Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 12(1): 4488, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301944

RESUMO

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Optogenética/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Opsinas/genética , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapses/genética , Sinapses/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
2.
Hum Mol Genet ; 30(19): 1811-1832, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34077522

RESUMO

Episodic ataxia type 2 (EA2) is a rare autosomal dominant disorder characterized by motor incoordination, paroxysmal dystonia, vertigo, nystagmus and more recently cognitive deficits. To date over 100 mutations in the CACNA1A gene have been identified in EA2 patients leading to a loss of P/Q-type channel activity, dysfunction of cerebellar Purkinje cells and motor incoordination. To determine if the cerebellum is contributing to these cognitive deficits, we examined two different EA2 mouse models for cognition impairments where CACNA1A was removed specifically from cerebellar Purkinje or granule cells postnatally. Both mutant mouse models showed anxiolytic behavior to lighted, open areas in the open field and light/dark place preference tests but enhanced anxiousness in the novel suppressed feeding test. However, EA2 mice continued to show augmented latencies in the light/dark preference test and when the arena was divided into two dark zones in the dark/dark preference test. Moreover, increased latencies were also displayed in the novel object recognition test, indicating that EA2 mice are indecisive and anxious to explore new territories and objects and may have memory recognition deficits. Exposure to a foreign mouse led to deficiencies in attention and sniffing as well as in social and genital sniffing. These data suggest that postnatal removal of the P/Q type calcium channel from the cerebellum regulates neuronal activity involved in anxiety, memory, decision making and social interactions. Our EA2 mice will provide a model to identify the mechanisms and therapeutic agents underlying cognitive and psychiatric disorders seen in EA2 patients.

3.
Front Cell Neurosci ; 15: 642521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679332

RESUMO

Astrocytes are the most abundant cell type within the central nervous system (CNS) with various functions. Furthermore, astrocytes show a regional and developmental heterogeneity traceable with specific markers. In this study, the influence of the low-density lipoprotein receptor-related protein 1 (LRP1) on astrocytic maturation within the hippocampus was analyzed during development. Previous studies mostly focused on the involvement of LRP1 in the neuronal compartment, where the deletion caused hyperactivity and motor dysfunctions in knockout animals. However, the influence of LRP1 on glia cells is less intensively investigated. Therefore, we used a newly generated mouse model, where LRP1 is specifically deleted from GLAST-positive astrocytes co-localized with the expression of the reporter tdTomato to visualize recombination and knockout events in vivo. The influence of LRP1 on the maturation of hippocampal astrocytes was assessed with immunohistochemical stainings against stage-specific markers as well as on mRNA level with RT-PCR analysis. The examination revealed that the knockout induction caused a significantly decreased number of mature astrocytes at an early developmental timepoint compared to control animals. Additionally, the delayed maturation of astrocytes also caused a reduced activity of neurons within the hippocampus. As previous studies showed that the glial specification and maturation of astrocytes is dependent on the signaling cascades Ras/Raf/MEK/Erk and PI3K/Akt, the phosphorylation of the signaling molecules Erk1/2 and Akt was analyzed. The hippocampal tissue of LRP1-deficient animals at P21 showed a significantly decreased amount of activated Erk in comparison to control tissue leading to the conclusion that the activation of this signaling cascade is dependent on LRP1 in astrocytes, which in turn is necessary for proper maturation of astrocytes. Our results showed that the deletion of LRP1 at an early developmental timepoint caused a delayed maturation of astrocytes in the hippocampus based on an altered activation of the Ras/Raf/MEK/Erk signaling pathway. However, with ongoing development these effects were compensated and the number of mature astrocytes was comparable as well as the activity of neurons. Therefore, LRP1 acts as an early regulator of the differentiation and maturation of astrocytes within the hippocampus.

4.
J Neurochem ; 156(5): 589-603, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32083308

RESUMO

Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.


Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Proteínas da Matriz Extracelular/deficiência , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/deficiência , Receptores de GABA-B/fisiologia , Serina Endopeptidases/deficiência , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Feminino , Agonistas dos Receptores de GABA-B/farmacologia , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Serina Endopeptidases/genética , Transdução de Sinais/efeitos dos fármacos
5.
Glia ; 68(12): 2517-2549, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32579270

RESUMO

The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.

