RESUMO
Optical recording of neuronal activity in three-dimensional (3D) brain circuits at cellular and millisecond resolution in vivo is essential for probing information flow in the brain. While random-access multiphoton microscopy permits fast optical access to neuronal targets in three dimensions, the method is challenged by motion artifacts when recording from behaving animals. Therefore, we developed three-dimensional custom-access serial holography (3D-CASH). Built on a fast acousto-optic light modulator, 3D-CASH performs serial sampling at 40 kHz from neurons at freely selectable 3D locations. Motion artifacts are eliminated by targeting each neuron with a size-optimized pattern of excitation light covering the cell body and its anticipated displacement field. Spike rates inferred from GCaMP6f recordings in visual cortex of awake mice tracked the phase of a moving bar stimulus with higher spike correlation between intra compared to interlaminar neuron pairs. 3D-CASH offers access to the millisecond correlation structure of in vivo neuronal activity in 3D microcircuits.
Assuntos
Holografia/instrumentação , Holografia/métodos , Imageamento Tridimensional/métodos , Córtex Visual/citologia , Animais , Comportamento Animal , Teste de Esforço , Feminino , Fluorescência , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Estimulação Luminosa , Imagem com Lapso de Tempo , Córtex Visual/fisiologiaRESUMO
High resolution optical microscopy is essential in neuroscience but suffers from scattering in biological tissues and therefore grants access to superficial brain layers only. Recently developed techniques use scattered photons for imaging by exploiting angular correlations in transmitted light and could potentially increase imaging depths. But those correlations ('angular memory effect') are of a very short range and should theoretically be only present behind and not inside scattering media. From measurements on neural tissues and complementary simulations, we find that strong forward scattering in biological tissues can enhance the memory effect range and thus the possible field-of-view by more than an order of magnitude compared to isotropic scattering for â¼1 mm thick tissue layers.
RESUMO
Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.
RESUMO
In the neocortex, neuronal selectivities for multiple sensorimotor modalities are often distributed in topographical maps thought to emerge during a restricted period in early postnatal development. Rodent barrel cortex contains a somatotopic map for vibrissa identity, but the existence of maps representing other tactile features has not been clearly demonstrated. We addressed the issue of the existence in the rat cortex of an intrabarrel map for vibrissa movement direction using in vivo two-photon imaging. We discovered that the emergence of a direction map in rat barrel cortex occurs long after all known critical periods in the somatosensory system. This map is remarkably specific, taking a pinwheel-like form centered near the barrel center and aligned to the barrel cortex somatotopy. We suggest that this map may arise from intracortical mechanisms and demonstrate by simulation that the combination of spike-timing-dependent plasticity at synapses between layer 4 and layer 2/3 and realistic pad stimulation is sufficient to produce such a map. Its late emergence long after other classical maps suggests that experience-dependent map formation and refinement continue throughout adult life.
Assuntos
Vias Aferentes/fisiologia , Mapeamento Encefálico , Córtex Somatossensorial/fisiologia , Vibrissas/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Eletroencefalografia/métodos , Imageamento Tridimensional , Estimulação Física/métodos , Ratos , Córtex Somatossensorial/crescimento & desenvolvimento , Vibrissas/inervaçãoRESUMO
We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 µM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.
Assuntos
Cálcio/análise , Ácido Egtázico/análogos & derivados , Corantes Fluorescentes/química , Indicadores e Reagentes/química , Fótons , Animais , Astrócitos/química , Astrócitos/efeitos dos fármacos , Ácido Egtázico/síntese química , Ácido Egtázico/química , Ácido Egtázico/farmacocinética , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacocinética , Indicadores e Reagentes/síntese química , Indicadores e Reagentes/farmacocinética , Camundongos , Camundongos Endogâmicos , Estrutura MolecularRESUMO
Two-photon laser scanning microscopy (2PLSM) is an important tool for in vivo tissue imaging with sub-cellular resolution, but the penetration depth of current systems is potentially limited by sample-induced optical aberrations. To quantify these, we measured the refractive index n' in the somatosensory cortex of 7 rats in vivo using defocus optimization in full-field optical coherence tomography (ff-OCT). We found n' to be independent of imaging depth or rat age. From these measurements, we calculated that two-photon imaging beyond 200 µm into the cortex is limited by spherical aberration, indicating that adaptive optics will improve imaging depth.
