagonist pimozide. Analysis of the dose-response curves indicated that tropicamide showed approximately the same potency as atropine for suppression of pilocarpine-induced jaw movements but was more potent than atropine on the suppression of pimozide-induced jaw movements. In contrast, atropine was more potent than tropicamide in terms of impairing performance on visual stimulus detection and delayed nonmatch-to-position tasks. CONCLUSIONS: These studies demonstrate that tropicamide, which currently is used clinically for ophthalmic purposes, can exert actions that are consistent with antiparkinsonian effects. Moreover, the different pattern of effects shown by tropicamide compared to those of atropine on motor vs cognitive tasks could be due to the modest M4 selectivity shown by tropicamide.
Asunto(s)
Antagonistas Muscarínicos/farmacología , Trastornos Parkinsonianos/tratamiento farmacológico , Receptor Muscarínico M4/efectos de los fármacos , Temblor/tratamiento farmacológico , Tropicamida/farmacología , Animales , Atropina/administración & dosificación , Atropina/farmacología , Modelos Animales de Enfermedad , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Maxilares/efectos de los fármacos , Maxilares/fisiopatología , Masculino , Memoria/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/administración & dosificación , Trastornos Parkinsonianos/fisiopatología , Pilocarpina , Pimozida , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M4/metabolismo , Temblor/inducido químicamente , Temblor/fisiopatología , Tropicamida/administración & dosificación , Percepción Visual/efectos de los fármacosRESUMEN
alpha2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to alpha2-AR analgesia would also destroy nNOS and M4 receptor expression. Male Sprague-Dawley rats underwent left L5 and L6 spinal nerve ligation. Lumbar spinal cord was removed and immunostained for M4 muscarinic receptors and nNOS alone and for co-expression. Others received intrathecal injection of saporin linked to an antibody to the neurotrophin receptor p75(NTR), which eliminates cells expressing this receptor and the analgesic effects of alpha2-AR agonists. nNOS staining of fibers in the superficial dorsal horn was dramatically increased after spinal nerve ligation, and this was abolished by saporin linked anti-p75(NTR) treatment. In contrast, nNOS staining in dorsal horn neurons was unaltered by these manipulations. M4 receptors were present on neurons in the dorsal horn, some of which co-expressed nNOS, but their pattern of expression was not altered by these manipulations. Peripheral nerve injury increases nNOS expression in fibers in the superficial dorsal horn, some of which likely express p75(NTR), and alpha2-AR agonists may reduce injury-induced sensitization by activation of nNOS in these fibers In contrast, changes in nNOS and M4 receptor location on spinal cord neurons are not responsible for increased analgesic potency of alpha2-AR agonists after nerve injury.
Asunto(s)
Ganglios Espinales/metabolismo , Neuronas Aferentes/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Raíces Nerviosas Espinales/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiopatología , Ligadura , Masculino , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Neuralgia/fisiopatología , Neuronas Aferentes/efectos de los fármacos , Neurotoxinas/farmacología , Óxido Nítrico/metabolismo , Nociceptores/citología , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismo , Receptor de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptor de Factor de Crecimiento Nervioso/metabolismo , Raíces Nerviosas Espinales/efectos de los fármacos , Raíces Nerviosas Espinales/fisiopatología , Nervios Espinales/lesiones , Nervios Espinales/metabolismo , Nervios Espinales/fisiopatología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
G-protein-coupled receptors (GPCRs) play critical roles in regulating brain function. Recent advances have greatly expanded our understanding of these receptors as complex signaling machines that can adopt numerous conformations and modulate multiple downstream signaling pathways. While agonists and antagonists have traditionally been pursued to target GPCRs, allosteric modulators provide several mechanistic advantages, including the ability to distinguish between closely related receptor subtypes. Recently, the discovery of allosteric ligands that confer bias and modulate some, but not all, of a given receptor's downstream signaling pathways can provide pharmacological modulation of brain circuitry with remarkable precision. In addition, allosteric modulators with unprecedented specificity have been developed that can differentiate between subpopulations of a given receptor subtype based on the receptor's dimerization state. These advances are not only providing insight into the biological roles of specific receptor populations, but hold great promise for treating numerous CNS disorders.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Antipsicóticos/farmacología , Receptores Dopaminérgicos/efectos de los fármacos , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores Muscarínicos/efectos de los fármacos , Esquizofrenia/tratamiento farmacológico , Animales , Antipsicóticos/uso terapéutico , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Cognición/efectos de los fármacos , Humanos , Corteza Prefrontal/efectos de los fármacos , Receptor del Glutamato Metabotropico 5/efectos de los fármacos , Receptor Muscarínico M4/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacosRESUMEN
In this work, we studied the in vitro G-protein activation induced by muscarinic agonists using [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiographic methods to characterize the M(2) and M(4) muscarinic subtypes response. Thus, we describe a detailed characterization of the increases in [(35)S]GTPgammaS binding elicited by carbachol (Cch) and oxotremorine (OXO) (binding in the presence minus binding in the absence of agonist) throughout the gerbil brain (Meriones unguiculatus). For both agonists, the strongest stimulations were found in the superficial gray layer of the superior colliculus, the anteroventral and anteromedial thalamic nuclei, the anterior paraventricular thalamic nucleus, and the caudate-putamen. The comparative study using OXO and Cch suggested that OXO is able to detect differences in the response of structures enriched in M(4) muscarinic receptors, showing a lower potency to stimulate these brain areas. Furthermore, using increasing concentrations of selective M(2) (AF-DX 116) and M(1)/M(4) (pirenzepine) antagonists to inhibit specific Cch- or OXO-induced [(35)S]GTPgammaS binding, significant differences were observed in M(2)-enriched structures but not in M(4)-enriched ones such as the caudate-putamen. These data indicate that appropriate muscarinic agonist stimulation, together with selective inhibition of this effect using functional autoradiography, can be used as a tool to unravel the M(2)- and M(4)-muscarinic subtype-mediated response.
