RESUMEN
BACKGROUND: Preclinical and some human data suggest allosteric modulation of the muscarinic M1 receptor (CHRM1) is a promising approach for the treatment of schizophrenia. However, it is suggested there is a subgroup of participants with schizophrenia who have profound loss of cortical CHRM1 (MRDS). This raises the possibility that some participants with schizophrenia may not respond optimally to CHRM1 allosteric modulation. Here we describe a novel methodology to measure positive allosteric modulation of CHRM1 in human CNS and the measurement of that response in the cortex, hippocampus, and striatum from participants with MRDS, non-MRDS and controls. METHODS: The cortex (Brodmann's area 6), hippocampus, and striatum from 40 participants with schizophrenia (20 MRDS and 20 non-MRDS) and 20 controls were used to measure benzyl quinolone carboxylic acid-mediated shift in acetylcholine displacement of [3H]N-methylscopolamine using a novel in situ radioligand binding with autoradiography methodology. RESULTS: Compared with controls, participants with schizophrenia had lower levels of specific [3H]N-methylscopolamine binding in all CNS regions, whilst benzyl quinolone carboxylic acid-modulated binding was less in the striatum, Brodmann's area 6, dentate gyrus, and subiculum. When divided by subgroup, only in MRDS was there lower specific [3H]N-methylscopolamine binding and less benzyl quinolone carboxylic acid-modulated binding in all cortical and subcortical regions studied. CONCLUSIONS: In a subgroup of participants with schizophrenia, there is a widespread decreased responsiveness to a positive allosteric modulator at the CHRM1. This finding may have ramifications it positive allosteric modulators of the CHRM1 are used in clinical trials to treat schizophrenia as some participants may not have an optimal response.
Asunto(s)
Regulación Alostérica , Receptor Muscarínico M1/agonistas , Esquizofrenia/metabolismo , Autorradiografía , Estudios de Casos y Controles , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Persona de Mediana Edad , N-Metilescopolamina/metabolismo , Unión Proteica/efectos de los fármacos , Quinolonas/metabolismo , Ensayo de Unión Radioligante/métodos , Receptor Muscarínico M1/deficiencia , Tritio/metabolismoRESUMEN
Muscarinic acetylcholine receptors have been implicated as potential neuroprotective targets for glaucoma. We tested the hypothesis that the lack of a single muscarinic receptor subtype leads to age-dependent neuron reduction in the retinal ganglion cell layer. Mice with targeted disruption of single muscarinic acetylcholine receptor subtype genes (M1 to M5) and wild-type controls were examined at two age categories, 5 and 15 months, respectively. We found no differences in intraocular pressure between individual mouse groups. Remarkably, in 15-month-old mice devoid of the M1 receptor, neuron number in the retinal ganglion cell layer and axon number in the optic nerve were markedly reduced. Moreover, mRNA expression for the prooxidative enzyme, NOX2, was increased, while mRNA expression for the antioxidative enzymes, SOD1, GPx1 and HO-1, was reduced in aged M1 receptor-deficient mice compared to age-matched wild-type mice. In line with these findings, the reactive oxygen species level was also elevated in the retinal ganglion cell layer of aged M1 receptor-deficient mice. In conclusion, M1 receptor deficiency results in retinal ganglion cell loss in aged mice via involvement of oxidative stress. Based on these findings, activation of M1 receptor signaling may become therapeutically useful to promote retinal ganglion cell survival.
Asunto(s)
Envejecimiento/metabolismo , Receptor Muscarínico M1/metabolismo , Células Ganglionares de la Retina/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Animales , Supervivencia Celular , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Noqueados , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Receptor Muscarínico M1/deficiencia , Células Ganglionares de la Retina/patologíaRESUMEN
To address the significance of enhancing myelination for functional recovery after white matter injury (WMI) in preterm infants, we characterized hypomyelination associated with chronic hypoxia and identified structural and functional deficits of excitatory cortical synapses with a prolonged motor deficit. We demonstrate that genetically delaying myelination phenocopies the synaptic and functional deficits observed in mice after hypoxia, suggesting that myelination may possibly facilitate excitatory presynaptic innervation. As a gain-of-function experiment, we specifically ablated the muscarinic receptor 1 (M1R), a negative regulator of oligodendrocyte differentiation in oligodendrocyte precursor cells. Genetically enhancing oligodendrocyte differentiation and myelination rescued the synaptic loss after chronic hypoxia and promoted functional recovery. As a proof of concept, drug-based myelination therapies also resulted in accelerated differentiation and myelination with functional recovery after chronic hypoxia. Together, our data indicate that myelination-enhancing strategies in preterm infants may represent a promising therapeutic approach for structural/functional recovery after hypoxic WMI.
