67 research outputs found
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Portable Scanless Microscope: Two- and One-photon Imaging and Photostimulation with Spatial Light Modulators
Imaging voltage in neurons
In the last decades, imaging membrane potential has become a fruitful approach to study neural circuits, especially in invertebrate preparations with large, resilient neurons. At the same time, particularly in mammalian preparations, voltage imaging methods suffer from poor signal to noise and secondary side effects, and they fall short of providing single-cell resolution when imaging of the activity of neuronal populations. As an introduction to these techniques, we briefly review different voltage imaging methods (including organic fluorophores, SHG chromophores, genetic indicators, hybrid, nanoparticles, and intrinsic approaches) and illustrate some of their applications to neuronal biophysics and mammalian circuit analysis. We discuss their mechanisms of voltage sensitivity, from reorientation, electrochromic, or electro-optical phenomena to interaction among chromophores or membrane scattering, and highlight their advantages and shortcomings, commenting on the outlook for development of novel voltage imaging methods
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Attenuation of Synaptic Potentials in Dendritic Spines
Dendritic spines receive the majority of excitatory inputs in many mammalian neurons, but their biophysical properties and exact role in dendritic integration are still unclear. Here, we study spine electrical properties in cultured hippocampal neurons using an improved genetically encoded voltage indicator (ArcLight) and two-photon glutamate uncaging. We find that back-propagating action potentials (bAPs) fully invade dendritic spines. However, uncaging excitatory post-synaptic potentials (uEPSPs) generated by glutamate photorelease, ranging from 4 to 27 mV in amplitude, are attenuated by up to 4-fold as they propagate to the parent dendrites. Finally, the simultaneous occurrence of bAPs and uEPSPs results in sublinear summation of membrane potential. Our results demonstrate that spines can behave as electric compartments, reducing the synaptic inputs injected into the cell, while receiving bAPs are unmodified. The attenuation of EPSPs by spines could have important repercussions for synaptic plasticity and dendritic integration
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Reliable and Elastic Propagation of Cortical Seizures In Vivo
Mapping the fine-scale neural activity that underlies epilepsy is key to identifying potential control targets of this frequently intractable disease. Yet, the detailed in vivo dynamics of seizure progression in cortical microcircuits remain poorly understood. We combine fast (30-Hz) two-photon calcium imaging with local field potential (LFP) recordings to map, cell by cell, the spread of locally induced (4-AP or picrotoxin) seizures in anesthetized and awake mice. Using single-layer and microprism-assisted multilayer imaging in different cortical areas, we uncover reliable recruitment of local neural populations within and across cortical layers, and we find layer-specific temporal delays, suggesting an initial supra-granular invasion followed by deep-layer recruitment during lateral seizure spread. Intriguingly, despite consistent progression pathways, successive seizures show pronounced temporal variability that critically depends on GABAergic inhibition. We propose an epilepsy circuit model resembling an elastic meshwork, wherein ictal progression faithfully follows preexistent pathways but varies flexibly in time, depending on the local inhibitory restraint
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Altered Cortical Ensembles in Mouse Models of Schizophrenia
In schizophrenia, brain-wide alterations have been identified at the molecular and cellular levels, yet how these phenomena affect cortical circuit activity remains unclear. We studied two mouse models of schizophrenia-relevant disease processes: chronic ketamine (KET) administration and Df(16)A+/-, modeling 22q11.2 microdeletions, a genetic variant highly penetrant for schizophrenia. Local field potential recordings in visual cortex confirmed gamma-band abnormalities similar to patient studies. Two-photon calcium imaging of local cortical populations revealed in both models a deficit in the reliability of neuronal coactivity patterns (ensembles), which was not a simple consequence of altered single neuron activity. This effect was present in ongoing and sensory-evoked activity and was not replicated by acute ketamine administration or pharmacogenetic parvalbumin-interneuron suppression. These results are consistent with the hypothesis that schizophrenia is an ‘‘attractor’’ disease and demonstrate that degraded neuronal ensembles are a common consequence of diverse genetic, cellular, and synaptic alterations seen in chronic schizophrenia
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Simultaneous two-photon imaging and two-photon optogenetics of cortical circuits in three dimensions
The simultaneous imaging and manipulating of neural activity could enable the functional dissection of neural circuits. Here we have combined two-photon optogenetics with simultaneous volumetric two-photon calcium imaging to measure and manipulate neural activity in mouse neocortex in vivo in three-dimensions (3D) with cellular resolution. Using a hybrid holographic approach, we simultaneously photostimulate more than 80 neurons over 150 μm in depth in layer 2/3 of the mouse visual cortex, while simultaneously imaging the activity of the surrounding neurons. We validate the usefulness of the method by photoactivating in 3D selected groups of interneurons, suppressing the response of nearby pyramidal neurons to visual stimuli in awake animals. Our all-optical approach could be used as a general platform to read and write neuronal activity
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RuBi-Glutamate: two-photon and visible-light photoactivation of neurons and dendritic spines
We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photo-activation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single spine, or single cell, precision
Upcoming Neurophotonics Status Report
Forthcoming status report articles provide updates on microscopy and on diffuse optical imaging in neurophotonics
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SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators
Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a significant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a “scanless” microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the flexibility of laser microscopy, as it can substitute traditional simple fixed lenses with any calculated lens function
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