6.
Elife ; 92020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32252889

RESUMO

Controlling gain of cortical activity is essential to modulate weights between internal ongoing communication and external sensory drive. Here, we show that serotonergic input has separable suppressive effects on the gain of ongoing and evoked visual activity. We combined optogenetic stimulation of the dorsal raphe nucleus (DRN) with wide-field calcium imaging, extracellular recordings, and iontophoresis of serotonin (5-HT) receptor antagonists in the mouse visual cortex. 5-HT1A receptors promote divisive suppression of spontaneous activity, while 5-HT2A receptors act divisively on visual response gain and largely account for normalization of population responses over a range of visual contrasts in awake and anesthetized states. Thus, 5-HT input provides balanced but distinct suppressive effects on ongoing and evoked activity components across neuronal populations. Imbalanced 5-HT1A/2A activation, either through receptor-specific drug intake, genetically predisposed irregular 5-HT receptor density, or change in sensory bombardment may enhance internal broadcasts and reduce sensory drive and vice versa.


Assuntos
Núcleo Dorsal da Rafe/fisiologia , Optogenética/métodos , Neurônios Serotoninérgicos/fisiologia , Córtex Visual/fisiologia , Animais , Linhagem Celular , Núcleo Dorsal da Rafe/efeitos dos fármacos , Luz , Estudos Longitudinais , Camundongos , Camundongos Transgênicos , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/fisiologia , Serotonina/fisiologia , Antagonistas da Serotonina/administração & dosagem , Córtex Visual/efeitos dos fármacos
7.
Bioelectrochemistry ; 133: 107487, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32120322

RESUMO

Carbon-based nanoelectrodes fabricated by means of pyrolysis of an alkane precursor gas purged through a glass capillary and subsequently etched with HF were modified with redox polymer/enzyme films for the detection of glucose at the single-cell level. Glucose oxidase (GOx) was immobilized and electrically wired by means of an Os-complex-modified redox polymer in a sequential dip coating process. For the synthesis of the redox polymer matrix, a poly(1-vinylimidazole-co-acrylamide)-based backbone was used that was first modified with the electron transfer mediator [Os(bpy)2Cl]+ (bpy = 2,2'-bipyridine) followed by the conversion of the amide groups within the acrylamide monomer into hydrazide groups in a polymer-analogue reaction. The hydrazide groups react readily with bifunctional epoxide-based crosslinkers ensuring high film stability. Insertion of the nanometre-sized polymer/enzyme modified electrodes into adherently growing single NG108-15 cells resulted in a positive current response correlating with the intracellular glucose concentration. Moreover, the nanosensors showed a stable current output without significant loss in performance after intracellular measurements.


Assuntos
Técnicas Biossensoriais/instrumentação , Carbono/química , Glucose/análise , Polímeros/química , Análise de Célula Única/instrumentação , Animais , Aspergillus niger/enzimologia , Linhagem Celular , Enzimas Imobilizadas/química , Glucose Oxidase/química , Camundongos , Microeletrodos
8.
J Mater Chem B ; 8(16): 3631-3639, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31942595

RESUMO

Carbon nanoelectrodes in the sub-micron range were modified with an enzyme cascade immobilized in a spatially separated polymer double layer system for the detection of glutamate at the cellular level. The enzyme cascade consists of glutamate oxidase (GlutOx) that was immobilized in a hydrophilic redox silent polymer on top of a horseradish peroxidase (HRP)/redox polymer layer. In the presence of O2, glutamate was oxidized under concomitant reduction of O2 to H2O2 at GlutOx. H2O2 is further reduced to water by means of HRP and electrons are shuttled via the redox polymer matrix that wires the HRP to the electrode surface, hence delivering a current response proportional to the glutamate concentration. The nanometer-sized sensors could be successfully used to measure glutamate release from primary mouse astrocytes in 10 mM HEPES buffer.


Assuntos
Aminoácido Oxirredutases/química , Carbono/química , Glutamatos/análise , Nanopartículas/química , Polímeros/química , Aminoácido Oxirredutases/metabolismo , Animais , Astrócitos/química , Técnicas Biossensoriais , Carbono/metabolismo , Células Cultivadas , Técnicas Eletroquímicas , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glutamatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Nanopartículas/metabolismo , Tamanho da Partícula , Polímeros/metabolismo , Streptomyces/enzimologia , Propriedades de Superfície
9.
Chembiochem ; 21(5): 612-617, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31468691

