RESUMO
Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (-2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Dopamina/metabolismo , Agitação Psicomotora/genética , Receptores de Dopamina D3/genética , Transmissão Sináptica/genética , Animais , Gânglios da Base/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Feminino , Heterozigoto , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Dopamina D3/deficiência , Regulação para Cima/genéticaRESUMO
Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Disfunção Cognitiva/etiologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Hipercinese/etiologia , Animais , Disfunção Cognitiva/metabolismo , Feminino , Técnicas de Inativação de Genes , Hipercinese/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.
Assuntos
Diferenciação Celular , Reprogramação Celular , Dopamina/metabolismo , Fibroblastos/citologia , Neurônios/citologia , Neurônios/metabolismo , Potenciais de Ação , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Homeodomínio LIM , Camundongos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Doença de Parkinson/patologia , Técnicas de Patch-Clamp , Medicina Regenerativa , Pele/citologia , Fatores de TranscriçãoRESUMO
The dopamine transporter (DAT) plays an important homeostatic role in the control of both the extracellular and intraneuronal concentrations of dopamine, thereby providing effective control over activity of dopaminergic transmission. Since brain dopamine is known to be involved in numerous neuropsychiatric disorders, investigations using mice with genetically altered DAT function and thus intensity of dopamine-mediated signaling have provided numerous insights into the pathology of these disorders and novel pathological mechanisms that could be targeted to provide new therapeutic approaches for these disorders. In this brief overview, we discuss recent investigations involving animals with genetically altered DAT function, particularly focusing on translational studies providing new insights into pathology and pharmacology of dopamine-related disorders. Perspective applications of these and newly developed models of DAT dysfunction are also discussed.
Assuntos
Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Animais , Camundongos , Pesquisa Translacional BiomédicaRESUMO
α-Synuclein is thought to regulate neurotransmitter release through multiple interactions with presynaptic proteins, cytoskeletal elements, ion channels, and synaptic vesicles membrane. α-Synuclein is abundant in the presynaptic compartment, and its release from neurons and glia has been described as responsible for spreading of α-synuclein-derived pathology. α-Synuclein-dependent dysregulation of neurotransmitter release might occur via its action on surface-exposed calcium channels. Here, we provide electrophysiological and biochemical evidence to show that α-synuclein, applied to rat neurons in culture or striatal slices, selectively activates Cav2.2 channels, and said activation correlates with increased neurotransmitter release. Furthermore, in vivo perfusion of α-synuclein into the striatum also leads to acute dopamine release. We further demonstrate that α-synuclein reduces the amount of plasma membrane cholesterol and alters the partitioning of Cav2.2 channels, which move from raft to cholesterol-poor areas of the plasma membrane. We provide evidence for a novel mechanism through which α-synuclein acts from the extracellular milieu to modulate neurotransmitter release and propose a unifying hypothesis for the mechanism of α-synuclein action on multiple targets: the reorganization of plasma membrane microdomains.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Dopamina/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Neurônios/citologia , alfa-Sinucleína/farmacologia , Compostos de Anilina/metabolismo , Animais , Anticorpos/farmacologia , Canais de Cálcio Tipo N/imunologia , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/farmacologia , Gânglio Cervical Superior/citologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Sinaptofisina/metabolismo , Xantenos/metabolismoRESUMO
Striatal dopamine D2 receptor (D2R) relies upon G protein- and ß-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gαi-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of ß-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Células HEK293 , Humanos , Proteína Huntingtina , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso , Proteínas Nucleares , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Several studies have reported the coupling of dopamine signaling to phospholipase C ß (PLCß) both in vitro and in vivo. However, the precise physiological relevance of this signaling pathway in mediating dopamine behaviors is still unclear. Here we report that stimulation of dopamine receptor signaling in vivo with systemic administration of apomorphine, amphetamine, and cocaine leads to increased production of inositol triphosphate (IP3) in the mouse striatum. Using selective antagonists and dopamine D1 and D2 receptor knock-out animals, we show that the production of IP3 is mediated by the D1 receptor, but not the D2 receptor. A selective blocker of PLCß, U73122, was used to assess the physiological relevance of D1-mediated IP3 production. We show that U73122 inhibits the locomotor-stimulating effects of apomorphine, amphetamine, cocaine, and SKF81297. Furthermore, U73122 also suppresses the spontaneous hyperactivity exhibited by dopamine transporter knock-out mice. Importantly, the effects of U73122 are selective to dopamine-mediated hyperactivity, as this compound does not affect hyperactivity induced by the glutamate NMDA receptor antagonist MK801. Finally, we present evidence showing that an imbalance of D1- and D2-mediated signaling following U73122 treatment modifies the locomotor output of animals from horizontal locomotor activity to vertical activity, further highlighting the importance of the PLCß pathway in the regulation of forward locomotion via dopamine receptors.
