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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.
FEBS J ; 2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33797854

RESUMO

What is the effect of activating a single modulatory neuronal receptor type on entire brain network dynamics? Can such effect be isolated at all? These are important questions because characterizing elementary neuronal processes that influence network activity across the given anatomical backbone is fundamental to guide theories of brain function. Here, we introduce the concept of the cortical 'receptome' taking into account the distribution and densities of expression of different modulatory receptor types across the brain's anatomical connectivity matrix. By modelling whole-brain dynamics in silico, we suggest a bidirectional coupling between modulatory neurotransmission and neuronal connectivity hardware exemplified by the impact of single serotonergic (5-HT) receptor types on cortical dynamics. As experimental support of this concept, we show how optogenetic tools enable specific activation of a single 5-HT receptor type across the cortex as well as in vivo measurement of its distinct effects on cortical processing. Altogether, we demonstrate how the structural neuronal connectivity backbone and its modulation by a single neurotransmitter system allow access to a rich repertoire of different brain states that are fundamental for flexible behaviour. We further propose that irregular receptor expression patterns-genetically predisposed or acquired during a lifetime-may predispose for neuropsychiatric disorders like addiction, depression and anxiety along with distinct changes in brain state. Our long-term vision is that such diseases could be treated through rationally targeted therapeutic interventions of high specificity to eventually recover natural transitions of brain states.

3.
Sci Rep ; 11(1): 6431, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742043

RESUMO

The schooling flashlight fish Anomalops katoptron can be found at dark nights at the water surface in the Indo-Pacific. Schools are characterized by bioluminescent blink patterns of sub-ocular light organs densely-packed with bioluminescent, symbiotic bacteria. Here we analyzed how blink patterns of A. katoptron are used in social interactions. We demonstrate that isolated specimen of A. katoptron showed a high motivation to align with fixed or moving artificial light organs in an experimental tank. This intraspecific recognition of A. katoptron is mediated by blinking light and not the body shape. In addition, A. katoptron adjusts its blinking frequencies according to the light intensities. LED pulse frequencies determine the swimming speed and the blink frequency response of A. katoptron, which is modified by light organ occlusion and not exposure. In the natural environment A. katoptron is changing its blink frequencies and nearest neighbor distance in a context specific manner. Blink frequencies are also modified by changes in the occlusion time and are increased from day to night and during avoidance behavior, while group cohesion is higher with increasing blink frequencies. Our results suggest that specific blink patterns in schooling flashlight fish A. katoptron define nearest neighbor distance and determine intraspecific communication.


Assuntos
Comunicação Animal , Peixes/fisiologia , Luminescência , Comportamento Social , Animais , Ecossistema , Natação
4.
Commun Biol ; 4(1): 100, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483632

RESUMO

Although optogenetics has revolutionized rodent neuroscience, it is still rarely used in other model organisms as the efficiencies of viral gene transfer differ between species and comprehensive viral transduction studies are rare. However, for comparative research, birds offer valuable model organisms as they have excellent visual and cognitive capabilities. Therefore, the following study establishes optogenetics in pigeons on histological, physiological, and behavioral levels. We show that AAV1 is the most efficient viral vector in various brain regions and leads to extensive anterograde and retrograde ChR2 expression when combined with the CAG promoter. Furthermore, transient optical stimulation of ChR2 expressing cells in the entopallium decreases pigeons' contrast sensitivity during a grayscale discrimination task. This finding demonstrates causal evidence for the involvement of the entopallium in contrast perception as well as a proof of principle for optogenetics in pigeons and provides the groundwork for various other methods that rely on viral gene transfer in birds.


Assuntos
Channelrhodopsins/metabolismo , Columbidae/genética , Dependovirus , Optogenética , Telencéfalo/metabolismo , Animais
5.
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
6.
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.