Assuntos
Encéfalo/fisiologia , Microscopia Confocal/métodos , Fótons , Refratometria , Tomografia de Coerência Óptica/métodos , Envelhecimento/fisiologia , Animais , Vasos Sanguíneos/anatomia & histologia , Masculino , Ratos , Ratos Wistar , Gravação em VídeoRESUMO
Fluorescence imaging in the brain of freely behaving mice is challenging due to severe miniaturization constraints. In particular, the ability to image a large field of view at high temporal resolution and with efficient out-of-focus background rejection still raises technical difficulties. Here, we present a novel fiberscope system that provides fast (up to 200 Hz) background-free fluorescence imaging in freely behaving mice over a field of view of diameter 230 µm. The fiberscope is composed of a custom-made multipoint-scanning confocal microscope coupled to the animal with an image guide and a micro-objective. By simultaneously registering a multipoint-scanning confocal image and a conventional widefield image, we subtracted the residual out-of-focus background and provided a background-free confocal image. Illumination and detection pinholes were created using a digital micromirror device, providing high adaptability to the sample structure and imaging conditions. Using this novel imaging tool, we demonstrated fast fluorescence imaging of microvasculature up to 120 µm deep in the mouse cortex, with an out-of-focus background reduced by two orders of magnitude compared with widefield microscopy. Taking advantage of the high acquisition rate (200 Hz), we measured red blood cell velocity in the cortical microvasculature and showed an increase in awake, unrestrained mice compared with anaesthetized animals.
Assuntos
Córtex Cerebral/diagnóstico por imagem , Tecnologia de Fibra Óptica/métodos , Microscopia Intravital/métodos , Microtecnologia/métodos , Animais , Velocidade do Fluxo Sanguíneo , Córtex Cerebral/irrigação sanguínea , Eritrócitos/fisiologia , Tecnologia de Fibra Óptica/instrumentação , Microscopia Intravital/instrumentação , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microtecnologia/instrumentação , Microvasos/diagnóstico por imagem , Modelos Animais , Fibras ÓpticasRESUMO
In the barrel cortex, several features of single-whisker stimuli are organized in functional maps. The barrel cortex also encodes spatio-temporal correlation patterns of multi-whisker inputs, but so far the cortical mapping of neurons tuned to such input statistics is unknown. Here we report that layer 2/3 of the rat barrel cortex contains an additional functional map based on neuronal tuning to correlated versus uncorrelated multi-whisker stimuli: neuron responses to uncorrelated multi-whisker stimulation are strongest above barrel centres, whereas neuron responses to correlated and anti-correlated multi-whisker stimulation peak above the barrel-septal borders, forming rings of multi-whisker synchrony-preferring cells.
Assuntos
Mapeamento Encefálico , Córtex Cerebral/fisiologia , Vibrissas/fisiologia , Animais , Cálcio/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/fisiologiaRESUMO
We analyze whether the "overstretched," or "S" form of double-stranded DNA consists of essentially separated, or essentially interacting, polynucleotide strands. Comparison of force-extension data for S-DNA and single-stranded DNA shows S-DNA to be distinct from both double helix and single-stranded forms. We use a simple thermodynamical model for tension-melted double-stranded DNA, which indicates that the overstretching transition near 65 piconewtons cannot be explained in terms of conversion of double helix to noninteracting polynucleotide strands. However, the single-strand-like response observed in some experiments can be explained in terms of "unpeeling" of large regions of one strand, starting from nicks on the original double helix. We show that S-DNA becomes unstable to unpeeling at large forces, and that at low ionic strength, or for weakly base-paired sequences, unpeeling can preempt formation of S-DNA. We also analyze the kinetics of unpeeling including the effect of sequence-generated free energy inhomogeneity. We find that strongly base-paired regions generate large barriers that stabilize DNA against unpeeling. For long genomic sequences, these barriers to unpeeling cannot be kinetically crossed until force exceeds approximately 150 piconewtons.
Assuntos
Dano ao DNA , DNA/química , Micromanipulação/métodos , Modelos Químicos , Modelos Moleculares , Cloreto de Sódio/química , Pareamento de Bases , Sequência de Bases , Líquidos Corporais/química , Simulação por Computador , Elasticidade , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Soluções , Estresse MecânicoRESUMO
Aberrations limit the resolution, signal intensity and achievable imaging depth in microscopy. Coherence-gated wavefront sensing (CGWS) allows the fast measurement of aberrations in scattering samples and therefore the implementation of adaptive corrections. However, CGWS has been demonstrated so far only in weakly scattering samples. We designed a new CGWS scheme based on a Linnik interferometer and a SLED light source, which is able to compensate dispersion automatically and can be implemented on any microscope. In the highly scattering rat brain tissue, where multiply scattered photons falling within the temporal gate of the CGWS can no longer be neglected, we have measured known defocus and spherical aberrations up to a depth of 400 µm.
RESUMO
Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-µm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.