RESUMO

Optogenetics uses light-sensitive proteins, so-called optogenetic tools, for highly precise spatiotemporal control of cellular states and signals. The major limitations of such tools include the overlap of excitation spectra, phototoxicity, and lack of sensitivity. The protein characterized in this study, the Japanese lamprey parapinopsin, which we named UVLamP, is a promising optogenetic tool to overcome these limitations. Using a hybrid strategy combining molecular, cellular, electrophysiological, and computational methods we elucidated a structural model of the dark state and probed the optogenetic potential of UVLamP. Interestingly, it is the first described bistable vertebrate opsin that has a charged amino acid interacting with the Schiff base in the dark state, that has no relevance for its photoreaction. UVLamP is a bistable UV-sensitive opsin that allows for precise and sustained optogenetic control of G protein-coupled receptor (GPCR) pathways and can be switched on, but more importantly also off within milliseconds via lowintensity short light pulses. UVLamP exhibits an extremely narrow excitation spectrum in the UV range allowing for sustained activation of the Gi/o pathway with a millisecond UV light pulse. Its sustained pathway activation can be switched off, surprisingly also with a millisecond blue light pulse, minimizing phototoxicity. Thus, UVLamP serves as a minimally invasive, narrow-bandwidth probe for controlling the Gi/o pathway, allowing for combinatorial use with multiple optogenetic tools or sensors. Because UVLamP activated Gi/o signals are generally inhibitory and decrease cellular activity, it has tremendous potential for health-related applications such as relieving pain, blocking seizures, and delaying neurodegeneration.


Assuntos
Proteínas de Peixes/metabolismo , Lampreias/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Células HEK293 , Humanos , Raios Ultravioleta
10.
Brain ; 143(1): 161-174, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31800012

RESUMO

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.


Assuntos
Ataxia/genética , Canais de Cálcio Tipo N/genética , Córtex Cerebral/metabolismo , Epilepsia Tipo Ausência/genética , Neurônios/metabolismo , Tálamo/metabolismo , Potenciais de Ação , Fatores Etários , Animais , Ataxia/metabolismo , Ataxia/fisiopatologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Epilepsia Tipo Ausência/metabolismo , Epilepsia Tipo Ausência/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Inibidores/genética , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Núcleos Talâmicos/citologia , Tálamo/fisiopatologia
11.
Commun Biol ; 2: 373, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31633064

RESUMO

Aggressive behavior in our modern, civilized society is often counterproductive and destructive. Identifying specific proteins involved in the disease can serve as therapeutic targets for treating aggression. Here, we found that overexpression of RGS2 in explicitly serotonergic neurons augments male aggression in control mice and rescues male aggression in Rgs2 -/- mice, while anxiety is not affected. The aggressive behavior is directly correlated to the immediate early gene c-fos induction in the dorsal raphe nuclei and ventrolateral part of the ventromedial nucleus hypothalamus, to an increase in spontaneous firing in serotonergic neurons and to a reduction in the modulatory action of Gi/o and Gq/11 coupled 5HT and adrenergic receptors in serotonergic neurons of Rgs2-expressing mice. Collectively, these findings specifically identify that RGS2 expression in serotonergic neurons is sufficient to drive male aggression in mice and as a potential therapeutic target for treating aggression.


Assuntos
Agressão/fisiologia , Proteínas RGS/metabolismo , Neurônios Serotoninérgicos/metabolismo , Potenciais de Ação , Animais , Ansiedade/metabolismo , Cálcio/metabolismo , Células Cultivadas , Depressão/metabolismo , Núcleo Dorsal da Rafe/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas RGS/genética , RNA Mensageiro/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Serotonina/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismo
12.
Front Neural Circuits ; 13: 51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447652

