RESUMO
The M1 muscarinic acetylcholine receptor (mAChR) subtype has been implicated in the underlying mechanisms of learning and memory and represents an important potential pharmacotherapeutic target for the cognitive impairments observed in neuropsychiatric disorders such as schizophrenia. Patients with schizophrenia show impairments in top-down processing involving conflict between sensory-driven and goal-oriented processes that can be modeled in preclinical studies using touchscreen-based cognition tasks. The present studies used a touchscreen visual pairwise discrimination task in which mice discriminated between a less salient and a more salient stimulus to assess the influence of the M1 mAChR on top-down processing. M1 mAChR knockout (M1 KO) mice showed a slower rate of learning, evidenced by slower increases in accuracy over 12 consecutive days, and required more days to acquire (achieve 80% accuracy) this discrimination task compared to wild-type mice. In addition, the M1 positive allosteric modulator BQCA enhanced the rate of learning this discrimination in wild-type, but not in M1 KO, mice when BQCA was administered daily prior to testing over 12 consecutive days. Importantly, in discriminations between stimuli of equal salience, M1 KO mice did not show impaired acquisition and BQCA did not affect the rate of learning or acquisition in wild-type mice. These studies are the first to demonstrate performance deficits in M1 KO mice using touchscreen cognitive assessments and enhanced rate of learning and acquisition in wild-type mice through M1 mAChR potentiation when the touchscreen discrimination task involves top-down processing. Taken together, these findings provide further support for M1 potentiation as a potential treatment for the cognitive symptoms associated with schizophrenia.
Assuntos
Colinérgicos/uso terapêutico , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/genética , Discriminação Psicológica/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Receptor Muscarínico M1/metabolismo , Análise de Variância , Animais , Condicionamento Operante/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estimulação Luminosa , Quinolonas/farmacologia , Quinolonas/uso terapêutico , RNA Mensageiro , Receptor Muscarínico M1/genética , Esquema de Reforço , Reforço Psicológico , Tato/fisiologiaRESUMO
Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1-M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect, most likely through the M1 receptor, and normalised the ATP response. M1, M4 and M5 receptor-deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptors are missing. None of the receptors is necessary for epithelial development.
Assuntos
Cílios/fisiologia , Receptores Muscarínicos/deficiência , Traqueia/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Cílios/ultraestrutura , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Depuração Mucociliar , Muscarina/farmacologia , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
It is generally accepted that cardiac sympathetic tone dominates the control of heart rate (HR) in mice. However, we have recently challenged this notion given that HR in the mouse is responsive to ambient temperature (T(a)) and that the housing T(a) is typically 21-23 degrees C, well below the thermoneutral zone ( approximately 30 degrees C) of this species. To specifically test the hypothesis that cardiac sympathetic tone is the primary mediator of HR control in the mouse, we first examined the metabolic and cardiovascular responses to rapid changes in T(a) to demonstrate the sensitivity of the mouse cardiovascular system to T(a). We then determined HR in 1) mice deficient in cardiac sympathetic tone ("beta-less" mice), 2) mice deficient in cardiac vagal tone [muscarinic M(2) receptor (M(2)R(-/-)) mice], and 3) littermate controls. At a T(a) of 30 degrees C, the HR of beta-less mice was identical to that of wild-type mice (351 +/- 11 and 363 +/- 10 beats/min, respectively). However, the HR of M(2)R(-/-) mice was significantly greater (416 +/- 7 beats/min), demonstrating that vagal tone predominates over HR control at this T(a). When these mice were calorically restricted to 70% of normal intake, HR fell equally in wild-type, beta-less, and M(2)R(-/-) mice (DeltaHR = 73 +/- 9, 76 +/- 3, and 73 +/- 7 beats/min, respectively), suggesting that the fall in intrinsic HR governs bradycardia of calorically restricted mice. Only when the T(a) was relatively cool, at 23 degrees C, did beta-less mice exhibit a HR (442 +/- 14 beats/min) that was different from that of littermate controls (604 +/- 10 beats/min) and M(2)R(-/-) mice (602 +/- 5 beats/min). These experiments conclusively demonstrate that in the absence of cold stress, regulation of vagal tone and modulation of intrinsic rate are important determinants of HR control in the mouse.
Assuntos
Sistema Nervoso Autônomo/metabolismo , Temperatura Corporal , Frequência Cardíaca , Coração/inervação , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta/metabolismo , Nervo Vago/metabolismo , Animais , Pressão Sanguínea , Bradicardia/etiologia , Bradicardia/metabolismo , Bradicardia/fisiopatologia , Restrição Calórica/efeitos adversos , Temperatura Baixa , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Muscarínico M2/deficiência , Receptor Muscarínico M2/genética , Receptores Adrenérgicos beta/deficiência , Receptores Adrenérgicos beta/genéticaRESUMO
The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.