Assuntos
Atividade Motora/fisiologia , Fosfolipase C beta/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/fisiologiaRESUMO
Chronic N-methyl-d-aspartate receptor (NMDAR) hypofunction has been proposed as a contributing factor to symptoms of schizophrenia. However, it is unclear how sustained NMDAR hypofunction throughout development affects other neurotransmitter systems that have been implicated in the disease. Dopamine neuron biochemistry and activity were examined to determine whether sustained NMDAR hypofunction causes a state of hyperdopaminergia. We report that a global, genetic reduction in NMDARs led to a remodeling of dopamine neurons, substantially affecting two key regulators of dopamine homeostasis, i.e., tyrosine hydroxylase and the dopamine transporter. In NR1 knockdown mice, dopamine synthesis and release were attenuated, and dopamine clearance was increased. Although these changes would have the effect of reducing dopamine transmission, we demonstrated that a state of hyperdopaminergia existed in these mice because dopamine D2 autoreceptors were desensitized. In support of this conclusion, NR1 knockdown dopamine neurons have higher tonic firing rates. Although the tonic firing rates are higher, phasic signaling is impaired, and dopamine overflow cannot be achieved with exogenous high-frequency stimulation that models phasic firing. Through the examination of several parameters of dopamine neurotransmission, we provide evidence that chronic NMDAR hypofunction leads to a state of elevated synaptic dopamine. Compensatory mechanisms to attenuate hyperdopaminergia also impact the ability to generate dopamine surges through phasic firing.
Assuntos
Encéfalo/fisiopatologia , Neurônios Dopaminérgicos/fisiologia , Proteínas do Tecido Nervoso/deficiência , Receptores de N-Metil-D-Aspartato/deficiência , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Dopamina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Estimulação Elétrica , Técnicas de Silenciamento de Genes , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Receptores de Dopamina D2/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Técnicas de Cultura de Tecidos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Although both cocaine and amphetamine mainly target the dopamine transporter (DAT) and cause psychomotor effects, they have very different mechanisms of actions. The authors examined whether responses to cocaine and amphetamine were affected differentially by changes in DAT expression levels using transgenic mice with different DAT expression levels. In the constitutive DAT knockdown mice, reduced DAT expression enhanced cocaine's locomotor stimulatory effects and at the same time diminished amphetamine's locomotor stimulatory effects. Similar effects were observed in the inducible DAT knockdown mice, ruling out the contribution of developmental compensations in DAT knockdown mice. Extracellular dopamine levels in response to psychostimulants were assessed by in vivo microdialysis. Whereas amphetamine-induced increase in extracellular dopamine was drastically diminished in constitutive DAT knockdown mice, cocaine-induced increase in extracellular dopamine had a faster onset in knockdown mice compared with wild-type controls. Postsynaptically, D1 agonist-stimulated c-fos expression was significantly attenuated in constitutive DAT knockdown mice compared with wild-type controls. The authors propose that responses to cocaine and amphetamine depend on psychostimulant drug type, drug dose, as well as DAT expression level. DAT expression level affects presynaptic responses to psychostimulants directly and postsynaptic responses to psychostimulants indirectly via changes in receptor signaling. These data imply that individual differences in DAT expression (either genetically or pharmacologically induced) may affect susceptibility to addiction of different types of psychostimulants.
