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1.
Neuron ; 109(18): 2914-2927.e5, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34534454

RESUMO

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.


Assuntos
Dendritos/fisiologia , Morfogênese/fisiologia , Neocórtex/citologia , Neocórtex/fisiologia , Células Piramidais/fisiologia , Transcriptoma/fisiologia , Potenciais de Ação/fisiologia , Adulto , Animais , Feminino , Humanos , Macaca nemestrina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodos
2.
Neuron ; 109(17): 2727-2739.e3, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34380016

RESUMO

Utilizing recent advances in machine learning, we introduce a systematic approach to characterize neurons' input/output (I/O) mapping complexity. Deep neural networks (DNNs) were trained to faithfully replicate the I/O function of various biophysical models of cortical neurons at millisecond (spiking) resolution. A temporally convolutional DNN with five to eight layers was required to capture the I/O mapping of a realistic model of a layer 5 cortical pyramidal cell (L5PC). This DNN generalized well when presented with inputs widely outside the training distribution. When NMDA receptors were removed, a much simpler network (fully connected neural network with one hidden layer) was sufficient to fit the model. Analysis of the DNNs' weight matrices revealed that synaptic integration in dendritic branches could be conceptualized as pattern matching from a set of spatiotemporal templates. This study provides a unified characterization of the computational complexity of single neurons and suggests that cortical networks therefore have a unique architecture, potentially supporting their computational power.


Assuntos
Córtex Cerebral/fisiologia , Aprendizado Profundo , Modelos Neurológicos , Células Piramidais/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dendritos/metabolismo , Dendritos/fisiologia , Humanos , Células Piramidais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
3.
Neuron ; 109(16): 2535-2544.e4, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34228959

RESUMO

Psilocybin is a serotonergic psychedelic with untapped therapeutic potential. There are hints that the use of psychedelics can produce neural adaptations, although the extent and timescale of the impact in a mammalian brain are unknown. In this study, we used chronic two-photon microscopy to image longitudinally the apical dendritic spines of layer 5 pyramidal neurons in the mouse medial frontal cortex. We found that a single dose of psilocybin led to ∼10% increases in spine size and density, driven by an elevated spine formation rate. The structural remodeling occurred quickly within 24 h and was persistent 1 month later. Psilocybin also ameliorated stress-related behavioral deficit and elevated excitatory neurotransmission. Overall, the results demonstrate that psilocybin-evoked synaptic rewiring in the cortex is fast and enduring, potentially providing a structural trace for long-term integration of experiences and lasting beneficial actions.


Assuntos
Dendritos/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Lobo Frontal/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Psilocibina/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Dendritos/fisiologia , Espinhas Dendríticas/fisiologia , Feminino , Masculino , Camundongos , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Transmissão Sináptica/efeitos dos fármacos
4.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299127

RESUMO

Reelin is a secretory protein involved in a variety of processes in forebrain development and function, including neuronal migration, dendrite growth, spine formation, and synaptic plasticity. Most of the function of Reelin is focused on excitatory neurons; however, little is known about its effects on inhibitory neurons and inhibitory synapses. In this study, we investigated the phosphatidylinositol 3-kinase/Akt pathway of Reelin in primary cortical and hippocampal neurons. Individual neurons were visualized using immunofluorescence to distinguish inhibitory neurons from excitatory neurons. Reelin-rich protein supplementation significantly induced the phosphorylation of Akt and ribosomal S6 protein in excitatory neurons, but not in most inhibitory neurons. In somatostatin-expressing inhibitory neurons, one of major subtypes of inhibitory neurons, Reelin-rich protein supplementation induced the phosphorylation of S6. Subsequently, we investigated whether or not Reelin-rich protein supplementation affected dendrite development in cultured inhibitory neurons. Reelin-rich protein supplementation did not change the total length of dendrites in inhibitory neurons in vitro. Finally, we examined the development of inhibitory synapses in primary hippocampal neurons and found that Reelin-rich protein supplementation significantly reduced the density of gephyrin-VGAT-positive clusters in the dendritic regions without changing the expression levels of several inhibitory synapse-related proteins. These findings indicate a new role for Reelin in specific groups of inhibitory neurons and the development of inhibitory synapses, which may contribute to the underlying cellular mechanisms of RELN-associated neurological disorders.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Potenciais Pós-Sinápticos Inibidores , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural , Plasticidade Neuronal , Neurônios/fisiologia , Serina Endopeptidases/metabolismo , Sinapses/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Hipocampo/citologia , Hipocampo/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/genética , Neurogênese , Neurônios/citologia , Serina Endopeptidases/genética , Transdução de Sinais
5.
Nat Commun ; 12(1): 4509, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301949