Asunto(s)
Hipoxia/metabolismo , Vaina de Mielina/fisiología , Neurogénesis/fisiología , Oligodendroglía/fisiología , Recuperación de la Función/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Enfermedad Crónica , Femenino , Hipoxia/genética , Hipoxia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vaina de Mielina/química , Vaina de Mielina/patología , Receptor Muscarínico M1/deficiencia , Sinapsis/química , Sinapsis/patologíaRESUMEN
Cholinergic nervous system regulates liver injury. However, the role of M1 muscarinic receptors (M1R) in modulating chronic liver injury is uncertain. To address this gap in knowledge we treated M1R-deficient and WT mice with azoxymethane (AOM) for six weeks and assessed liver injury responses 14 weeks after the last dose of AOM. Compared to AOM-treated WT mice, M1R-deficient mice had attenuated liver nodularity, fibrosis and ductular proliferation, α-SMA staining, and expression of α1 collagen, Tgfß-R, Pdgf-R, Mmp-2, Timp-1 and Timp-2. In hepatocytes, these findings were associated with reductions of cleaved caspase-3 staining and Tnf-α expression. In response to AOM treatment, M1R-deficient mice mounted a vigorous anti-oxidant response by upregulating Gclc and Nqo1 expression, and attenuating peroxynitrite generation. M1R-deficient mouse livers had increased expression of Trail-R2, a promotor of stellate cell apoptosis; dual staining for TUNNEL and α-SMA revealed increased stellate cells apoptosis in livers from M1R-deficient mice compared to those from WT. Finally, pharmacological inhibition of M1R reduced H2O2-induced hepatocyte apoptosis in vitro. These results indicate that following liver injury, anti-oxidant response in M1R-deficient mice attenuates hepatocyte apoptosis and reduces stellate cell activation, thereby diminishing fibrosis. Therefore, targeting M1R expression and activation in chronic liver injury may provide therapeutic benefit.
Asunto(s)
Azoximetano/efectos adversos , Hepatopatías/etiología , Receptor Muscarínico M1/deficiencia , Enfermedad Aguda , Animales , Apoptosis/genética , Conductos Biliares/metabolismo , Conductos Biliares/patología , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Fibrosis , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Hiperplasia , Hepatopatías/metabolismo , Hepatopatías/patología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Estrés Oxidativo , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with ß2-adrenergic (ß2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating ß2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.
Asunto(s)
Miedo/fisiología , Canales de Potasio KCNQ/metabolismo , Memoria/fisiología , Receptor Muscarínico M1/fisiología , Fosfolipasas de Tipo C/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/efectos adversos , Inhibidores Enzimáticos/farmacología , Miedo/efectos de los fármacos , Femenino , Canales de Potasio KCNQ/efectos de los fármacos , Masculino , Moduladores del Transporte de Membrana/farmacología , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Agonistas Muscarínicos/farmacología , Procaterol/farmacología , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/deficiencia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the muscarinic M3 receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the muscarinic M3 receptor. The objective of this work was to study the contribution of the muscarinic M3 receptor to allergen-induced remodeling using muscarinic M3 receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the muscarinic M3 receptor, and not via M1 or M2 receptors. No stimulatory role for muscarinic M3 receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.
