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1.
Nature ; 488(7411): 375-8, 2012 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-22820253

RESUMO

One defining characteristic of the mammalian brain is its neuronal diversity. For a given region, substructure, layer or even cell type, variability in neuronal morphology and connectivity persists. Although it is well known that such cellular properties vary considerably according to neuronal type, the substantial biophysical diversity of neurons of the same morphological class is typically averaged out and ignored. Here we show that the amplitude of hyperpolarization-evoked sag of membrane potential recorded in olfactory bulb mitral cells is an emergent, homotypic property of local networks and sensory information processing. Simultaneous whole-cell recordings from pairs of cells show that the amount of hyperpolarization-evoked sag potential and current (Ih) is stereotypic for mitral cells belonging to the same glomerular circuit. This is corroborated by a mosaic, glomerulus-based pattern of expression of the HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel 2) subunit of the Ih channel. Furthermore, inter-glomerular differences in both membrane potential sag and HCN2 protein are diminished when sensory input to glomeruli is genetically and globally altered so that only one type of odorant receptor is universally expressed. Population diversity in this intrinsic property therefore reflects differential expression between local mitral cell networks processing distinct odour-related information.


Assuntos
Rede Nervosa/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/fisiologia , Olfato/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Canais de Potássio , Receptores Odorantes/metabolismo
2.
Nature ; 473(7345): 87-91, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21478872

RESUMO

Neuronal connectivity is fundamental to information processing in the brain. Therefore, understanding the mechanisms of sensory processing requires uncovering how connection patterns between neurons relate to their function. On a coarse scale, long-range projections can preferentially link cortical regions with similar responses to sensory stimuli. But on the local scale, where dendrites and axons overlap substantially, the functional specificity of connections remains unknown. Here we determine synaptic connectivity between nearby layer 2/3 pyramidal neurons in vitro, the response properties of which were first characterized in mouse visual cortex in vivo. We found that connection probability was related to the similarity of visually driven neuronal activity. Neurons with the same preference for oriented stimuli connected at twice the rate of neurons with orthogonal orientation preferences. Neurons responding similarly to naturalistic stimuli formed connections at much higher rates than those with uncorrelated responses. Bidirectional synaptic connections were found more frequently between neuronal pairs with strongly correlated visual responses. Our results reveal the degree of functional specificity of local synaptic connections in the visual cortex, and point to the existence of fine-scale subnetworks dedicated to processing related sensory information.


Assuntos
Sinapses Elétricas/fisiologia , Rede Nervosa/fisiologia , Córtex Visual/fisiologia , Animais , Cálcio/química , Sinalização do Cálcio/fisiologia , Simulação por Computador , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Estimulação Luminosa , Células Piramidais/fisiologia
3.
Neurophotonics ; 11(1): 015006, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38322022

RESUMO

Significance: Two-photon optogenetics combines nonlinear excitation with noninvasive activation of neurons to enable the manipulation of neural circuits with a high degree of spatial precision. Combined with two-photon population calcium imaging, these approaches comprise a flexible platform for all-optical interrogation of neural circuits. However, a multitude of optical and biological factors dictate the exact precision of this approach in vivo, where it is most usefully applied. Aim: We aimed to assess how the optical point spread function (OPSF) contributes to the spatial precision of two-photon photostimulation in neurobiology. Approach: We altered the axial spread of the OPSF of the photostimulation beam using a spatial light modulator. Subsequently, calcium imaging was used to monitor the axial spatial precision of two-photon photostimulation of layer 2 neurons in the mouse neocortex. Results: We found that optical resolution is not always the limiting factor of the spatial precision of two-photon optogenetic photostimulation and, by doing so, reveal the key factors that must be improved to achieve maximal precision. Conclusions: Our results enable future work to focus on the optimal factors by providing key insight from controlled experiments in a manner not previously reported. This research can be applied to advance the state-of-the-art of all-optical interrogation, extending the toolkit for neuroscience research to achieve spatiotemporal precision at the crucial levels in which neural circuits operate.