Assuntos
Glicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Receptor Muscarínico M3/fisiologia , Acetilcolina , Animais , Diabetes Mellitus Tipo 2/metabolismo , Glucose/administração & dosagem , Intolerância à Glucose/metabolismo , Homeostase , Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/metabolismoRESUMO
BACKGROUND AND PURPOSE: The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes. EXPERIMENTAL APPROACH: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M(2)-knockout (KO), M(3)-KO, M(2)/M(3)-double KO, and wild-type mice. KEY RESULTS: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M(2)-KO or M(3)-KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M(2)-KO or M(3)-KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K(+)-induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M(3)-KO but not in M(2)-KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M(2)/M(3)-double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains. CONCLUSION AND IMPLICATIONS: These results demonstrate that M(2) and M(3) receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M(2) and M(3) receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.
Assuntos
Músculo Liso/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/fisiologia , Anestésicos Locais/farmacologia , Animais , Atropina/farmacologia , Capsaicina/farmacologia , Estimulação Elétrica , Feminino , Íleo/efeitos dos fármacos , Contração Isométrica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Sistema Nervoso Parassimpático/efeitos dos fármacos , Toxina Pertussis/farmacologia , Receptor Muscarínico M2/efeitos dos fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/efeitos dos fármacos , Receptor Muscarínico M3/genética , Tetrodotoxina/farmacologiaRESUMO
The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine (ACh) release at frog and mouse neuromuscular junctions was studied by measuring the dependency of the amount (m) of ACh release on the level of presynaptic depolarization. Addition of methoctramine (a blocker of M2 muscarinic receptors), or of acetylcholinesterase (AChE), increased release in a voltage-dependent manner; enhancement of release declined as the depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from about 7 in the control to about 4 in the presence of methoctramine or AChE. In M2 muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (about 4) and was not further reduced by addition of either methoctramine or AChE. The effect of a brief (0.1 ms), but strong (-1.2 microA) depolarizing prepulse on the dependency of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and AChE. In particular, it enhanced release of test pulses in a voltage-dependent manner and reduced the slope of log m/log PA from about 7 to about 4. Methoctramine + AChE occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic ACh release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M2 muscarinic receptors.
Assuntos
Acetilcolina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptor Muscarínico M2/fisiologia , Acetilcolinesterase/farmacologia , Animais , Diaminas/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Técnicas In Vitro , Modelos Lineares , Camundongos , Camundongos Knockout , Junção Neuromuscular/metabolismo , Junção Neuromuscular/efeitos da radiação , Parassimpatolíticos/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos da radiação , Rana ridibunda , Receptor Muscarínico M2/deficiência , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Transmissão Sináptica/efeitos da radiação , Fatores de TempoRESUMO
A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.
Assuntos
Receptores Muscarínicos/genética , Animais , Comportamento Animal/fisiologia , Epilepsia/genética , Coração/fisiologia , Camundongos , Camundongos Knockout , Músculo Liso/fisiologia , Receptores Muscarínicos/fisiologiaRESUMO
Among the five different muscarinic receptors that have been cloned and characterized, M2 and M4 receptors are localized both post- and presynaptically and are believed to have a pronounced autoreceptor role. The functional importance of these receptors in the regulation of acetylcholine release in the hippocampus and in cognitive processes was investigated by using M2 and M4 receptor single knockout (KO) as well as M2/M4 receptor double KO mice. We found profound alterations in acetylcholine homeostasis in the hippocampus of both M2- and M4-KO mice as well as of the combined M2/M4-KOs, as assessed by in vivo microdialysis. Basal acetylcholine efflux in the hippocampus was significantly increased in M4-KO and was elevated further in M2/M4-KOs. The increase in hippocampal acetylcholine induced by local administration of scopolamine was markedly reduced in M2-KO and completely abolished in M2/M4-KOs. In M2-KO and much more in M2/M4-KOs, the increase in hippocampal acetylcholine triggered by exposure to a novel environment was more pronounced both in amplitude and duration, with a similar trend observed for M4-KOs. Dysregulation of cholinergic function in the hippocampus, as it could result from perturbed autoreceptor function, may be associated with cognitive deficits. Importantly, M2- and M2/M4-KO, but not M4-KO, animals showed an impaired performance in the passive avoidance test. Together these results suggest a crucial role for muscarinic M2 and M4 receptors in the tonic and phasic regulation of acetylcholine efflux in the hippocampus as well as in cognitive processes.
Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M4/fisiologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Cognição/fisiologia , Cruzamentos Genéticos , Meio Ambiente , Habituação Psicofisiológica/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Knockout , Microdiálise , Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M2/deficiência , Receptor Muscarínico M2/efeitos dos fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M4/deficiência , Receptor Muscarínico M4/efeitos dos fármacos , Receptor Muscarínico M4/genética , Escopolamina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.
Assuntos
Piridinas/metabolismo , Receptores Muscarínicos/deficiência , Tiazóis/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Radioisótopos de Flúor/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Muscarínico M1 , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Receptor Muscarínico M4 , Receptores Muscarínicos/genéticaRESUMO
We have previously suggested that presynaptic M(2)-muscarinic receptors (M(2)R) are involved in the control of the time course of evoked acetylcholine release in the frog neuromuscular junction. The availability of knockout mice lacking functional M(2)R (M(2)-KO) enabled us to address this issue in a more direct way. Using the phrenic diaphragm preparation, we show that in wild-type (WT) mice experimental manipulations known to affect Ca(2+) entry and removal, greatly affected the amount of acetylcholine released (quantal content). However, the time course of release remained unaltered under all these experimental treatments. On the other hand, in the M(2)-KO mice, similar experimental treatments affected both the quantal content and the time course of release. In general, a larger quantal content was accompanied by a longer duration of release. Similarly, the rise time of the postsynaptic current produced by axon stimulation was sensitive to changes in [Ca(2+)](o) or [Mg(2+)](o) in M(2)-KO mice but not in WT mice. Measurements of Ca(2+) currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg(2+)](o) in M(2)-KO mice was not produced by a shorter wave of the presynaptic Ca(2+) current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M(2)-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.
Assuntos
Acetilcolina/metabolismo , Cálcio/farmacocinética , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Espaço Extracelular/metabolismo , Camundongos , Camundongos Knockout , Terminações Pré-Sinápticas/metabolismo , Receptor Muscarínico M2 , Sódio/metabolismoRESUMO
Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions in the central and peripheral nervous system. Due to the lack of receptor subtype-selective ligands, however, the physiological roles of individual muscarinic receptor subtypes remain to be determined. In this study, we examined the effects of the muscarinic M(2)/M(4) receptor-preferring agonist [5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.2.1]-octane (BuTAC) on serum corticosterone levels in M(2) and M(4) receptor single knockout (KO) and M(2,4) receptor double KO mice. Responses were compared with those obtained with the corresponding wild-type (WT) mice. BuTAC (0.03-0.3 mg/kg s.c.) dose dependently and significantly increased serum corticosterone concentrations in WT mice to 5-fold or greater levels compared with vehicle controls. In muscarinic M(2) and M(2,4) KO mice, however, BuTAC had no significant effect on corticosterone concentrations at doses of 0.1, 0.3, and 1 mg/kg s.c. In both WT and muscarinic M(4) KO mice increases in serum corticosterone concentrations induced by BuTAC (0.1 and 0.3 mg/kg) were not significantly different and were blocked by scopolamine. In summary, the muscarinic M(2,4)-preferring agonist BuTAC had no effect on corticosterone levels in mice lacking functional muscarinic M(2) receptors. These data suggest that the muscarinic M(2) receptor subtype mediates muscarinic agonist-induced activation of the hypothalamic-pituitary-adrenocortical axis in mice.
Assuntos
Corticosterona/sangue , Agonistas Muscarínicos/farmacologia , Receptores Muscarínicos/fisiologia , Tropanos/farmacologia , Animais , Cinética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Pergolida/farmacologia , Receptor Muscarínico M2 , Receptor Muscarínico M4 , Receptores Muscarínicos/deficiência , Receptores Muscarínicos/genética , Escopolamina/farmacologiaRESUMO
The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice). M5R(-/-) mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M(5) receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M(5) receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M(5) receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R(-/-) mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R(-/-) mice. Our findings provide direct evidence that M(5) muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M(5) receptors may represent an attractive therapeutic target.