RESUMO

The cerebellar involvement in cognitive functions such as attention, language, working memory, emotion, goal-directed behavior and spatial navigation is constantly growing. However, an exact connectivity map between the hippocampus and cerebellum in mice is still unknown. Here, we conducted a tracing study to identify the sequence of transsynaptic, cerebellar-hippocampal connections in the mouse brain using combinations of Recombinant adeno-associated virus (rAAV) and pseudotyped deletion-mutant rabies (RABV) viruses. Stereotaxic injection of a primarily anterograde rAAV-WGA (wheat germ agglutinin)-Cre tracer virus in the deep cerebellar nuclei (DCN) of a Cre-dependent tdTomato reporter mouse resulted in strong tdTomato labeling in hippocampal CA1 neurons, retrosplenial cortex (RSC), rhinal cortex (RC) as well as thalamic and cerebellar areas. Whereas hippocampal injections with the retrograde tracer virus rAAV-TTC (tetanus toxin C fragment)-eGFP, displayed eGFP positive cells in the rhinal cortex and subiculum. To determine the sequence of mono-transsynaptic connections between the cerebellum and hippocampus, we used the retrograde tracer RABVΔG-eGFP(EnvA). The tracing revealed a direct connection from the dentate gyrus (DG) in the hippocampus to the RSC, RC and subiculum (S), which are monosynaptically connected to thalamic laterodorsal and ventrolateral areas. These thalamic nuclei are directly connected to cerebellar fastigial (FN), interposed (IntP) and lateral (Lat) nuclei, discovering a new projection route from the fastigial to the laterodorsal thalamic nucleus in the mouse brain. Collectively, our findings suggest a new cerebellar-hippocampal connection via the laterodorsal and ventrolateral thalamus to RSC, RC and S. These results strengthen the notion of the cerebellum's involvement in cognitive functions such as spatial navigation via a polysynaptic circuitry.


Assuntos
Núcleos Cerebelares/fisiologia , Hipocampo/fisiologia , Núcleos Laterais do Tálamo/fisiologia , Rede Nervosa/fisiologia , Núcleos Talâmicos/fisiologia , Núcleos Ventrais do Tálamo/fisiologia , Animais , Núcleos Cerebelares/química , Feminino , Células HEK293 , Hipocampo/química , Humanos , Núcleos Laterais do Tálamo/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/química , Núcleos Talâmicos/química , Núcleos Ventrais do Tálamo/química
13.
Chembiochem ; 20(14): 1766-1771, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30920724

RESUMO

The primary goal of optogenetics is the light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein-coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on G protein-coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.


Assuntos
Opsinas de Bastonetes/química , Opsinas de Bastonetes/efeitos da radiação , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Luz , Camundongos , Mutação , Optogenética/métodos , Estudo de Prova de Conceito , Engenharia de Proteínas , Células de Purkinje/metabolismo , Células de Purkinje/efeitos da radiação , Opsinas de Bastonetes/genética , Alinhamento de Sequência , Transdução de Sinais/efeitos da radiação
14.
Commun Biol ; 2: 60, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30793039

RESUMO

The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.


Assuntos
Técnicas Biossensoriais , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Optogenética/métodos , Receptor 5-HT2A de Serotonina/genética , Opsinas de Bastonetes/genética , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Eletrodos Implantados , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Transporte Proteico , Ratos , Ratos Long-Evans , Receptor 5-HT2A de Serotonina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Opsinas de Bastonetes/metabolismo , Técnicas Estereotáxicas
15.
Nat Commun ; 10(1): 826, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778063

RESUMO

Synaptic heterogeneity is widely observed but its underpinnings remain elusive. We addressed this issue using mature calyx of Held synapses whose numbers of bouton-like swellings on stalks of the nerve terminals inversely correlate with release probability (Pr). We examined presynaptic Ca2+ currents and transients, topology of fluorescently tagged knock-in Ca2+ channels, and Ca2+ channel-synaptic vesicle (SV) coupling distance using Ca2+ chelator and inhibitor of septin cytomatrix in morphologically diverse synapses. We found that larger clusters of Ca2+ channels with tighter coupling distance to SVs elevate Pr in stalks, while smaller clusters with looser coupling distance lower Pr in swellings. Septin is a molecular determinant of the differences in coupling distance. Supported by numerical simulations, we propose that varying the ensemble of two morphological modules containing distinct Ca2+ channel-SV topographies diversifies Pr in the terminal, thereby establishing a morpho-functional continuum that expands the coding capacity within a single synapse population.

16.
PLoS One ; 13(7): e0198765, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29995896

RESUMO

Bioluminescence is a fascinating phenomenon and can be found in many different organisms including fish. It has been suggested that bioluminescence is used for example for defense, prey attraction, and for intraspecific communication to attract for example sexual partners. The flashlight fish, Anomalops katoptron (A. katoptron), is a nocturnal fish that produces bioluminescence and lives in shallow waters, which makes it ideal for laboratory studies. In order to understand A. katoptron's ability to detect bioluminescent light (480 to 490 nm) at night, we characterized the visual system adaptation of A. katoptron using phylogenetic, electrophysiological and behavioral studies. We found that the retinae of A. katoptron contain rods and sparse cones. A. katoptron retinae express two main visual pigments, rhodopsin (RH1), and to a lesser extent, rhodopsin-like opsin (RH2). Interestingly, recombinant RH1 and RH2 are maximally sensitive to a wavelength of approximately 490 nm light (λmax), which correspond to the spectral peak of in vivo electroretinogram (ERG) measurements. In addition, behavioral assays revealed that A. katoptron is attracted by low intensity blue but not red light. Collectively, our results suggest that the A. katoptron visual system is optimized to detect blue light in the frequency range of its own bioluminescence and residual starlight.