4.
bioRxiv ; 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38328224

RESUMO

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

5.
Proc Natl Acad Sci U S A ; 106(35): 15049-54, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19706480

RESUMO

Eye-opening represents a turning point in the function of the visual cortex. Before eye-opening, the visual cortex is largely devoid of sensory inputs and neuronal activities are generated intrinsically. After eye-opening, the cortex starts to integrate visual information. Here we used in vivo two-photon calcium imaging to explore the developmental changes of the mouse visual cortex by analyzing the ongoing spontaneous activity. We found that before eye-opening, the activity of layer 2/3 neurons consists predominantly of slow wave oscillations. These waves were first detected at postnatal day 8 (P8). Their initial very low frequency (0.01 Hz) gradually increased during development to approximately 0.5 Hz in adults. Before eye-opening, a large fraction of neurons (>75%) was active during each wave. One day after eye-opening, this dense mode of recruitment changed to a sparse mode with only 36% of active neurons per wave. This was followed by a progressive decrease during the following weeks, reaching 12% of active neurons per wave in adults. The possible role of visual experience for this process of sparsification was investigated by analyzing dark-reared mice. We found that sparsification also occurred in these mice, but that the switch from a dense to a sparse activity pattern was delayed by 3-4 days as compared with normally-reared mice. These results reveal a modulatory contribution of visual experience during the first days after eye-opening, but an overall dominating role of intrinsic factors. We propose that the transformation in network activity from dense to sparse is a prerequisite for the changed cortical function at eye-opening.


Assuntos
Neurônios/fisiologia , Visão Ocular/fisiologia , Córtex Visual/crescimento & desenvolvimento , Córtex Visual/fisiologia , Animais , Cálcio/metabolismo , Fenômenos Eletrofisiológicos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenômenos Fisiológicos do Sistema Nervoso , Técnicas de Patch-Clamp
6.
Nature ; 426(6962): 74-8, 2003 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-14603320

RESUMO

The neurotrophin receptor TrkB is essential for normal function of the mammalian brain. It is expressed in three splice variants. Full-length receptors (TrkB(FL)) possess an intracellular tyrosine kinase domain and are considered as those TrkB receptors that mediate the crucial effects of brain-derived neurotrophic factor (BDNF) or neurotrophin 4/5 (NT-4/5). By contrast, truncated receptors (TrkB-T1 and TrkB-T2) lack tyrosine kinase activity and have not been reported to elicit rapid intracellular signalling. Here we show that astrocytes predominately express TrkB-T1 and respond to brief application of BDNF by releasing calcium from intracellular stores. The calcium transients are insensitive to the tyrosine kinase blocker K-252a and persist in mutant mice lacking TrkB(FL). By contrast, neurons produce rapid BDNF-evoked signals through TrkB(FL) and the Na(v)1.9 channel. Expression of antisense TrkB messenger RNA strongly reduces BDNF-evoked calcium signals in glia. Thus, our results show that, unexpectedly, TrkB-T1 has a direct signalling role in mediating inositol-1,4,5-trisphosphate-dependent calcium release; in addition, they identify a previously unknown mechanism of neurotrophin action in the brain.


Assuntos
Processamento Alternativo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Receptor trkB/química , Receptor trkB/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Eletrofisiologia , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor trkB/genética , Deleção de Sequência
7.
J Neurosci Methods ; 222: 69-81, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24200507

RESUMO

BACKGROUND: Two-photon microscopy is widely used to study brain function, but conventional microscopes are too slow to capture the timing of neuronal signalling and imaging is restricted to one plane. Recent development of acousto-optic-deflector-based random access functional imaging has improved the temporal resolution, but the utility of these technologies for mapping 3D synaptic activity patterns and their performance at the excitation wavelengths required to image genetically encoded indicators have not been investigated. NEW METHOD: Here, we have used a compact acousto-optic lens (AOL) two-photon microscope to make high speed [Ca(2+)] measurements from spines and dendrites distributed in 3D with different excitation wavelengths (800-920 nm). RESULTS: We show simultaneous monitoring of activity from many synaptic inputs distributed over the 3D arborisation of a neuronal dendrite using both synthetic as well as genetically encoded indicators. We confirm the utility of AOL-based imaging for fast in vivo recordings by measuring, simultaneously, visually evoked responses in 100 neurons distributed over a 150 µm focal depth range. Moreover, we explore ways to improve the measurement of timing of neuronal activation by choosing specific regions within the cell soma. COMPARISON WITH EXISTING METHODS: These results establish that AOL-based 3D random access two-photon microscopy has a wider range of neuroscience applications than previously shown. CONCLUSIONS: Our findings show that the compact AOL microscope design has the speed, spatial resolution, sensitivity and wavelength flexibility to measure 3D patterns of synaptic and neuronal activity on individual trials.


