RESUMO
Abnormal dopamine neurotransmission is a common trait of some psychiatric diseases, like schizophrenia or bipolar disorder. Excessive dopaminergic tone in subcortical brain regions is associated with psychotic episodes, while reduced prefrontal dopaminergic activity is associated with impaired cognitive performance and reduced motivation, among other symptoms. Inhibitory interneurons expressing the calcium binding protein parvalbumin are particularly affected in both schizophrenia and bipolar disorder, as they set a fine-tuned physiological inhibitory/excitatory balance. Parvalbumin and somatostatin interneuron subtypes, are born from the medial ganglionic eminence and require the sequential expression of specific transcription factors for their specification, such as Nkx6.2. Here, we aimed at characterizing in detail interneuron subtypes derived from Nkx6.2 expressing progenitors by the generation of an Nkx6.2 Cre transgenic mouse line. We show that Nkx6.2 specifies over a third part of the total population of cortical somatostatin interneurons, preferentially at early developmental time points, whereas at late developmental stages, Nkx6.2 expressing progenitors shift to parvalbumin interneuron specification. Dopamine D2 receptor deletion from Nkx6.2 expressing progenitors causes abnormal phenotypes restricted to cognitive, motivation and anxiety domains. Our results show that Nkx6.2 have the potential to specify both somatostatin and parvalbumin interneurons in an opposite timed program and that DRD2 expression is required in Nkx6.2 expressing progenitors to avoid impaired phenotypes commonly associated to the pathophysiology of psychiatric diseases.
Assuntos
Motivação , Parvalbuminas , Animais , Camundongos , Ansiedade/genética , Cognição , Interneurônios/metabolismo , Camundongos Transgênicos , Parvalbuminas/metabolismo , Fenótipo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Somatostatina/genética , Somatostatina/metabolismoRESUMO
BACKGROUND: Schizophrenia is a disease diagnosed by visible signs and symptoms from late adolescence to early adulthood. The etiology of this disease remains unknown. An objective diagnostic approach is required. Here, we used a mouse model that shows schizophrenia-like phenotypes to study brain glucose metabolism and presynaptic dopaminergic functioning by positron emission tomography (PET) and immunohistochemistry. PET scannings were performed on mice after the administration of [18F]-FDG or [18F]-F-DOPA. Glucose metabolism was evaluated in basal conditions and after the induction of a hyperdopaminergic state. RESULTS: Mutant animals show reduced glucose metabolism in prefrontal cortex, amygdala, and nucleus reuniens under the hyperdopaminergic state. They also show reduced [18F]-F-DOPA uptake in prefrontal cortex, substantia nigra reticulata, raphe nucleus, and ventral striatum but increased [18F]-F-DOPA uptake in dorsal striatum. Mutant animals also show reduced tyrosine hydroxylase expression on midbrain neurons. CONCLUSIONS: Dopamine D2 mutant animals show reduced glucose metabolism and impaired presynaptic dopaminergic functioning, in line with reports from human studies. This mouse line may be a valuable model of schizophrenia, useful to test novel tracers for PET scanning diagnostic.
RESUMO
The process of locomotion is controlled by fine-tuned dopaminergic neurons in the Substantia Nigra pars-compacta (SNpc) that projects their axons to the dorsal striatum regulating cortical innervations of medium spiny neurons. Dysfunction in dopaminergic neurotransmission within the striatum leads to movement impairments, gaiting defects, and hypo-locomotion. Due to their high polarity and extreme axonal arborization, neurons depend on molecular motor proteins and microtubule-based transport for their normal function. Transport defects have been associated with neurodegeneration since axonopathies, axonal clogging, microtubule destabilization, and lower motor proteins levels were described in the brain of patients with Parkinson's Disease and other neurodegenerative disorders. However, the contribution of specific motor proteins to the regulation of the nigrostriatal network remains unclear. Here, we generated different conditional knockout mice for the kinesin heavy chain 5B subunit (Kif5b) of Kinesin-1 to unravel its contribution to locomotion. Interestingly, mice with neuronal Kif5b deletion showed hypo-locomotion, movement initiation deficits, and coordination impairments. High pressure liquid chromatography determined that dopamine (DA) metabolism is impaired in neuronal Kif5b-KO, while no dopaminergic cell loss was observed. However, the deletion of Kif5b only in dopaminergic neurons is not sufficient to induce locomotor defects. Noteworthy, pharmacological stimulation of DA release together with agonist or antagonist of DA receptors revealed selective D2-dependent movement initiation defects in neuronal Kif5b-KO. Finally, subcellular fractionation from striatum showed that Kif5b deletion reduced the amount of dopamine D2 receptor in synaptic plasma membranes. Together, these results revealed an important role for Kif5b in the modulation of the striatal network that is relevant to the overall locomotor response. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
Assuntos
Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Cinesinas/metabolismo , Locomoção/fisiologia , Receptores de Dopamina D2/metabolismo , Animais , Camundongos , Camundongos KnockoutRESUMO
Excessive dopamine neurotransmission underlies psychotic episodes as observed in patients with some types of bipolar disorder and schizophrenia. The dopaminergic hypothesis was postulated after the finding that antipsychotics were effective to halt increased dopamine tone. However, there is little evidence for dysfunction within the dopaminergic system itself. Alternatively, it has been proposed that excessive afferent activity onto ventral tegmental area dopaminergic neurons, particularly from the ventral hippocampus, increase dopamine neurotransmission, leading to psychosis. Here, we show that selective dopamine D2 receptor deletion from parvalbumin interneurons in mouse causes an impaired inhibitory activity in the ventral hippocampus and a dysregulated dopaminergic system. Conditional mutant animals show adult onset of schizophrenia-like behaviors and molecular, cellular, and physiological endophenotypes as previously described from postmortem brain studies of patients with schizophrenia. Our findings show that dopamine D2 receptor expression on parvalbumin interneurons is required to modulate and limit pyramidal neuron activity, which may prevent the dysregulation of the dopaminergic system.