Assuntos
Adaptação Fisiológica , Proteínas de Peixes/genética , Opsinas/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/genética , Sequência de Aminoácidos , Animais , Eletrorretinografia , Proteínas de Peixes/metabolismo , Peixes , Expressão Gênica , Células HEK293 , Humanos , Luz , Luminescência , Medições Luminescentes/métodos , Opsinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Rodopsina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
17.
PLoS One ; 12(2): e0170489, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28178297

RESUMO

Bioluminescence is a fascinating phenomenon occurring in numerous animal taxa in the ocean. The reef dwelling splitfin flashlight fish (Anomalops katoptron) can be found in large schools during moonless nights in the shallow water of coral reefs and in the open surrounding water. Anomalops katoptron produce striking blink patterns with symbiotic bacteria in their sub-ocular light organs. We examined the blink frequency in A. katoptron under various laboratory conditions. During the night A. katoptron swims in schools roughly parallel to their conspecifics and display high blink frequencies of approximately 90 blinks/minute with equal on and off times. However, when planktonic prey was detected in the experimental tank, the open time increased compared to open times in the absence of prey and the frequency decreased to 20% compared to blink frequency at night in the absence of planktonic prey. During the day when the school is in a cave in the reef tank the blink frequency decreases to approximately 9 blinks/minute with increasing off-times of the light organ. Surprisingly the non-luminescent A. katoptron with non-functional light organs displayed the same blink frequencies and light organ open/closed times during the night and day as their luminescent conspecifics. In the presence of plankton non-luminescent specimens showed no change in the blink frequency and open/closed times compared to luminescent A. katoptron. Our experiments performed in a coral reef tank show that A. katoptron use bioluminescent illumination to detect planktonic prey and that the blink frequency of A. katoptron light organs follow an exogenous control by the ambient light.


Assuntos
Escuridão , Peixes/fisiologia , Luminescência , Comportamento Predatório , Animais , Peixes/anatomia & histologia , Luz
18.
Biochem Biophys Res Commun ; 483(4): 1040-1050, 2017 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-27392710

RESUMO

Calcium is a key signaling molecule and ion involved in a variety of diverse processes in our central nervous system (CNS) which include gene expression, synaptic transmission and plasticity, neuronal excitability and cell maintenance. Proper control of calcium signaling is not only vital for neuronal physiology but also cell survival. Mutations in fundamental channels, transporters and second messenger proteins involved in orchestrating the balance of our calcium homeostasis can lead to severe neurodegenerative disorders, such as Spinocerebellar (SCA) and Episodic (EA) ataxias. Hereditary ataxias make up a remarkably diverse group of neurological disorders clinically characterized by gait ataxia, nystagmus, dysarthria, trunk and limb ataxia and often atrophy of the cerebellum. The largest family of hereditary ataxias is SCAs which consists of a growing family of 42 members. A relatively smaller family of 8 members compose the EAs. The gene mutations responsible for half of the EA members and over 35 of the SCA subtypes have been identified, and several have been found to be responsible for cerebellar atrophy, abnormal intracellular calcium levels, dysregulation of Purkinje cell pacemaking, altered cerebellar synaptic transmission and/or ataxia in mouse models. Although the genetic diversity and affected cellular pathways of hereditary ataxias are broad, one common theme amongst these genes is their effects on maintaining calcium balance in primarily the cerebellum. There is emerging evidence that the pathogenesis of hereditary ataxias may be caused by imbalances in intracellular calcium due to genetic mutations in calcium-mediating proteins. In this review we will discuss the current evidence supporting the role of deranged calcium as the culprit to neurodegenerative diseases with a primary focus on SCAs and EAs.