Asunto(s)
Acetilcolina/metabolismo , Remodelación de las Vías Aéreas (Respiratorias) , Alérgenos , Pulmón/metabolismo , Músculo Liso/metabolismo , Ovalbúmina , Neumonía/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Matriz Extracelular/metabolismo , Femenino , Células Caliciformes/metabolismo , Células Caliciformes/patología , Mediadores de Inflamación/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Metaplasia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso/patología , Músculo Liso/fisiopatología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Neumonía/inducido químicamente , Neumonía/patología , Neumonía/fisiopatología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/deficiencia , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/deficiencia , Receptor Muscarínico M3/genéticaRESUMEN
Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin-immunoreactive (PV) neurons in macaque V1 express the m1-type (pirenzepine-sensitive, Gq-coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine-sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV- and mAChR-immunoreactive somata by dual-immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV-ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only â¼25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq-coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neuronas/metabolismo , Parvalbúminas/metabolismo , Receptor Muscarínico M1/metabolismo , Corteza Visual/citología , Adulto , Animales , Recuento de Células , Femenino , Hurones , Regulación de la Expresión Génica/genética , Cobayas , Humanos , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptor Muscarínico M1/deficiencia , Especificidad de la Especie , Transducción GenéticaRESUMEN
We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 µM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 µM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation.
Asunto(s)
Calcio/metabolismo , Páncreas/metabolismo , Receptor Muscarínico M1/fisiología , Receptor Muscarínico M3/fisiología , Acetilcolina/farmacología , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Páncreas/citología , Páncreas/efectos de los fármacos , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M3/deficiencia , Receptor Muscarínico M3/efectos de los fármacosRESUMEN
Cholinergic receptors have been implicated in schizophrenia, Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, to better target therapeutically the appropriate receptor subsystems, we need to understand more about the functions of those subsystems. In the current series of experiments, we assessed the functional role of M(1) receptors in cognition by testing M(1) receptor-deficient mice (M1R(-/-)) on the five-choice serial reaction time test of attentional and response functions, carried out using a computer-automated touchscreen test system. In addition, we tested these mice on several tasks featuring learning, memory and perceptual challenges. An advantage of the touchscreen method is that each test in the battery is carried out in the same task setting, using the same types of stimuli, responses and feedback, thus providing a high level of control and task comparability. The surprising finding, given the predominance of the M(1) receptor in cortex, was the complete lack of effect of M(1) deletion on measures of attentional function per se. Moreover, M1R(-/-) mice performed relatively normally on tests of learning, memory and perception, although they were impaired in object recognition memory with, but not without an interposed delay interval. They did, however, show clear abnormalities on a variety of response measures: M1R(-/-) mice displayed fewer omissions, more premature responses, and increased perseverative responding compared to wild-types. These data suggest that M1R(-/-) mice display abnormal responding in the face of relatively preserved attention, learning and perception.
Asunto(s)
Atención/fisiología , Condicionamiento Clásico/fisiología , Tiempo de Reacción/genética , Receptor Muscarínico M1/deficiencia , Percepción Visual/genética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estimulación Luminosa/métodos , Tiempo de Reacción/fisiologíaRESUMEN
OBJECTIVE: Altered muscarinic acetylcholine receptor levels and receptor-coupled signaling processes have been reported in mood disorders. M(1) , one of five muscarinic receptor subtypes, couples to the phospholipase C/protein kinase C and extracellular signal-regulated kinase (ERK) pathways. Mood stabilizers regulate these pathways. MicroRNAs (miRNAs) are small noncoding RNAs that suppress translation in a sequence-selective manner. Lithium downregulates several miRNAs, including let-7b and let-7c. One predicted target of let-7b and let-7c is the M(1) receptor. We hypothesized that miRNAs regulate M(1) receptor translation, and that disrupted M(1) expression leads to aberrant behaviors and disrupted downstream signaling pathways that are rescued by lithium treatment. METHODS: The effects of miRNAs and chronic treatment with mood stabilizers on M(1) levels were tested in primary cultures and in rat frontal cortex. Effects of M(1) ablation and chronic treatment with mood stabilizers on several signaling cascades and M(1) -modulated behaviors were examined in wild-type and M(1) knockout mice. RESULTS: Let-7b, but not let-7c, negatively regulated M(1) levels. Chronic treatment with lithium, but not valproate, increased M(1) levels in the rat cortex. M(1) knockout mice exhibit ERK pathway deficits and behavioral hyperactivity; chronic treatment with lithium attenuated these deficits and hyperactivity. CONCLUSIONS: Lithium treatment can affect M(1) receptor function through intracellular signaling enhancement and, in situations without M(1) ablation, concomitant receptor upregulation via mechanisms involving miRNAs. Muscarinic dysfunction may contribute to mood disorders, while M(1) receptors and the downstream ERK pathway may serve as potential therapeutic targets for alleviating manic symptoms such as psychomotor hyperactivity.