Assuntos
Anfetamina/farmacologia , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Administração Oral , Inibidores da Captação Adrenérgica/farmacologia , Animais , Benzazepinas/farmacologia , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Relação Dose-Resposta a Droga , Doxiciclina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , N-Metil-3,4-Metilenodioxianfetamina/farmacologiaRESUMO
The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and ß-phenylethylamine, has proven to be an important modulator of the dopaminergic system and is considered a promising target for the treatment of neuropsychiatric disorders. To decipher the brain functions of TAAR1, a selective TAAR1 agonist, RO5166017, was engineered. RO5166017 showed high affinity and potent functional activity at mouse, rat, cynomolgus monkey, and human TAAR1 stably expressed in HEK293 cells as well as high selectivity vs. other targets. In mouse brain slices, RO5166017 inhibited the firing frequency of dopaminergic and serotonergic neurons in regions where Taar1 is expressed (i.e., the ventral tegmental area and dorsal raphe nucleus, respectively). In contrast, RO5166017 did not change the firing frequency of noradrenergic neurons in the locus coeruleus, an area devoid of Taar1 expression. Furthermore, modulation of TAAR1 activity altered the desensitization rate and agonist potency at 5-HT(1A) receptors in the dorsal raphe, suggesting that TAAR1 modulates not only dopaminergic but also serotonergic neurotransmission. In WT but not Taar1(-/-) mice, RO5166017 prevented stress-induced hyperthermia and blocked dopamine-dependent hyperlocomotion in cocaine-treated and dopamine transporter knockout mice as well as hyperactivity induced by an NMDA antagonist. These results tie TAAR1 to the control of monoamine-driven behaviors and suggest anxiolytic- and antipsychotic-like properties for agonists such as RO5166017, opening treatment opportunities for psychiatric disorders.
Assuntos
Monoaminas Biogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transmissão Sináptica/fisiologia , Animais , Benzodioxóis/farmacologia , Dopamina/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Transtornos Mentais , Camundongos , Fenilpropionatos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiênciaRESUMO
Parkinson's disease is the second most common neurodegenerative pathology. Due to the limitations of existing therapeutic approaches, novel anti-parkinsonian medicines with non-dopamine mechanisms of action are clearly needed. One of the promising pharmacological targets for anti-Parkinson drug development is phosphodiesterase (PDE) 10A. The stimulating motor effects of PDE10A inhibition were detected only under the conditions of partial dopamine depletion. The results raise the question of whether PDE10A inhibitors are able to restore locomotor activity when dopamine levels are very low. To address this issue, we (1) developed and validated the rat model of acute severe dopamine deficiency and (2) tested the action of PDE10A inhibitor MP-10 in this model. All experiments were performed in dopamine transporter knockout (DAT-KO) rats. A tyrosine hydroxylase inhibitor, α-Methyl-DL-tyrosine (αMPT), was used as an agent to cause extreme dopamine deficiency. In vivo tests included estimation of locomotor activity and catalepsy levels in the bar test. Additionally, we evaluated the tissue content of dopamine in brain samples by HPLC analysis. The acute administration of αMPT to DAT-KO rats caused severe depletion of dopamine, immobility, and catalepsy (Dopamine-Deficient DAT-KO (DDD) rats). As expected, treatment with the L-DOPA and carbidopa combination restored the motor functions of DDD rats. Strikingly, administration of MP-10 also fully reversed immobility and catalepsy in DDD rats. According to neurochemical studies, the action of MP-10, in contrast to L-DOPA + carbidopa, seems to be dopamine-independent. These observations indicate that targeting PDE10A may represent a new promising approach in the development of non-dopamine therapies for Parkinson's disease.