Assuntos
Cálcio/metabolismo , Homeostase , Animais , Sinalização do Cálcio , Humanos , Camundongos , Mutação , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
19.
Curr Biol ; 26(9): 1206-12, 2016 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-27068418

RESUMO

G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. Therefore, various strategies have been developed to analyze the function of GPCRs involving pharmaco- and optogenetic approaches [1, 2]. However, a tool that combines precise control of the activation and deactivation of GPCR pathways and/or neuronal firing with limited phototoxicity is still missing. We compared the biophysical properties and optogenetic application of a human and a mouse melanopsin variant (hOpn4L and mOpn4L) on the control of Gi/o and Gq pathways in heterologous expression systems and mouse brain. We found that GPCR pathways can be switched on/off by blue/yellow light. The proteins differ in their kinetics and wavelength dependence to activate and deactivate G protein pathways. Whereas mOpn4L is maximally activated by very short light pulses, leading to sustained G protein activation, G protein responses of hOpn4L need longer light pulses to be activated and decline in amplitude. Based on the different biophysical properties, brief light activation of mOpn4L is sufficient to induce sustained neuronal firing in cerebellar Purkinje cells (PC), whereas brief light activation of hOpn4L induces AP firing, which declines in frequency over time. Most importantly, mOpn4L-induced sustained firing can be switched off by yellow light. Based on the biophysical properties, hOpn4L and mOpn4L represent the first GPCR optogenetic tools, which can be used to switch GPCR pathways/neuronal firing on an off with temporal precision and limited phototoxicity. We suggest to name these tools moMo and huMo for future optogenetic applications.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/fisiologia , Variação Genética , Humanos , Camundongos , Mutação , Células de Purkinje/fisiologia , Opsinas de Bastonetes/genética
20.
J Neurosci ; 36(2): 405-18, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26758833

RESUMO

UNLABELLED: Generalized spike-wave seizures involving abnormal synchronization of cortical and underlying thalamic circuitry represent a major category of childhood epilepsy. Inborn errors of Cacna1a, the P/Q-type voltage-gated calcium channel α subunit gene, expressed throughout the brain destabilize corticothalamic rhythmicity and produce this phenotype. To determine the minimal cellular lesion required for this network disturbance, we used neurotensin receptor 1 (Ntsr1) cre-driver mice to ablate floxed Cacna1a in layer VI pyramidal neurons, which supply the sole descending cortical synaptic input to thalamocortical relay cells and reticular interneurons and activate intrathalamic circuits. Targeted Cacna1a ablation in layer VI cells resulted in mice that display a robust spontaneous spike-wave absence seizure phenotype accompanied by behavioral arrest and inhibited by ethosuximide. To verify the selectivity of the molecular lesion, we determined that P/Q subunit proteins were reduced in corticothalamic relay neuron terminal zones, and confirmed that P/Q-mediated glutamate release was reduced at these synapses. Spike-triggered exocytosis was preserved by N-type calcium channel rescue, demonstrating that evoked release at layer VI terminals relies on both P/Q and N-type channels. Whereas intrinsic excitability of the P/Q channel depleted layer VI neurons was unaltered, T-type calcium currents in the postsynaptic thalamic relay and reticular cells were dramatically elevated, favoring rebound bursting and seizure generation. We find that an early P/Q-type release defect, limited to synapses of a single cell-type within the thalamocortical circuit, is sufficient to remodel synchronized firing behavior and produce a stable generalized epilepsy phenotype. SIGNIFICANCE STATEMENT: This study dissects a critical component of the corticothalamic circuit in spike-wave epilepsy and identifies the developmental importance of P/Q-type calcium channel-mediated presynaptic glutamate release at layer VI pyramidal neuron terminals. Genetic ablation of Cacna1a in layer VI neurons produced synchronous spike-wave discharges in the cortex and thalamus that were inhibited by ethosuximide. These mice also displayed N-type calcium channel compensation at descending thalamic synapses, and consistent with other spike-wave models increased low-threshold T-type calcium currents within postsynaptic thalamic relay and reticular neurons. These results demonstrate, for the first time, that preventing the developmental homeostatic switch from loose to tightly coupled synaptic release at a single class of deep layer cortical excitatory output neurons results in generalized spike-wave epilepsy.


Assuntos
Canais de Cálcio Tipo N/deficiência , Epilepsia Tipo Ausência/patologia , Neurônios/patologia , Tálamo/patologia , Córtex Visual/patologia , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Canais de Cálcio Tipo N/genética , Modelos Animais de Doenças , Epilepsia Tipo Ausência/tratamento farmacológico , Epilepsia Tipo Ausência/genética , Etossuximida/uso terapêutico , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Motores/etiologia , Transtornos Motores/genética , Mutação/genética , Tempo de Reação/genética , Receptores de Neurotensina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...