Asunto(s)
Antimaníacos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Litio/farmacología , Receptor Muscarínico M1/metabolismo , Anfetamina , Análisis de Varianza , Animales , Células Cultivadas , Corteza Cerebral/citología , Interacciones Farmacológicas , Conducta Exploratoria/efectos de los fármacos , Lóbulo Frontal/efectos de los fármacos , Proteína GAP-43/metabolismo , Regulación de la Expresión Génica/genética , Hipercinesia/inducido químicamente , Hipercinesia/tratamiento farmacológico , Relaciones Interpersonales , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Aprendizaje por Laberinto/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/farmacología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Endogámicas WKY , Receptor Muscarínico M1/deficiencia , Natación/psicologíaRESUMEN
Alzheimer's disease (AD) is a progressive neurological disorder that causes dementia and poses a major public health crisis as the population ages. Aberrant processing of the amyloid precursor protein (APP) is strongly implicated as a proximal event in AD pathophysiology, but the neurochemical signals that regulate APP processing in the brain are not completely understood. Activation of muscarinic acetylcholine receptors (mAChRs) has been shown to affect APP processing and AD pathology, but less is known about the roles of specific mAChR subtypes. In this study, we used M(1) mAChR knock-out mice (M(1)KO) to isolate the effects of the M(1) mAChR on APP processing in primary neurons and on the development of amyloid pathology in a transgenic mouse model of AD. We demonstrate that the loss of M(1) mAChRs increases amyloidogenic APP processing in neurons, as evidenced by decreased agonist-regulated shedding of the neuroprotective APP ectodomain APPsalpha and increased production of toxic Abeta peptides. Expression of M(1) mAChRs on the M(1)KO background rescued this phenotype, indicating that M(1) mAChRs are sufficient to modulate nonamyloidogenic APP processing. In APP(Swe/Ind) transgenic mice, the loss of M(1) mAChRs resulted in increased levels of brain Abeta and greater accumulation of amyloid plaque pathology. Analysis of APP metabolites in APP(Swe/Ind) brain tissue indicates that the loss of M(1) mAChRs increases amyloidogenic APP processing. These results indicate that the M(1) mAChR is an important regulator of amyloidogenesis in the brain and provide strong support for targeting the M(1) mAChR as a therapeutic candidate in AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/patología , Regulación de la Expresión Génica/fisiología , Neuronas/metabolismo , Receptor Muscarínico M1/deficiencia , Proteínas ADAM/metabolismo , Proteína ADAM10 , Actinas/metabolismo , Factores de Edad , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Carbacol/farmacología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Agonistas Colinérgicos/farmacología , Embrión de Mamíferos , Ensayo de Inmunoadsorción Enzimática/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/metabolismoRESUMEN
Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called "volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M(1), the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M(1) was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M(1) was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M(1) and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M(1) is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M(1)-mediated cortical cholinergic signaling.
Asunto(s)
Corteza Cerebral/citología , Dendritas/ultraestructura , Espinas Dendríticas/metabolismo , Células Piramidales/ultraestructura , Receptor Muscarínico M1/metabolismo , Animales , Calbindina 2 , Dendritas/metabolismo , Glutamato Descarboxilasa/metabolismo , Hipocampo/citología , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión/métodos , Microscopía Inmunoelectrónica/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptido Y/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M1/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
To investigate the role of M1 muscarininc acetylcholine receptors (m1 receptors) in metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), we produced mouse lines in which deletion of the m1 gene is restricted to the forebrain (FB-m1KO) or hippocampal CA3 pyramidal neurons (CA3-m1KO). Stimulation in FB-m1KO hippocampal slices resulted in excitatory postsynaptic potentials and long-term synaptic plasticity (long-term potentiation and LTD) similar to controls. The mice were deficient in (S)-3,5-dihydroxyphenylglycine hydrate (DHPG)-induced mGluR LTD, which correlated with a presynaptic increase in the release of neurotransmitters. Protein kinase C (PKC) activity, which is downstream from both mGluRs and m1 receptors, was reduced in CA3 but not in CA1. The presynaptic requirement of m1 receptors was confirmed by the lack of DHPG-induced mGluR LTD in the CA1 of slices from CA3-m1KO mice. mGluR LTD was rescued by stimulating PKC activity pharmacologically in CA3-m1KO mice. These data confirm a role for PKC activation in presynaptic induction of mGluR LTD and distinguish between the roles of mGluRs and m1 receptors.