Assuntos
Levodopa , Doença de Parkinson , Animais , Ratos , Carbidopa , Catalepsia/complicações , Catalepsia/tratamento farmacológico , Dopamina , Levodopa/farmacologia , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologiaRESUMO
Although normal dopaminergic tone has been shown to be essential for the induction of cortico-striatal and mesolimbic theta oscillatory activity, the influence of norepinephrine on these brain networks remains relatively unknown. To address this question, we simultaneously recorded local field potentials and single-neuron activity across 10 interconnected brain areas (ventral striatum, frontal association cortex, hippocampus, primary motor cortex, orbital frontal cortex, prelimbic cortex, dorsal lateral striatum, medial dorsal nucleus of thalamus, substantia nigra pars reticularis, and ventral tegmental area) in a combined genetically and pharmacologically induced mouse model of hyponoradrenergia. Our results show that norepinephrine (NE) depletion induces a novel state in male mice characterized by a profound disruption of coherence across multiple cortico-striatal circuits and an increase in mesolimbic cross-structural coherence. Moreover, this brain state is accompanied by a complex behavioral phenotype consisting of transient hyperactivity, stereotypic behaviors, and an acute 12-fold increase in grooming. Notably, treatment with a norepinephrine precursors (l-3,4-dihydroxyphenylalanine at 100 mg/kg or l-threo-dihydroxyphenylserine at 5 mg/kg) or a selective serotonin reuptake inhibitor (fluoxetine at 20 mg/kg) attenuates the abnormal behaviors and selectively reverses the circuit changes observed in NE-depleted mice. Together, our results demonstrate that norepinephrine modulates the dynamic tuning of coherence across cortico-striato-thalamic circuits, and they suggest that changes in coherence across these circuits mediate the abnormal generation of hyperactivity and repetitive behaviors.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiologia , Vias Neurais/fisiologia , Norepinefrina/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ritmo Delta/efeitos dos fármacos , Di-Hidroxifenilalanina/farmacologia , Droxidopa/farmacologia , Fluoxetina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Norepinefrina/deficiência , Norepinefrina/genética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Ritmo Teta/efeitos dos fármacosRESUMO
The ability of dopamine receptors to interact with other receptor subtypes may provide mechanisms for modulating dopamine-related functions and behaviors. In particular, there is evidence suggesting that the trace amine-associated receptor 1 (TAAR1) affects the dopaminergic system by regulating the firing rate of dopaminergic neurons or by altering dopamine D2 receptor (D2R) responsiveness to ligands. TAAR1 is a Gα(s) protein-coupled receptor that is activated by biogenic amines, "trace amines," such as ß-phenylethylamine (ß-PEA) and tyramine that are normally found at low concentrations in the mammalian brain. In the present study, we investigated the biochemical mechanism of interaction between TAAR1 and D2R and the role this interaction plays in D2R-related signaling and behaviors. Using a bioluminescence resonance energy transfer biosensor for cAMP, we demonstrated that the D2R antagonists haloperidol, raclopride, and amisulpride were able to enhance selectively a TAAR1-mediated ß-PEA increase of cAMP. Moreover, TAAR1 and D2R were able to form heterodimers when coexpressed in human embryonic kidney 293 cells, and this direct interaction was disrupted in the presence of haloperidol. In addition, in mice lacking TAAR1, haloperidol-induced striatal c-Fos expression and catalepsy were significantly reduced. Taken together, these data suggest that TAAR1 and D2R have functional and physical interactions that could be critical for the modulation of the dopaminergic system by TAAR1 in vivo.
Assuntos
Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Dimerização , Imunofluorescência , Haloperidol/farmacologia , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Dysregulation of brain serotonin (5-HT) neurotransmission is thought to underlie mental conditions as diverse as depression, anxiety disorders, bipolar disorder, autism, and schizophrenia. Despite treatment of these conditions with serotonergic drugs, the molecular mechanisms by which 5-HT is involved in the regulation of aberrant emotional behaviors are poorly understood. Here, we generated knockin mice expressing a mutant form of the brain 5-HT synthesis enzyme, tryptophan hydroxylase 2 (Tph2). This mutant is equivalent to a rare human variant (R441H) identified in few individuals with unipolar major depression. Expression of mutant Tph2 in mice results in markedly reduced ( approximately 80%) brain 5-HT production and leads to behavioral abnormalities in tests assessing 5-HT-mediated emotional states. This reduction in brain 5-HT levels is accompanied by activation of glycogen synthase kinase 3beta (GSK3beta), a signaling molecule modulated by many psychiatric therapeutic agents. Importantly, inactivation of GSK3beta in Tph2 knockin mice, using pharmacological or genetic approaches, alleviates the aberrant behaviors produced by 5-HT deficiency. These findings establish a critical role of Tph2 in the maintenance of brain serotonin homeostasis and identify GSK3beta signaling as an important pathway through which brain 5-HT deficiency induces abnormal behaviors. Targeting GSK3beta and related signaling events may afford therapeutic advantages for the management of certain 5-HT-related psychiatric conditions.