Asunto(s)
Unión Competitiva/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Muscarínicos/efectos de los fármacos , Acetilcolina/metabolismo , Animales , Unión Competitiva/fisiología , Encéfalo/metabolismo , Química Encefálica/fisiología , Carbacol/farmacología , Relación Dosis-Respuesta a Droga , Gerbillinae , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Masculino , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Oxotremorina/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Muscarínicos/metabolismo , Radioisótopos de Azufre , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
The aim of this study was to examine the influence of different fat diets on muscarinic acetylcholine receptor binding. Nineteen male Sprague-Dawley rats were divided into four groups and fed a diet of either high saturated fat, n-6 polyunsaturated fatty acid (PUFA), n-3 PUFA or low fat (control) for 8 weeks. Using quantitative autoradiography, [(3)H]pirenzepine binding to muscarinic M1/M4 receptors and [(3)H]AF-DX384 binding to M2/M4 receptors were measured throughout the brain in all four groups. The main findings were that compared to the low fat control group, M2/M4 receptor binding was significantly reduced in the dorsolateral, dorsomedial and ventromedial parts of the caudate putamen (61-64%, p < 0.05), anterior cingulate cortex (59%, p < 0.01), dentate gyrus and CA1-3 fields of the hippocampus (32-43%, p < 0.01) of rats on a high n-6 PUFA diet; however, no differences in M1/M4 receptor binding densities between the four groups were observed. These results suggest that a diet high in n-6 PUFA, but not of n-3 PUFAs or saturated fat, may selectively alter M2/M4 receptor-mediated signal transduction in the rat brain.
Asunto(s)
Unión Competitiva/fisiología , Encéfalo/metabolismo , Ácidos Grasos Omega-6/farmacología , Alimentos Formulados , Alimentos Fortificados , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animales , Ácido Araquidónico/metabolismo , Unión Competitiva/efectos de los fármacos , Encéfalo/efectos de los fármacos , Mapeo Encefálico , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-6/metabolismo , Masculino , Antagonistas Muscarínicos/metabolismo , Parasimpatolíticos/metabolismo , Pirenzepina/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismo , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
The present study aimed to determine the effect of two M2/M4-selective muscarinic receptor antagonists on blocking the hydrolysis of carbachol (CCh) stimulated phospho-inositide (PI) breakdown in order to address the possibility that a muscarinic receptor other than the M(3) receptor is involved in PI hydrolysis in this tissue. Gallbladder tissue slices labeled with myo-[2-3H] inositol were incubated with increasing concentrations of antagonists and agonist. After the reactions were terminated by the addition of chloroform/methanol, labeled inositol phosphates were separated using anion exchange chromatography. Muscarinic M2 antagonists methoctramine and gallamine both inhibited carbachol-induced PI breakdown at high concentrations, with log IC50 values of -5.145 and -6.049, respectively. Gallamine at 10(-5)M concentration failed to displace the dose-response curve for carbachol-induced accumulation of inositol triphosphate (IP3). Our data suggest that M(3) receptors play a major role in stimulation of PI hydrolysis in the guinea-pig gallbladder.
Asunto(s)
Diaminas/farmacología , Trietyoduro de Galamina/farmacología , Vesícula Biliar/metabolismo , Antagonistas Muscarínicos/farmacología , Fosfatidilinositoles/metabolismo , Animales , Carbacol/antagonistas & inhibidores , Carbacol/farmacología , Depresión Química , Femenino , Vesícula Biliar/efectos de los fármacos , Cobayas , Hidrólisis , Técnicas In Vitro , Masculino , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M4/efectos de los fármacosRESUMEN
1. Muscarinic agonist specificity is limited, making it difficult to match receptor subtypes with signal transduction cascades that mediate ion channel modulation. We have characterized the inhibitory effects of two muscarinic agonists, oxotremorine-M (Oxo-M) and bethanechol chloride (BeCh), on Ca(2+) currents in neonatal rat superior cervical ganglion neurons. 2. Oxo-M-mediated (10 micro M) inhibition occurred via two signaling pathways. The first pathway inhibited whole cell peak currents, consisting primarily of N-type current, but not FPL 64176-induced, long-lasting tail currents, comprised entirely of L-type current. Inhibited currents displayed slowed activation kinetics and voltage dependence, characteristics of membrane-delimited inhibition. Current inhibition was blocked by the selective M(2) receptor antagonist, methoctramine (METH; 100 nM), or following pertussis toxin (PTX) pretreatment. 3. Activation of the second pathway inhibited both peak and long-lasting tail currents. This pathway was voltage-independent, PTX-insensitive, but sensitive to internal Ca(2+) chelator concentration. Muscarinic toxin 7 (MT-7, 100 nM), an irreversible M(1) receptor antagonist, eliminated this inhibition. Oxo-M (100 micro M) decreased L- and N-type channel activities in cell-attached patches, indicating that a diffusible second messenger is involved. 4. BeCh (100 micro M) also inhibited whole cell currents via the membrane-delimited pathway. Blocking M(4) receptors with 100 nM pirenzepine (in the presence of MT-7) had no effect, while antagonizing M(2) receptors with METH abolished inhibition. Concentrations of BeCh as high as 3 mM failed to inhibit either peak or long-lasting tail currents following PTX pretreatment. 5. These results indicate that BeCh may be an effective tool for selectively activating M(2) receptor stimulation of the membrane-delimited pathway.