Assuntos
Imagem Molecular/métodos , Bainha de Mielina/química , Tomografia de Coerência Óptica/métodos , Animais , Córtex Cerebral/química , Córtex Cerebral/ultraestrutura , Proteína 2 de Resposta de Crescimento Precoce/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Microscopia , Bainha de Mielina/ultraestrutura , Ratos , Ratos Wistar , Nervo Isquiático/química , Nervo Isquiático/ultraestruturaRESUMO
Sustained firing is necessary for the persistent activity associated with working memory. The relative contributions of the reverberation of excitation and of the temporal dynamics of the excitatory postsynaptic potential (EPSP) to the maintenance of activity are difficult to evaluate in classical preparations. We used simplified models of synchronous excitatory networks, hippocampal autapses and pairs, to study the synaptic mechanisms underlying firing at low rates. Calcium imaging and cell attached recordings showed that these neurons spontaneously fired bursts of action potentials that lasted for seconds over a wide range of frequencies. In 2-wk-old cells, the median firing frequency was low (11 +/- 8.8 Hz), whereas in 3- to 4-wk-old cells, it decreased to a very low value (2 +/- 1.3 Hz). In both cases, we have shown that the slowest synaptic component supported firing. In 2-wk-old autapses, antagonists of N-methyl-d-aspartate receptors (NMDARs) induced rare isolated spikes showing that the NMDA component of the EPSP was essential for bursts at low frequency. In 3- to 4-wk-old neurons, the very low frequency firing was maintained without the NMDAR activation. However EGTA-AM or alpha-methyl-4-carboxyphenylglycine (MCPG) removed the very slow depolarizing component of the EPSP and prevented the sustained firing at very low rate. A metabotropic glutamate receptor (mGluR)-activated calcium sensitive conductance is therefore responsible for a very slow synaptic component associated with firing at very low rate. In addition, our observations suggested that the asynchronous release of glutamate might participate also in the recurring bursting.
Assuntos
Potenciais de Ação/fisiologia , Ácido Egtázico/análogos & derivados , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/fisiologia , Dinâmica não Linear , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Diagnóstico por Imagem/métodos , Interações Medicamentosas , Ácido Egtázico/metabolismo , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Ratos , Transmissão Sináptica/efeitos dos fármacos , Fatores de TempoRESUMO
Neocortical neurons in vivo are embedded in networks with intensive ongoing activity. How this network activity affects the neurons' integrative properties and what function this may imply at the network level remain largely unknown. Most of our knowledge regarding synaptic communication and integration is based on recordings in vitro, where network activity is strongly diminished or even absent. Here, we present results from two complementary series of experiments based on intracellular in vivo recordings in anesthetized rat frontal cortex. Specifically, we measured 1) the relationship between the excursions of a neuron's membrane potential and the spiking activity in the surrounding network and 2) how the summation of several inputs to a single neuron changes with the different levels of its membrane potential excursions and the associated states of network activity. The combination of these measurements enables us to assess how the level of network activity influences synaptic integration. We present direct evidence that integration of synaptic inputs in frontal cortex is linear, independent of the level of network activity. However, during periods of high network activity, the neurons' response to synaptic input is markedly reduced in both amplitude and duration. This results in a drastic shortening of its window for temporal integration, requiring more precise coordination of presynaptic spike discharges to reliably drive the neuron to spike under conditions of high network activity. We conclude that ongoing activity, as present in the active brain, emphasizes the need for neuronal cooperation at the network level, and cannot be ignored in the exploration of cortical function.
Assuntos
Neocórtex/citologia , Rede Nervosa/fisiologia , Córtex Pré-Frontal/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Limiar Diferencial/fisiologia , Limiar Diferencial/efeitos da radiação , Impedância Elétrica , Estimulação Elétrica/métodos , Eletrodos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Modelos Neurológicos , Neocórtex/fisiologia , Condução Nervosa/fisiologia , Condução Nervosa/efeitos da radiação , Células Piramidais/efeitos da radiação , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Sinapses/efeitos da radiação , Transmissão Sináptica/efeitos da radiação , Fatores de TempoRESUMO
PURPOSE: The substantia nigra pars reticulata (SNpr) is assumed to be involved in the control of several kinds of epileptic seizures, an assumption based mostly on neuropharmacologic evidence. However, only very few neurophysiological recordings from the basal ganglia support neuropharmacologic data. We investigated the electrophysiologic activity of SNpr neurons in rats with genetic absence epilepsy. METHODS: Electrocorticography (ECoG) and multi-unit recordings using permanently implanted tetrodes were obtained in freely behaving rats. After spike sorting, auto- and cross-correlation analysis was used to detect oscillatory neuronal activities and synchronizations. RESULTS: During interictal periods, neither oscillation nor synchronization could be observed in the firing patterns of SNpr neurons. At the beginning of the absence seizure, the firing rate increased significantly. The SNpr neurons started firing in bursts of action potentials. Bursts were highly correlated to the spike-and-wave discharges (SWDs) in the ECoG, mainly after the spike component of the cortical spike-and-wave complex. Moreover, pairs of SNpr neurons tended to fire synchronously. Before the end of the seizure, the firing rate decreased progressively, and the burst-firing pattern ended at or before the end of the SWDs. Once the SWDs had stopped, the SNpr neurons resumed their basal firing pattern as before the seizure onset. CONCLUSIONS: These results provide electrophysiologic evidence that firing patterns and synchronization of SNpr neurons are in phase with the occurrence of SWDs. The findings support the concept that nigral control mechanisms are involved in modulating the propagation of an ongoing generalized seizure.