RESUMO

The capacity of the brain to encode multiple types of sensory input is key to survival. Yet, how neurons integrate information from multiple sensory pathways and to what extent this influences behavior is largely unknown. Using two-photon Ca2+ imaging, optogenetics and electrophysiology in vivo and in vitro, we report the influence of auditory input on sensory encoding in the somatosensory cortex and show its impact on goal-directed behavior. Monosynaptic input from the auditory cortex enhanced dendritic and somatic encoding of tactile stimulation in layer 2/3 (L2/3), but not layer 5 (L5), pyramidal neurons in forepaw somatosensory cortex (S1). During a tactile-based goal-directed task, auditory input increased dendritic activity and reduced reaction time, which was abolished by photoinhibition of auditory cortex projections to forepaw S1. Taken together, these results indicate that dendrites of L2/3 pyramidal neurons encode multisensory information, leading to enhanced neuronal output and reduced response latency during goal-directed behavior.


Assuntos
Potenciais de Ação/fisiologia , Córtex Auditivo/fisiologia , Dendritos/fisiologia , Células Piramidais/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Córtex Auditivo/citologia , Estimulação Elétrica , Eletromiografia/métodos , Objetivos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética/métodos , Técnicas de Patch-Clamp , Células Piramidais/citologia , Córtex Somatossensorial/citologia , Tato/fisiologia
6.
Nat Commun ; 12(1): 3689, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140486

RESUMO

Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging.


Assuntos
Interfaces Cérebro-Computador , Cálcio/metabolismo , Dendritos/fisiologia , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Córtex Motor/diagnóstico por imagem , Imagem Multimodal/métodos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Dendritos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Implantes Experimentais , Macaca mulatta , Masculino , Modelos Neurológicos , Atividade Motora/fisiologia , Córtex Motor/fisiologia , Neurônios/fisiologia , Fótons
7.
Nat Commun ; 12(1): 4005, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183661

RESUMO

Synaptic inputs on cortical dendrites are organized with remarkable subcellular precision at the micron level. This organization emerges during early postnatal development through patterned spontaneous activity and manifests both locally where nearby synapses are significantly correlated, and globally with distance to the soma. We propose a biophysically motivated synaptic plasticity model to dissect the mechanistic origins of this organization during development and elucidate synaptic clustering of different stimulus features in the adult. Our model captures local clustering of orientation in ferret and receptive field overlap in mouse visual cortex based on the receptive field diameter and the cortical magnification of visual space. Including action potential back-propagation explains branch clustering heterogeneity in the ferret and produces a global retinotopy gradient from soma to dendrite in the mouse. Therefore, by combining activity-dependent synaptic competition and species-specific receptive fields, our framework explains different aspects of synaptic organization regarding stimulus features and spatial scales.


Assuntos
Visão Ocular/fisiologia , Córtex Visual/fisiologia , Percepção Visual/fisiologia , Potenciais de Ação/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dendritos/fisiologia , Furões , Camundongos , Modelos Neurológicos , Fatores de Crescimento Neural/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Córtex Visual/anatomia & histologia
8.
Nat Commun ; 12(1): 3558, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117238

RESUMO

Hippocampal place cells contribute to mammalian spatial navigation and memory formation. Numerous models have been proposed to explain the location-specific firing of this cognitive representation, but the pattern of excitatory synaptic input leading to place firing is unknown, leaving no synaptic-scale explanation of place coding. Here we used resonant scanning two-photon microscopy to establish the pattern of synaptic glutamate input received by CA1 place cells in behaving mice. During traversals of the somatic place field, we found increased excitatory dendritic input, mainly arising from inputs with spatial tuning overlapping the somatic field, and functional clustering of this input along the dendrites over ~10 µm. These results implicate increases in total excitatory input and co-activation of anatomically clustered synaptic input in place firing. Since they largely inherit their fields from upstream synaptic partners with similar fields, many CA1 place cells appear to be part of multi-brain-region cell assemblies forming representations of specific locations.