7.
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
8.
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
9.
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
10.
Biochem Biophys Res Commun ; 527(2): 325-330, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31982136

RESUMO

Optogenetics is a biological technique that combines the advantageous spatial-temporal resolution of optics and genetic cell targeting to control cellular activity with unprecedented precision. It has found vast applications both in neurosciences and therapy, particularly in view of its application to restore vision in blind patients. Optogenetics requires the ectopic expression of a so-called opsin to render neurons sensitive to light. There are two types of opsins for modulating membrane potential of neurons: (i) microbial opsins from unicellular organisms that respond to a light stimulus by mediating a flow of ions across the membrane (ii) animal opsins that are naturally present in mammalian retinas that initiate G protein coupled signaling in response to light. The former category has been extensively employed for vision restoration in the past decade with two ongoing clinical trials employing microbial opsins to restore light sensation in retinitis pigmentosa patients. The latter subtype of animal opsins is emerging more recently as strong candidates to restore vision with the promise of greater light sensitivity and tolerability. In this review we will discuss each approach in view of its utility for vision restoration in retinal blindness.


Assuntos
Cegueira/terapia , Opsinas/genética , Optogenética/métodos , Degeneração Retiniana/terapia , Animais , Cegueira/genética , Cegueira/fisiopatologia , Humanos , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Visão Ocular
11.
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
12.
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
13.
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
14.
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
15.
Brain Stimul ; 12(5): 1177-1186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040077

RESUMO

BACKGROUND: Cerebellar transcranial direct current stimulation (ctDCS) is increasingly used to modulate cerebellar excitability and plasticity in healthy subjects and various patient populations. ctDCS parameters are poorly standardized, and its physiology remains little understood. Our aim was to compare the physiological effects of three different non-target electrode positions (buccinator muscle, supraorbital region, deltoid muscle). METHODS: In the first experiment, physiological after-effects of ctDCS were compared based on cerebellar-brain inhibition (CBI) in a group of 15 healthy right-handed participants. In the second experiment, CBI after-effects of ctDCS were assessed using different transcranial magnetic stimulation (TMS) intensities in 14 participants (CBI recruitment curve). The electric field distribution was calculated for each of the electrode montages based on a single anatomically accurate head model. RESULTS: Anodal and cathodal ctDCS polarities significantly decreased cerebellar-brain inhibition (CBI) with no substantial differences between the montages. Lower cerebellar TMS intensities resulted in decreased CBI following cathodal and increased CBI after anodal ctDCS. Computational modeling revealed minor differences in the electric field distribution between non-target electrode positions based on the effect size. CONCLUSION: Our results show that the non-target electrode position has no significant impact on modeling results and physiological ctDCS after-effects. The recruitment of the cerebellar-M1 connection, however, varied depending on ctDCS polarity and cerebellar transcranial magnetic stimulation intensity, possibly due to diverse effects on different cell populations in the cerebellar cortex. This may be one of the reasons why ctDCS effects on functional measures are difficult to predict.


Assuntos
Cerebelo/fisiologia , Inibição Neural/fisiologia , Estimulação Transcraniana por Corrente Contínua/métodos , Adulto , Feminino , Humanos , Masculino , Distribuição Aleatória , Adulto Jovem
16.
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
17.
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
18.
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.

19.
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
20.
Nat Commun ; 8(1): 1606, 2017 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-29151577

RESUMO

Emotionally salient information activates orexin neurons in the lateral hypothalamus, leading to increase in sympathetic outflow and vigilance level. How this circuit alters animals' behavior remains unknown. Here we report that noradrenergic neurons in the locus coeruleus (NALC neurons) projecting to the lateral amygdala (LA) receive synaptic input from orexin neurons. Pharmacogenetic/optogenetic silencing of this circuit as well as acute blockade of the orexin receptor-1 (OX1R) decreases conditioned fear responses. In contrast, optogenetic stimulation of this circuit potentiates freezing behavior against a similar but distinct context or cue. Increase of orexinergic tone by fasting also potentiates freezing behavior and LA activity, which are blocked by pharmacological blockade of OX1R in the LC. These findings demonstrate the circuit involving orexin, NALC and LA neurons mediates fear-related behavior and suggests inappropriate excitation of this pathway may cause fear generalization sometimes seen in psychiatric disorders, such as PTSD.


Assuntos
Medo , Locus Cerúleo/metabolismo , Neurônios/metabolismo , Orexinas/metabolismo , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transtornos de Estresse Pós-Traumáticos/psicologia , Neurônios Adrenérgicos/metabolismo , Animais , Comportamento Animal , Humanos , Masculino , Camundongos , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/genética , Transtornos de Estresse Pós-Traumáticos/genética
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