Assuntos
Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica , Neurônios/fisiologia , Sinapses/fisiologia , Potenciais de Ação , Animais , Cálcio/metabolismo , Córtex Cerebral/fisiologia , Dendritos/fisiologia , Espinhas Dendríticas/fisiologia , Eletroporação , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Transmissão Sináptica/fisiologia , Tempo , Percepção Visual/fisiologia , Imagens com Corantes Sensíveis à Voltagem/instrumentação , Imagens com Corantes Sensíveis à Voltagem/métodos
8.
Nat Commun ; 4: 2100, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23820818

RESUMO

Lesion experiments suggest that odour input to the olfactory bulb contains significant redundant signal such that rodents can discern odours using minimal stimulus-related information. Here we investigate the dependence of odour-quality perception on the integrity of glomerular activity by comparing odour-evoked activity maps before and after epithelial lesions. Lesions prevent mice from recognizing previously experienced odours and differentially delay discrimination learning of unrecognized and novel odour pairs. Poor recognition results not from mice experiencing an altered concentration of an odour but from perception of apparent novel qualities. Consistent with this, relative intensity of glomerular activity following lesions is altered compared with maps recorded in shams and by varying odour concentration. Together, these data show that odour recognition relies on comprehensively matching input patterns to a previously generated stimulus template. When encountering novel odours, access to all glomerular activity ensures rapid generation of new templates to perform accurate perceptual judgements.


Assuntos
Julgamento/fisiologia , Odorantes , Percepção/fisiologia , Olfato/fisiologia , Animais , Aprendizagem por Discriminação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Nasal/patologia , Estimulação Física , Reconhecimento Psicológico , Sulfato de Zinco
9.
Nat Neurosci ; 14(4): 527-32, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21336272

RESUMO

Single-cell genetic manipulation is expected to substantially advance the field of systems neuroscience. However, existing gene delivery techniques do not allow researchers to electrophysiologically characterize cells and to thereby establish an experimental link between physiology and genetics for understanding neuronal function. In the mouse brain in vivo, we found that neurons remained intact after 'blind' whole-cell recording, that DNA vectors could be delivered through the patch-pipette during such recordings and that these vectors drove protein expression in recorded cells for at least 7 d. To illustrate the utility of this approach, we recorded visually evoked synaptic responses in primary visual cortical cells while delivering DNA plasmids that allowed retrograde, monosynaptic tracing of each neuron's presynaptic inputs. By providing a biophysical profile of a cell before its specific genetic perturbation, this combinatorial method captures the synaptic and anatomical receptive field of a neuron.


Assuntos
Técnicas de Rastreamento Neuroanatômico/métodos , Neurônios/fisiologia , Transfecção/métodos , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Vetores Genéticos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Rastreamento Neuroanatômico/tendências , Neurônios/citologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodos , Técnicas de Patch-Clamp/tendências , Transfecção/tendências
10.
Nat Neurosci ; 14(8): 1045-52, 2011 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-21765421

RESUMO

Neuronal responses during sensory processing are influenced by both the organization of intracortical connections and the statistical features of sensory stimuli. How these intrinsic and extrinsic factors govern the activity of excitatory and inhibitory populations is unclear. Using two-photon calcium imaging in vivo and intracellular recordings in vitro, we investigated the dependencies between synaptic connectivity, feature selectivity and network activity in pyramidal cells and fast-spiking parvalbumin-expressing (PV) interneurons in mouse visual cortex. In pyramidal cell populations, patterns of neuronal correlations were largely stimulus-dependent, indicating that their responses were not strongly dominated by functionally biased recurrent connectivity. By contrast, visual stimulation only weakly modified co-activation patterns of fast-spiking PV cells, consistent with the observation that these broadly tuned interneurons received very dense and strong synaptic input from nearby pyramidal cells with diverse feature selectivities. Therefore, feedforward and recurrent network influences determine the activity of excitatory and inhibitory ensembles in fundamentally different ways.


Assuntos
Inibição Neural/fisiologia , Neurônios/fisiologia , Córtex Visual/citologia , Vias Visuais/fisiologia , Análise de Variância , Animais , Cálcio/metabolismo , Potenciais Evocados Visuais/genética , Potenciais Evocados Visuais/fisiologia , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/classificação , Compostos Orgânicos/metabolismo , Parvalbuminas/genética , Técnicas de Patch-Clamp , Estimulação Luminosa/métodos , Tempo de Reação/fisiologia , Estatística como Assunto , Sinapses/fisiologia
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