Assuntos
Comportamento Animal/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Serotonina/deficiência , Serotonina/fisiologia , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Imobilização/fisiologia , Relações Interpessoais , Locomoção/genética , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese Sítio-Dirigida , Serotonina/biossíntese , Transdução de Sinais/genética , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/fisiologiaRESUMO
The dopamine transporter (DAT) plays a key role in the regulation of dopaminergic signaling wherein it controls both the spatial and temporal actions of dopamine. Here we evaluated the behavioral and neurochemical consequences of increased DAT function by generating DAT transgenic mice (DAT-tg) that overexpress the transporter. These mice were generated by pronuclear injection of a bacterial artificial chromosome containing the mouse DAT locus, yielding an anatomical expression pattern of DAT-tg identical to WT. In DAT-tg mice there is a 3-fold increase in the levels of total and membrane-expressed DAT, but synaptic plasma membrane fractions of DAT-tg mice show only a 30% increase in transporter levels. Functional studies reveal that in the DAT-tg animals there is a 50% increase in the rate of dopamine (DA) uptake resulting in extracellular levels of DA that are decreased by approximately 40%. Behaviorally, DAT-tg animals display similar locomotor stimulation when treated with DAT blockers such as GBR12909, methylphenidate, and cocaine. However, these mice demonstrate markedly increased locomotor responses to amphetamine compared with WT animals. Furthermore, compared with controls, there is a 3-fold greater increase in the amount of DA released by amphetamine in DAT-tg mice that correlates with the 3-fold increase in protein expression. Finally, DAT-tg animals show reduced operant responding for natural reward while displaying preference for amphetamine at much lower doses (0.2 and 0.5 mg/kg) than WT mice (2 mg/kg). These results suggest that overexpression of DAT leads to a marked increase in sensitivity to psychomotor and rewarding properties of amphetamine.
Assuntos
Anfetamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Regulação da Expressão Gênica , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica , Atividade MotoraRESUMO
Trace amine-associated receptors (TAARs) are a class of sensory G protein-coupled receptors that detect biogenic amines, products of decarboxylation of amino acids. The majority of TAARs (TAAR2-TAAR9) have been described mainly in the olfactory epithelium and considered to be olfactory receptors sensing innate odors. However, there is recent evidence that one of the members of this family, TAAR5, is expressed also in the limbic brain areas receiving projection from the olfactory system and involved in the regulation of emotions. In this study, we further characterized a mouse line lacking TAAR5 (TAAR5 knockout, TAAR5-KO mice) that express beta-galactosidase mapping TAAR5 expression. We found that in TAAR5-KO mice the number of dopamine neurons, the striatal levels of dopamine and its metabolites, as well as striatal levels of GDNF mRNA, are elevated indicating a potential increase in dopamine neuron proliferation. Furthermore, an analysis of TAAR5 beta-galactosidase expression revealed that TAAR5 is present in the major neurogenic areas of the brain such as the subventricular zone (SVZ), the subgranular zone (SGZ) and the less characterized potentially neurogenic zone surrounding the 3rd ventricle. Direct analysis of neurogenesis by using specific markers doublecortin (DCX) and proliferating cell nuclear antigen (PCNA) revealed at least 2-fold increase in the number of proliferating neurons in the SVZ and SGZ of TAAR5-KO mice, but no such markers were detected in mutant or control mice in the areas surrounding the 3rd ventricle. These observations indicate that TAAR5 involved not only in regulation of emotional status but also adult neurogenesis and dopamine transmission. Thus, future TAAR5 antagonists may exert not only antidepressant and/or anxiolytic action but may also provide new treatment opportunity for neurodegenerative disorders such as Parkinson's disease.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Transmissão Sináptica/fisiologia , Fatores Etários , Animais , Encéfalo/crescimento & desenvolvimento , Neurônios Dopaminérgicos/metabolismo , Proteína Duplacortina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/genéticaRESUMO
Dopaminergic dysregulation can cause motor dysfunction, but the mechanisms underlying dopamine-related motor disorders remain under debate. We used an inducible and reversible pharmacogenetic approach in dopamine transporter knockout mice to investigate the simultaneous activity of neuronal ensembles in the dorsolateral striatum and primary motor cortex during hyperdopaminergia ( approximately 500% of controls) with hyperkinesia, and after rapid and profound dopamine depletion (<0.2%) with akinesia in the same animal. Surprisingly, although most cortical and striatal neurons ( approximately 70%) changed firing rate during the transition between dopamine-related hyperkinesia and akinesia, the overall cortical firing rate remained unchanged. Conversely, neuronal oscillations and ensemble activity coordination within and between cortex and striatum did change rapidly between these periods. During hyperkinesia, corticostriatal activity became largely asynchronous, while during dopamine-depletion the synchronicity increased. Thus, dopamine-related disorders like Parkinson's disease may not stem from changes in the overall levels of cortical activity, but from dysfunctional activity coordination in corticostriatal circuits.