Asunto(s)
Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo , Receptor Muscarínico M1/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Ratones , Ratones Noqueados , Proteína Quinasa C/metabolismo , Receptor Muscarínico M1/deficiencia , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The forebrain cholinergic system promotes higher brain function in part by signaling through the M(1) muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M(1) receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M(1) mAChR. BQCA reduces the concentration of ACh required to activate M(1) up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 microM. Furthermore studies in M(1)(-/-) mice demonstrates that BQCA requires M(1) to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M(1) allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces beta-arrestin recruitment to M(1), suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M(1) receptor and represents a promising therapeutic strategy for cognitive disorders.
Asunto(s)
Receptor Muscarínico M1/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Señalización del Calcio/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Circulación Cerebrovascular/fisiología , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Cricetinae , Cricetulus , Perros , Miedo/efectos de los fármacos , Miedo/fisiología , Humanos , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Macaca mulatta , Ratones , Ratones Noqueados , Modelos Moleculares , Estructura Terciaria de Proteína , Quinolonas/farmacología , Ensayo de Unión Radioligante , Ratas , Receptor Muscarínico M1/química , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sueño/efectos de los fármacos , Sueño/fisiologíaRESUMEN
The muscarinic acetylcholine receptor (mAChR) agonist, xanomeline, attenuates amphetamine-induced activity in WT mice. This effect is abolished in mice lacking the M(4) muscarinic acetylcholine receptor (M(4) mAChR KO) and partially attenuated in mice lacking M(1) muscarinic acetylcholine receptor (M(1) mAChR KO). Collectively, these data suggest that the efficacy exhibited by xanomeline in the mouse amphetamine-induced hyperactivity model, is mediated predominantly by M(4) muscarinic acetylcholine receptors, and that M(1) muscarinic acetylcholine receptors may play a more minor role. This supports the hypothesis that activation of M(4), and to a lesser extent M(1) muscarinic acetylcholine receptors, may represent a potential target for the treatment of psychosis seen in schizophrenia.
Asunto(s)
Anfetamina/toxicidad , Agonistas Muscarínicos/farmacología , Piridinas/farmacología , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M1/genética , Receptor Muscarínico M4/deficiencia , Receptor Muscarínico M4/genética , Tiadiazoles/farmacología , Animales , Hipercinesia/inducido químicamente , Hipercinesia/tratamiento farmacológico , Ratones , Ratones Noqueados , Agonistas Muscarínicos/uso terapéutico , Piridinas/uso terapéutico , Especificidad por Sustrato , Tiadiazoles/uso terapéuticoRESUMEN
Dopamine-depleting lesions of the striatum that mimic Parkinson's disease induce a profound pruning of spines and glutamatergic synapses in striatopallidal medium spiny neurons, leaving striatonigral medium spiny neurons intact. The mechanisms that underlie this cell type-specific loss of connectivity are poorly understood. The Kir2 K(+) channel is an important determinant of dendritic excitability in these cells. Here we show that opening of these channels is potently reduced by signaling through M1 muscarinic receptors in striatopallidal neurons, but not in striatonigral neurons. This asymmetry could be attributed to differences in the subunit composition of Kir2 channels. Dopamine depletion alters the subunit composition further, rendering Kir2 channels in striatopallidal neurons even more susceptible to modulation. Reduced opening of Kir2 channels enhances dendritic excitability and synaptic integration. This cell type-specific enhancement of dendritic excitability is an essential trigger for synaptic pruning after dopamine depletion, as pruning was prevented by genetic deletion of M1 muscarinic receptors.