Assuntos
Hipocampo/fisiologia , Células de Lugar/fisiologia , Memória Espacial/fisiologia , Sinapses/fisiologia , Potenciais de Ação/fisiologia , Animais , Comportamento Animal , Região CA1 Hipocampal , Dendritos/fisiologia , Ácido Glutâmico , Hipocampo/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Plasticidade Neuronal/fisiologia , Neurotransmissores
9.
PLoS Comput Biol ; 17(6): e1009073, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34106921

RESUMO

Neurons rely on localized mitochondria to fulfill spatially heterogeneous metabolic demands. Mitochondrial aging occurs on timescales shorter than the neuronal lifespan, necessitating transport of fresh material from the soma. Maintaining an optimal distribution of healthy mitochondria requires an interplay between a stationary pool localized to sites of high metabolic demand and a motile pool capable of delivering new material. Interchange between these pools can occur via transient fusion / fission events or by halting and restarting entire mitochondria. Our quantitative model of neuronal mitostasis identifies key parameters that govern steady-state mitochondrial health at discrete locations. Very infrequent exchange between stationary and motile pools optimizes this system. Exchange via transient fusion allows for robust maintenance, which can be further improved by selective recycling through mitophagy. These results provide a framework for quantifying how perturbations in organelle transport and interactions affect mitochondrial homeostasis in neurons, a key aspect underlying many neurodegenerative disorders.


Assuntos
Dinâmica Mitocondrial/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Animais , Transporte Axonal/fisiologia , Axônios/fisiologia , Biologia Computacional , Dendritos/fisiologia , Homeostase , Humanos , Mitofagia/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/fisiopatologia , Processos Estocásticos
10.
J Vis Exp ; (170)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33970138

RESUMO

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.


Assuntos
Dendritos/fisiologia , Microscopia Confocal/métodos , Tecido Nervoso/fisiologia , Neurônios/fisiologia , Animais , Camundongos
11.
Nature ; 592(7854): 414-420, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33828296

RESUMO

Critical periods-brief intervals during which neural circuits can be modified by activity-are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood1,2. Here we define a critical period in a developing Drosophila motor circuit and identify astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure3, and astrocyte ablation prolonged the critical period. Finally, we used a genetic screen to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses4; we show here that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour.


Assuntos
Astrócitos/fisiologia , Período Crítico Psicológico , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Vias Eferentes/fisiologia , Neurônios Motores/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/fisiologia , Feminino , Locomoção/fisiologia , Masculino , Microtúbulos/metabolismo , Neurópilo/fisiologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Sinapses/fisiologia , Fatores de Tempo
12.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925434

RESUMO

The investigation of synaptic functions remains one of the most fascinating challenges in the field of neuroscience and a large number of experimental methods have been tuned to dissect the mechanisms taking part in the neurotransmission process. Furthermore, the understanding of the insights of neurological disorders originating from alterations in neurotransmission often requires the development of (i) animal models of pathologies, (ii) invasive tools and (iii) targeted pharmacological approaches. In the last decades, additional tools to explore neurological diseases have been provided to the scientific community. A wide range of computational models in fact have been developed to explore the alterations of the mechanisms involved in neurotransmission following the emergence of neurological pathologies. Here, we review some of the advancements in the development of computational methods employed to investigate neuronal circuits with a particular focus on the application to the most diffuse neurological disorders.


Assuntos
Modelos Neurológicos , Doenças do Sistema Nervoso/etiologia , Transmissão Sináptica/fisiologia , Doença de Alzheimer/etiologia , Animais , Dendritos/fisiologia , Epilepsia/etiologia , Humanos , Doenças do Sistema Nervoso/fisiopatologia , Doença de Parkinson/etiologia , Esquizofrenia/etiologia , Sinapses/fisiologia
13.
J Vis Exp ; (169)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33818570

RESUMO

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.


Assuntos
Retina/fisiologia , Imagem com Lapso de Tempo/métodos , Animais , Dendritos/fisiologia , Camundongos , Camundongos Transgênicos
14.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33783472

RESUMO

Macroautophagy (hereafter "autophagy") is a lysosomal degradation pathway that is important for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. Little is known, however, about the molecular details of how autophagy is regulated with synaptic activity. Here, we used live-cell confocal microscopy to define the autophagy pathway in primary hippocampal neurons under various paradigms of synaptic activity. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampens AV motility, whereas silencing synaptic activity induces AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. On the basis of our findings, we propose a model whereby synaptic activity locally controls AV dynamics and function within dendrites that may regulate the synaptic proteome.


Assuntos
Autofagia , Movimento Celular , Dendritos/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Vacúolos/fisiologia , Animais , Autofagossomos/fisiologia , Axônios/fisiologia , Hipocampo/citologia , Lisossomos/fisiologia , Camundongos , Neurônios/citologia , Ratos , Ratos Sprague-Dawley
15.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671638

RESUMO

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC.


Assuntos
MicroRNAs/genética , Células de Purkinje/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Cerebelo/citologia , Cerebelo/fisiologia , Dendritos/fisiologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Microdissecção e Captura a Laser , Masculino , Técnicas de Cultura de Órgãos , Células de Purkinje/efeitos dos fármacos , Ratos Wistar , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
16.
J Vis Exp ; (169)2021 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-33749674

RESUMO

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 µm (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.