Assuntos
Antipsicóticos/farmacologia , Resistência a Medicamentos , Interneurônios/metabolismo , Parvalbuminas/metabolismo , Receptores de Dopamina D2/fisiologia , Esquizofrenia/etiologia , Animais , Masculino , Camundongos , Camundongos Knockout , Parvalbuminas/genética , Fenótipo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Transmissão SinápticaRESUMO
Competition between adult males for limited resources such as food and receptive females is shaped by the male pattern of pituitary growth hormone (GH) secretion that determines body size and the production of urinary pheromones involved in male-to-male aggression. In the brain, dopamine (DA) provides incentive salience to stimuli that predict the availability of food and sexual partners. Although the importance of the GH axis and central DA neurotransmission in social dominance and fitness is clearly appreciated, the two systems have always been studied unconnectedly. Here we conducted a cell-specific genetic dissection study in conditional mutant mice that selectively lack DA D2 receptors (D2R) from pituitary lactotropes (lacDrd2KO) or neurons (neuroDrd2KO). Whereas lacDrd2KO mice developed a normal GH axis, neuroDrd2KO mice displayed fewer somatotropes; reduced hypothalamic Ghrh expression, pituitary GH content, and serum IGF-I levels; and exhibited reduced body size and weight. As a consequence of a GH axis deficit, neuroDrd2KO adult males excreted low levels of major urinary proteins and their urine failed to promote aggression and territorial behavior in control male challengers, in contrast to the urine taken from control adult males. These findings reveal that central D2Rs mediate a neuroendocrine-exocrine cascade that controls the maturation of the GH axis and downstream signals that are critical for fitness, social dominance, and competition between adult males.
Assuntos
Tamanho Corporal/fisiologia , Hormônio do Crescimento/metabolismo , Hipófise/metabolismo , Prolactina/metabolismo , Receptores de Dopamina D2/metabolismo , Análise de Variância , Animais , Benzamidas/farmacocinética , Tamanho Corporal/efeitos dos fármacos , Tamanho Corporal/genética , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Estudos de Casos e Controles , Catatonia/induzido quimicamente , Catatonia/metabolismo , Antagonistas de Dopamina/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Feminino , Haloperidol/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas de Filamentos Intermediários/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Nestina , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Feromônios/urina , Hipófise/efeitos dos fármacos , Prolactina/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas/metabolismo , Radioimunoensaio , Receptores de Dopamina D2/deficiência , Receptores de Dopamina D2/genética , Predomínio Social , Territorialidade , Trítio/farmacocinéticaRESUMO
The proopiomelanocortin gene (POMC) is expressed in a group of neurons present in the arcuate nucleus of the hypothalamus. Neuron-specific POMC expression in mammals is conveyed by two distal enhancers, named nPE1 and nPE2. Previous transgenic mouse studies showed that nPE1 and nPE2 independently drive reporter gene expression to POMC neurons. Here, we investigated the evolutionary mechanisms that shaped not one but two neuron-specific POMC enhancers and tested whether nPE1 and nPE2 drive identical or complementary spatiotemporal expression patterns. Sequence comparison among representative genomes of most vertebrate classes and mammalian orders showed that nPE1 is a placental novelty. Using in silico paleogenomics we found that nPE1 originated from the exaptation of a mammalian-apparent LTR retrotransposon sometime between the metatherian/eutherian split (147 Mya) and the placental mammal radiation (≈ 90 Mya). Thus, the evolutionary origin of nPE1 differs, in kind and time, from that previously demonstrated for nPE2, which was exapted from a CORE-short interspersed nucleotide element (SINE) retroposon before the origin of prototherians, 166 Mya. Transgenic mice expressing the fluorescent markers tomato and EGFP driven by nPE1 or nPE2, respectively, demonstrated coexpression of both reporter genes along the entire arcuate nucleus. The onset of reporter gene expression guided by nPE1 and nPE2 was also identical and coincidental with the onset of Pomc expression in the presumptive mouse diencephalon. Thus, the independent exaptation of two unrelated retroposons into functional analogs regulating neuronal POMC expression constitutes an authentic example of convergent molecular evolution of cell-specific enhancers.