Assuntos
Acetilcolina/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Acetilcolina/metabolismo , Animais , Artérias/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Artérias Cerebrais/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dopamina/metabolismo , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Receptor Muscarínico M5 , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Salivação/efeitos dos fármacos , TremorRESUMO
Members of the muscarinic acetylcholine receptor family are thought to play key roles in the regulation of a large number of important functions of the CNS. However, the precise roles of the individual muscarinic receptor subtypes in modulating these processes are not well understood at present, primarily because of the lack of ligands with sufficient receptor subtype selectivity. To investigate the behavioral significance of the M(1) muscarinic receptor (M(1)R), which is abundantly expressed in the forebrain, we subjected M(1) receptor-deficient mice (M(1)R(-/-) mice) to a battery of behavioral tests. M(1)R(-/-) mice showed no significant impairments in neurological reflexes, motor coordination, pain sensitivity, and prepulse inhibition. Strikingly, however, M(1)R(-/-) mice consistently exhibited a pronounced increase in locomotor activity in various tests, including open field, elevated plus maze, and light/dark transition tests. Moreover, M(1)R(-/-) mice showed reduced immobilization in the Porsolt forced swim test and reduced levels of freezing after inescapable footshocks, suggesting that M(1)R(-/-) mice are hyperactive under stressful conditions as well. An increased number of social contacts was observed in a social interaction test. Surprisingly, M(1)R(-/-) mice displayed no significant cognitive impairments in the Morris water maze and in contextual fear conditioning. M(1)R(-/-) mice showed slight performance deficits in auditory-cued fear conditioning and in an eight-arm radial maze, most likely because of the hyperactivity phenotype displayed by the M(1)R(-/-) mice. Our results indicate that M(1) muscarinic receptors play an important role in the regulation of locomotor activity but appear to be less critical for cognitive processes, as generally assumed.
Assuntos
Hipocampo/fisiologia , Hipercinese , Aprendizagem/fisiologia , Receptores Muscarínicos/deficiência , Animais , Aprendizagem da Esquiva/fisiologia , Comportamento Animal/fisiologia , Corpo Estriado/metabolismo , Medo/fisiologia , Homozigoto , Hipercinese/fisiopatologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Reflexo de Sobressalto/fisiologia , Comportamento Social , Comportamento Espacial/fisiologiaRESUMO
Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.
Assuntos
Analgesia , Química Encefálica , Marcação de Genes , Atividade Motora , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Receptores Muscarínicos/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Acetilcolina/metabolismo , Analgésicos/farmacologia , Animais , Apomorfina/farmacologia , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Atividade Motora/efeitos dos fármacos , Medição da Dor , Quimpirol/farmacologia , Ensaio Radioligante , Receptor Muscarínico M2 , Receptor Muscarínico M4 , Receptores Dopaminérgicos/metabolismo , Receptores Muscarínicos/genética , Vasodilatadores/metabolismoRESUMO
Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.
Assuntos
Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Receptores Muscarínicos/fisiologia , Animais , Carbacol/farmacologia , Córtex Cerebral/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotermia/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Agonistas Muscarínicos/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Oxotremorina/metabolismo , Ensaio Radioligante , Receptor Muscarínico M2 , Receptor Muscarínico M4 , Receptores Muscarínicos/genética , Salivação/efeitos dos fármacos , Salivação/fisiologia , Tremor/induzido quimicamenteRESUMO
Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.
Assuntos
Canais de Cálcio/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Muscarínicos/metabolismo , Gânglio Cervical Superior/citologia , Animais , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Camundongos , Camundongos Knockout , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Oxotremorina/farmacologia , Isoformas de Proteínas/genética , Ratos , Receptores Muscarínicos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/efeitos dos fármacos , Gânglio Cervical Superior/fisiologia , Fatores de TempoRESUMO
Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.
Assuntos
Doença de Alzheimer/fisiopatologia , Neurônios/metabolismo , Receptores Muscarínicos/metabolismo , Esquizofrenia/fisiopatologia , Animais , Fracionamento Celular , Linhagem Celular , Membrana Celular/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Masculino , Memória/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Fenciclidina/farmacologia , Ensaio Radioligante/métodos , Receptores Muscarínicos/genética , Transdução de Sinais/fisiologiaRESUMO
The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.
Assuntos
Receptores Muscarínicos/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Western Blotting , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/genética , Humanos , Ligantes , Dados de Sequência Molecular , Plasmídeos/genética , Ensaio Radioligante , Receptor Muscarínico M1 , Receptor Muscarínico M3 , Receptor Muscarínico M5 , Receptores Muscarínicos/genéticaRESUMO
To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.
Assuntos
Arginina Vasopressina/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Vasopressinas/química , Receptores de Vasopressinas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Bovinos , Membrana Celular/fisiologia , Clonagem Molecular , Primers do DNA , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Biblioteca Gênica , Proteínas Heterotriméricas de Ligação ao GTP/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Subunidades Proteicas , Receptores de Vasopressinas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Members of the muscarinic acetylcholine receptor family (M1-M5) have central roles in the regulation of many fundamental physiological functions. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R-/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake, are significantly reduced in M3R-/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R-/- mice. However, M3R-/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.