Asunto(s)
Cuerpo Estriado/citología , Dendritas/fisiología , Neuronas/ultraestructura , Canales de Potasio de Rectificación Interna/fisiología , Receptor Muscarínico M1/fisiología , Animales , Animales Recién Nacidos , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Oxidopamina/farmacología , Técnicas de Placa-Clamp/métodos , Receptor Muscarínico M1/deficiencia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Reserpina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca(2+)-signaling and up-regulation of c-fos expression; moreover, M1 mAChRs play a crucial role in the differentiation of CD8(+) T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M1 and M5 mAChR knockout (M1/M5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG1 were significantly lower in M1/M5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M1/M5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M1/M5 KO mice. These results suggest that M1 and/or M5 mAChRs are involved in regulating cytokine (e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.
Asunto(s)
Acetilcolinesterasa/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Inmunoglobulina G/sangre , Interleucina-6/metabolismo , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M5/deficiencia , Acetilcolinesterasa/genética , Animales , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Interferón gamma/metabolismo , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
RATIONALE: Muscarinic acetylcholine receptors (M1-M5) modulate the activity of the central nervous system and an array of physiological functions. Recent evidence has also implicated muscarinic receptors in behavioral effects of drugs of abuse such as morphine and cocaine. However, the genetic similarity between muscarinic receptors and the coexpression of multiple subtypes in most cells has impeded the development of selective antagonists and the determination of the role of each muscarinic receptor subtype in morphine's and cocaine's behavioral effects. OBJECTIVE: The present studies employ mice deficient in the M1 receptor subtype (M1 KO) to assess morphine antinociception (2.5, 5.0, 10, or 20 mg/kg) and the conditioned rewarding effects of morphine and cocaine (2.5, 5.0, or 10 mg/kg). METHODS: M1 KO and their wild-type (WT) littermates were tested using a 56 degrees C hotplate assay and a conditioned place preference procedure. Parallel studies using the M1 receptor antagonist, pirenzepine, were also conducted in the background strain C57BL6 mice. RESULTS: The results demonstrate that M1 KO mice display a greater antinociceptive effect of morphine in the hotplate assay; however, the effects of morphine as well as cocaine were attenuated in the conditioned place preference procedure. Comparable results were obtained with the pharmacological antagonism of the M1 receptor by pirenzepine. CONCLUSIONS: These results suggest a modulatory role of the M1 muscarinic receptor in opioid antinociception and conditioned drug reward, and demonstrate the utility of M1 receptor knockout models for the determination of the role of the M1 subtype in the behavioral effects of morphine and cocaine.
Asunto(s)
Analgésicos Opioides/farmacología , Conducta Animal/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Cocaína/farmacología , Morfina/farmacología , Receptor Muscarínico M1/metabolismo , Animales , Condicionamiento Psicológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas Muscarínicos/farmacología , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Pirenzepina/farmacología , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M1/genética , RecompensaRESUMEN
The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.
Asunto(s)
Cognición/fisiología , Hormonas/metabolismo , Receptor Muscarínico M1/deficiencia , Receptor Muscarínico M2/deficiencia , Receptor Muscarínico M3/deficiencia , Animales , Carbacol/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/genéticaRESUMEN
Cholinergically induced intestinal anion secretion is generally believed to be caused by stimulation of epithelial muscarinic M3 receptors, whereas muscarinic M1 receptors are thought to be localized primarily on enteric neurons. In order to test this assumption, carbachol-stimulated Cl- secretion across distal colon, measured as increase in short-circuit current (I(sc)), was compared between M1-knockout (M1R-KO) and M3-knockout (M3R-KO) mice. Surprisingly, the maximal increase in I(sc) evoked by carbachol was more than twice as large in M3R-KO compared to M1R-KO mice. This difference was not due to a reduced secretory capacity of the epithelium from M3R-KO animals, as forskolin stimulated a similar maximal I(sc) in both types of animals. The neurotoxin tetrodotoxin diminished, but did not abolish the secretory response evoked by carbachol in M3R-KO distal colon, suggesting the existence of epithelial muscarinic receptors other than the type M3. Furthermore, in muscarinic receptor wild-type animals, the muscarinic M1 receptor antagonist pirenzepine inhibited the carbachol-stimulated I(sc) by more than 70% suggesting the presence of epithelial muscarinic M1 receptors; a conclusion, which was confirmed by the identification of mRNA for muscarinic M1 receptors in isolated crypts from wild-type colon. Consequently, epithelial muscarinic receptors from the type M1 contribute to cholinergically induced ion secretion in mouse colon.