Assuntos
Retina/metabolismo , Coloração e Rotulagem/métodos , Células Amácrinas/fisiologia , Animais , Dendritos/fisiologia , Neurônios Dopaminérgicos/fisiologia , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Confocal/métodos , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/metabolismo , Refratometria
17.
Nat Commun ; 12(1): 1374, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654091

RESUMO

In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale 'non-synaptic' mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a 'tripartite' structure facilitates a 'multi-directed' form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.


Assuntos
Acetilcolina/metabolismo , Sistema Nervoso Central/fisiologia , Transmissão Sináptica/fisiologia , Células Amácrinas/fisiologia , Células Amácrinas/ultraestrutura , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Cinética , Camundongos Endogâmicos C57BL , Fótons , Células Ganglionares da Retina/ultraestrutura
18.
Nat Commun ; 12(1): 1068, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594066

RESUMO

A graphdiyne-based artificial synapse (GAS), exhibiting intrinsic short-term plasticity, has been proposed to mimic biological signal transmission behavior. The impulse response of the GAS has been reduced to several millivolts with competitive femtowatt-level consumption, exceeding the biological level by orders of magnitude. Most importantly, the GAS is capable of parallelly processing signals transmitted from multiple pre-neurons and therefore realizing dynamic logic and spatiotemporal rules. It is also found that the GAS is thermally stable (at 353 K) and environmentally stable (in a relative humidity up to 35%). Our artificial efferent nerve, connecting the GAS with artificial muscles, has been demonstrated to complete the information integration of pre-neurons and the information output of motor neurons, which is advantageous for coalescing multiple sensory feedbacks and reacting to events. Our synaptic element has potential applications in bioinspired peripheral nervous systems of soft electronics, neurorobotics, and biohybrid systems of brain-computer interfaces.


Assuntos
Grafite/farmacologia , Neurônios Eferentes/fisiologia , Sinapses/fisiologia , Dendritos/efeitos dos fármacos , Dendritos/fisiologia , Teoria da Densidade Funcional , Difusão , Íons , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Plasticidade Neuronal , Neurônios Eferentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Temperatura
19.
Neural Netw ; 138: 126-139, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33639581

RESUMO

In spiking neural P (SN P) systems, neurons are interconnected by means of synapses, and they use spikes to communicate with each other. However, in biology, the complex structure of dendritic tree is also an important part in the communication scheme between neurons since these structures are linked to advanced neural process such as learning and memory formation. In this work, we present a new variant of the SN P systems inspired by diverse dendrite and axon phenomena such as dendritic feedback, dendritic trunk, dendritic delays and axonal delays, respectively. This new variant is referred to as a spiking neural P system with dendritic and axonal computation (DACSN P system). Specifically, we include experimentally proven biological features in the current SN P systems to reduce the computational complexity of the soma by providing it with stable firing patterns through dendritic delays, dendritic feedback and axonal delays. As a consequence, the proposed DACSN P systems use the minimum number of synapses and neurons with simple and homogeneous standard spiking rules. Here, we study the computational capabilities of a DACSN P system. In particular, we prove that DACSN P systems with dendritic and axonal behavior are universal as both number-accepting/generating devices. In addition, we constructed a small universal SN P system using 39 neurons with standard spiking rules to compute any Turing computable function.


Assuntos
Retroalimentação , Modelos Neurológicos , Redes Neurais de Computação , Sinapses/fisiologia , Potenciais de Ação , Axônios/fisiologia , Dendritos/fisiologia , Humanos , Tempo de Reação
20.
Neural Netw ; 136: 40-53, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33445004

RESUMO

A typical feature of hyperbox-based dendrite morphological neurons (DMN) is the generation of sharp and rough decision boundaries that inaccurately track the distribution shape of classes of patterns. This feature is because the minimum and maximum activation functions force the decision boundaries to match the faces of the hyperboxes. To improve the DMN response, we introduce a dendritic model that uses smooth maximum and minimum functions to soften the decision boundaries. The classification performance assessment is conducted on nine synthetic and 28 real-world datasets. Based on the experimental results, we demonstrate that the smooth activation functions improve the generalization capacity of DMN. The proposed approach is competitive with four machine learning techniques, namely, Multilayer Perceptron, Radial Basis Function Network, Support Vector Machine, and Nearest Neighbor algorithm. Besides, the computational complexity of DMN training is lower than MLP and SVM classifiers.


Assuntos
Dendritos , Aprendizado de Máquina , Redes Neurais de Computação , Neurônios , Máquina de Vetores de Suporte , Algoritmos , Dendritos/fisiologia , Humanos , Neurônios/fisiologia
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