Assuntos
Doenças dos Gânglios da Base/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Córtex Motor/metabolismo , Transtornos dos Movimentos/metabolismo , Vias Neurais/metabolismo , Potenciais de Ação/fisiologia , Doença Aguda , Animais , Doenças dos Gânglios da Base/fisiopatologia , Relógios Biológicos/fisiologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Regulação para Baixo/fisiologia , Hipercinese/metabolismo , Hipercinese/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Motor/fisiopatologia , Transtornos dos Movimentos/fisiopatologia , Vias Neurais/fisiopatologia , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Transmissão Sináptica/fisiologia , Fatores de TempoRESUMO
An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.
Assuntos
Encéfalo/metabolismo , Doenças da Junção Neuromuscular/etiologia , Junção Neuromuscular/metabolismo , Reconhecimento Psicológico/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/deficiência , Acetilcolina/análise , Acetilcolina/metabolismo , Animais , Northern Blotting , Southern Blotting , Encéfalo/patologia , Encéfalo/fisiopatologia , Química Encefálica , Cromatografia Líquida de Alta Pressão , Feminino , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Microdiálise , Atividade Motora/fisiologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Doenças da Junção Neuromuscular/patologia , Doenças da Junção Neuromuscular/fisiopatologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Transmissão Sináptica/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
Dopamine (DA) plays crucial roles in the cognitive functioning of the prefrontal cortex (PFC), which, to a large degree, depends on lasting neural traces formed in prefrontal networks. The establishment of these permanent traces requires changes in cortical synaptic efficacy. DA, via the D(1)-class receptors, is thought to gate or facilitate synaptic plasticity in the PFC, with little role recognized for the D(2)-class receptors. Here we show that, when significantly elevated, DA erodes, rather than facilitates, the induction of long-term potentiation (LTP) in the PFC by acting at the far less abundant cortical D(2)-class receptors through a dominant coupling to the protein phosphatase 1 (PP1) activity in postsynaptic neurons. In mice with persistently elevated extracellular DA, resulting from inactivation of the DA transporter (DAT) gene, LTP in layer V PFC pyramidal neurons cannot be established, regardless of induction protocols. Acute increase of dopaminergic transmission by DAT blockers or overstimulation of D(2) receptors in normal mice have similar LTP shutoff effects. LTP in mutant mice can be rescued by a single in vivo administration of D(2)-class antagonists. Suppression of postsynaptic PP1 mimics and occludes the D(2)-mediated rescue of LTP in mutant mice and prevents the acute erosion of LTP by D(2) agonists in normal mice. Our studies reveal a mechanistically unique heterosynaptic PP1 gate that is constitutively driven by background DA to influence LTP induction. By blocking prefrontal synaptic plasticity, excessive DA may prevent storage of lasting memory traces in PFC networks and impair executive functions.
Assuntos
Dopamina/metabolismo , Potenciação de Longa Duração/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Proteína Fosfatase 1/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Antagonistas dos Receptores de Dopamina D2 , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Espaço Extracelular/metabolismo , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Receptores de Dopamina D2/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Dysregulation of central serotonin neurotransmission has been widely suspected as an important contributor to major depression. Here, we identify a (G1463A) single nucleotide polymorphism (SNP) in the rate-limiting enzyme of neuronal serotonin synthesis, human tryptophan hydroxylase-2 (hTPH2). The functional SNP in hTPH2 replaces the highly conserved Arg441 with His, which results in approximately 80% loss of function in serotonin production when hTPH2 is expressed in PC12 cells. Strikingly, SNP analysis in a cohort of 87 patients with unipolar major depression revealed that nine patients carried the mutant (1463A) allele, while among 219 controls, three subjects carried this mutation. In addition, this functional SNP was not found in a cohort of 60 bipolar disorder patients. Identification of a loss-of-function mutation in hTPH2 suggests that defect in brain serotonin synthesis may represent an important risk factor for unipolar major depression.