All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins
01 Jun 2014-
TL;DR: The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes and revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons.
Abstract: All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.
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1,063 citations
TL;DR: This review describes and assess different classes of fluorescent protein indicators of neural activity and focuses on how indicator characteristics relate to their use in living animals and on likely areas of future progress.
Abstract: Genetically encoded indicators of neuronal activity have diversified and improved in performance in recent years, becoming essential tools for neuroscientists. Lin and Schnitzer review indicators for pH, neurotransmitter, voltage and calcium, with an emphasis on quantifying key indicator attributes and relating them to their applications in neuroscience.
528 citations
TL;DR: This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mouse brain in vivo.
Abstract: We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. We performed simultaneous two-photon optogenetic activation and calcium imaging by coexpression of a red-shifted opsin and a genetically encoded calcium indicator. A spatial light modulator allows tens of user-selected neurons to be targeted for spatiotemporally precise concurrent optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical cross-talk. Proof-of-principle experiments in mouse barrel cortex demonstrate interrogation of the same neuronal population during different behavioral states and targeting of neuronal ensembles based on their functional signature. This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mouse brain in vivo.
511 citations
TL;DR: It is shown that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity to the biological sample.
Abstract: Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution-e.g., magnetic resonance imaging methods and magnetoencephalography-or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector.
509 citations
TL;DR: This review provides both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
464 citations
Cites background from "All-optical electrophysiology in ma..."
...With the rapid adoption of optogenetic approaches and recent advances towards achieving all optical observation and manipulation of biological pathways [159], the potential utilization of FPs for simultaneous observation and manipulation may further revolutionize the way intracellular events are studied in the coming decades....
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References
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TL;DR: In this paper, the authors adapted the naturally occurring algal protein Channelrhodopsin-2, a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons.
Abstract: Temporally precise, noninvasive control of activity in well-defined neuronal populations is a long-sought goal of systems neuroscience. We adapted for this purpose the naturally occurring algal protein Channelrhodopsin-2, a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons. We demonstrate reliable, millisecond-timescale control of neuronal spiking, as well as control of excitatory and inhibitory synaptic transmission. This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events, yielding a widely applicable tool for neuroscientists and biomedical engineers.
4,411 citations
TL;DR: These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.
Abstract: A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.
3,785 citations
TL;DR: Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium and yielded thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization.
3,086 citations
TL;DR: The action potential of the squid giant axon is formed by just two voltage-dependent conductances in the cell membrane, yet mammalian central neurons typically express more than a dozen different types of voltage- dependent ion channels.
Abstract: The action potential of the squid giant axon is formed by just two voltage- dependent conductances in the cell membrane, yet mammalian central neurons typically express more than a dozen different types of voltage-dependent ion channels. This rich repertoire of channels allows neurons to encode information by generating action potentials with a wide range of shapes, frequencies and patterns. Recent work offers an increasingly detailed understanding of how the expression of particular channel types underlies the remarkably diverse firing behaviour of various types of neurons.
1,426 citations
Book•
01 Jan 1998
TL;DR: A user's guide to types of nerve cell cultures, their advantages and limitations, and tissue culture methods for the study of myelination.
Abstract: Part 1 A user's guide: getting started, Gary Banker, Kimberly Goslin. Part 2 General principles: types of nerve cell cultures, their advantages and limitations, Gary Banker, Kimberly Goslin primary dissociated cell cultures, Dennis Higgins, Gary Banker transfecting cultured neurons, Ann Marie Craig characterizing and studying neuronal cultures, Ginger S. Withers, Gary Banker. Part 3 Culture of specific cell types - choosing the right system: culture and experimental use of the PC12 rat pheochromocytoma cell line, Lloyd A. Greene et al neuronlike cells derived in culture from P19 embryonal carcinoma and embryonic stem cells, Gerard Bain et al culturing the large neurons of aplasia, Daniel J. Goldberg, Samuel Schacher culturing spinal neurons and muscle cells from xenopus embryos, Nacira Tabti et el cultures from chick peripheral ganglia, Carolyn L. Smith culturing mammalian sympathoadrenal derivatives, Nagesh K. Mahanthappa, Paul H. Patterson mass cultures and microislands of neurons from postnatal rat brain, Michael M. Segal et al rat hippocampal neurons in low-density culture, Kimberley Goslin et al rat striatal neurons in low-density, serum-free culture, Roseann Ventimiglia, Ronald M. Lindsay cell culture of cholinergic and cholinoceptive neurons from the medial habenulae, Raul Krauss, Gerald D. Fischbach the cerebellum - purification and coculture of identified cell populations, Mary Beth Hatten et al organotypic slice cultures of neural tissue, Beat H. Gahwiler et al culture of astrocytes, oligodendrocytes, and O-2A progenitor cells, Mark Noble, Margot Mayer-Proschel tissue culture methods for the study of myelination, Naomi Kleitman et al.
1,396 citations