Assuntos
Elementos Facilitadores Genéticos/genética , Evolução Molecular , Mamíferos/genética , Neurônios/metabolismo , Retroelementos/genética , Animais , Sequência de Bases , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neurônios/citologia , Filogenia , Placenta/metabolismo , Gravidez , Pró-Opiomelanocortina/genética , Fatores de TempoRESUMO
Dopamine (DA) D2 receptors expressed in DA neurons (D2 autoreceptors) exert a negative feedback regulation that reduces DA neuron firing, DA synthesis and DA release. As D2 receptors are mostly expressed in postsynaptic neurons, pharmacological and genetic approaches have been unable to definitively address the in vivo contribution of D2 autoreceptors to DA-mediated behaviors. We found that midbrain DA neurons from mice deficient in D2 autoreceptors (Drd2(loxP/loxP); Dat(+/IRES-cre), referred to as autoDrd2KO mice) lacked DA-mediated somatodendritic synaptic responses and inhibition of DA release. AutoDrd2KO mice displayed elevated DA synthesis and release, hyperlocomotion and supersensitivity to the psychomotor effects of cocaine. The mice also exhibited increased place preference for cocaine and enhanced motivation for food reward. Our results highlight the importance of D2 autoreceptors in the regulation of DA neurotransmission and demonstrate that D2 autoreceptors are important for normal motor function, food-seeking behavior, and sensitivity to the locomotor and rewarding properties of cocaine.
Assuntos
Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Motivação/fisiologia , Receptores de Dopamina D2/deficiência , Recompensa , Análise de Variância , Animais , Autorradiografia , Baclofeno/farmacologia , Comportamento de Escolha/fisiologia , Condicionamento Operante , Di-Hidroxifenilalanina/metabolismo , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Comportamento Exploratório/fisiologia , Alimentos , Agonistas dos Receptores de GABA-B/farmacologia , Hidrazinas/farmacologia , Hipercinese/tratamento farmacológico , Hipercinese/fisiopatologia , Técnicas In Vitro , Aprendizagem em Labirinto/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Atividade Motora/genética , Neurônios/fisiologia , Quimpirol/farmacologia , Esquema de Reforço , Sulpirida/farmacologiaRESUMO
Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1(-/-) animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1(-/-) mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1(-/-) sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1(+/+) and Crisp1(-/-) sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1(-/-) sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family.
Assuntos
Fertilização/fisiologia , Glicoproteínas de Membrana/deficiência , Espermatozoides/fisiologia , Reação Acrossômica , Animais , Fertilidade , Marcação de Genes , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Capacitação EspermáticaRESUMO
To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.
Assuntos
Catecolaminas/metabolismo , Marcação de Genes , Engenharia Genética , Integrases/metabolismo , Neurônios/metabolismo , Recombinação Genética , Proteínas Virais/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Química Encefálica/genética , Catecolaminas/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Humanos , Imuno-Histoquímica , Integrases/genética , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Gravidez , Regiões Promotoras Genéticas , Ratos , Distribuição Tecidual , Transgenes , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Virais/genéticaRESUMO
The prefrontal cortex receives a major dopaminergic input from the ventral tegmental area, which plays an important role in the integration of neuronal signals influencing behavioural responses to stressful environmental stimuli. The dopamine D4 receptor (D4R) is expressed at highest levels in the prefrontal cortex and is the predominant D2-like receptor localized in this brain area. To investigate the functional significance of D4Rs in dopamine-mediated responses we have analysed a strain of mice lacking this receptor subtype (Drd4-/-). Wild-type and Drd4-/- mice were challenged in two different approach/avoidance conflict paradigms: the elevated plus maze and the light/dark preference exploration test. By these behavioural measures Drd4-/- mice showed heightened avoidance to the more fear-provoking areas of each maze as demonstrated by reduced exploration of the open arms of the plus maze and longer latencies to explore the illuminated compartment of the light/dark shuttle box. These exaggerated avoidance behaviours were further enhanced by an additional handling stress but completely prevented by anxiolytic agents such as the benzodiazepine midazolam and ethanol. Although Drd4-/- mice displayed heightened anxiety, they exhibited normal ethanol preference and consumption in a two-bottle choice test. Learned fear responses evaluated by contextual, cued and instrumental fear-conditioning tests showed no difference between wild-type and Drd4-/- mice. Taken together these results indicate that the absence of D4Rs increases avoidance behaviour to unconditioned stimuli and does not impair behavioural reactions to Pavlovian fear-conditioning, suggesting that the D4R could play a key role in the dopaminergic modulation of cortical signals triggered by environmental stimuli.