Genetically-encoded Voltage Research Articles

Fluorescence Imaging of Cell Membrane Potential: From Relative Changes to Absolute Values.

Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.

Optimal control methods for nonlinear parameter estimation in biophysical neuron models.

Functional forms of biophysically-realistic neuron models are constrained by neurobiological and anatomical considerations, such as cell morphologies and the presence of known ion channels. Despite these constraints, neuron models still contain unknown static parameters which must be inferred from experiment. This inference task is most readily cast into the framework of state-space models, which systematically takes into account partial observability and measurement noise. Inferring only dynamical state variables such as membrane voltages is a well-studied problem, and has been approached with a wide range of techniques beginning with the well-known Kalman filter. Inferring both states and fixed parameters, on the other hand, is less straightforward. Here, we develop a method for joint parameter and state inference that combines traditional state space modeling with chaotic synchronization and optimal control. Our methods are tailored particularly to situations with considerable measurement noise, sparse observability, very nonlinear or chaotic dynamics, and highly uninformed priors. We illustrate our approach both in a canonical chaotic model and in a phenomenological neuron model, showing that many unknown parameters can be uncovered reliably and accurately from short and noisy observed time traces. Our method holds promise for estimation in larger-scale systems, given ongoing improvements in calcium reporters and genetically-encoded voltage indicators.

Evoked Cortical Depolarizations Before and After the Amyloid Plaque Accumulation: Voltage Imaging Study.

In Alzheimer's disease (AD), synaptic dysfunction is thought to occur many years before the onset of cognitive decline. Detecting synaptic dysfunctions at the earliest stage of AD would be desirable in both clinic and research settings. Population voltage imaging allows monitoring of synaptic depolarizations, to which calcium imaging is relatively blind. We developed an AD mouse model (APPswe/PS1dE9 background) expressing a genetically-encoded voltage indicator (GEVI) in the neocortex. GEVI was restricted to the excitatory pyramidal neurons (unlike the voltage-sensitive dyes). Expression of GEVI did not disrupt AD model formation of amyloid plaques. GEVI expression was stable in both AD model mice and Control (healthy) littermates (CTRL) over 247 days postnatal. Brain slices were stimulated in layer 2/3. From the evoked voltage waveforms, we extracted several parameters for comparison AD versus CTRL. Some parameters (e.g., temporal summation, refractoriness, and peak latency) were weak predictors, while other parameters (e.g., signal amplitude, attenuation with distance, and duration (half-width) of the evoked transients) were stronger predictors of the AD condition. Around postnatal age 150 days (P150) and especially at P200, synaptically-evoked voltage signals in brain slices were weaker in the AD groups versus the age- and sex-matched CTRL groups, suggesting an AD-mediated synaptic weakening that coincides with the accumulation of plaques. However, at the youngest ages examined, P40 and P80, the AD groups showed differentially stronger signals, suggesting "hyperexcitability" prior to the formation of plaques. Our results indicate bidirectional alterations in cortical physiology in AD model mice; occurring both prior (P40-80), and after (P150-200) the amyloid deposition.

Simultaneous two-photon imaging of action potentials and subthreshold inputs in vivo

To better understand the input-output computations of neuronal populations, we developed ArcLight-ST, a genetically-encoded voltage indicator, to specifically measure subthreshold membrane potentials. We combined two-photon imaging of voltage and calcium, and successfully discriminated subthreshold inputs and spikes with cellular resolution in vivo. We demonstrate the utility of the method by mapping epileptic seizures progression through cortical circuits, revealing divergent sub- and suprathreshold dynamics within compartmentalized epileptic micronetworks. Two-photon, two-color imaging of calcium and voltage enables mapping of inputs and outputs in neuronal populations in living animals.

Abstract 14161: Drug Compounds Combination Screening Using Human Induced Pluripotent Stem Cell-Derived Cardiac Tissues

Introduction: The introduction of human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) technology provided an important tool for the field of safety pharmacology (QT screening). This approach has been limited, however, mostly to the assessment of a single drug. We present a large scale EP phenotyping system comprised of hiPSC-derived cardiac cell sheets (hiPSC-CCSs), fluorescent voltage sensors and actuators, and an optical mapping system; with the aim of performing drug combination screening. Methods and Results: hIPSCs-CMs were seeded as hiPSC-CCSs in multi-well plates. Tissues were treated with varying concentrations of drug compounds’ combinations. Optical signals were acquired following loading of the hiPSC-CCSs with voltage-sensitive dyes or expression of genetically-encoded voltage indicator (ArcLight). EP parameters such as conduction velocity (CV) and action potential duration (APD) were extracted. ATX2 (late sodium current activator) and Dofetilide (IKr blocker) were chosen as representative APD prolonging reagents. Example of the effects of 16 different drug doses combinations on APD (% change) prolongation is depicted in Fig.1a. Similarly, examples of the restitution plots obtained at 4 different drug combinations are presented in Fig.1b-c. Notice that drug compound's effect on prolonging APD showed greater effect as compared to the cumulative effect of each drug alone. Figure 1: (a) Percentage of change in APD 80 15 minutes post treatment (b, c) APD 80 per condition before and after treatment, respectively Conclusion: These results highlight the potential of the hiPSC-CCS as a model for combinational drug screening. This may bring a unique value to the fields of safety pharmacology, pathophysiology testing and drug screening applications.

Cholinergic modulation of sensory processing in awake mouse cortex

Cholinergic modulation of brain activity is fundamental for awareness and conscious sensorimotor behaviours, but deciphering the timing and significance of acetylcholine actions for these behaviours is challenging. The widespread nature of cholinergic projections to the cortex means that new insights require access to specific neuronal populations, and on a time-scale that matches behaviourally relevant cholinergic actions. Here, we use fast, voltage imaging of L2/3 cortical pyramidal neurons exclusively expressing the genetically-encoded voltage indicator Butterfly 1.2, in awake, head-fixed mice, receiving sensory stimulation, whilst manipulating the cholinergic system. Altering muscarinic acetylcholine function re-shaped sensory-evoked fast depolarisation and subsequent slow hyperpolarisation of L2/3 pyramidal neurons. A consequence of this re-shaping was disrupted adaptation of the sensory-evoked responses, suggesting a critical role for acetylcholine during sensory discrimination behaviour. Our findings provide new insights into how the cortex processes sensory information and how loss of acetylcholine, for example in Alzheimer’s Disease, disrupts sensory behaviours.

Population imaging discrepancies between a genetically-encoded calcium indicator (GECI) versus a genetically-encoded voltage indicator (GEVI)

Genetically-encoded calcium indicators (GECIs) are essential for studying brain function, while voltage indicators (GEVIs) are slowly permeating neuroscience. Fundamentally, GECI and GEVI measure different things, but both are advertised as reporters of “neuronal activity”. We quantified the similarities and differences between calcium and voltage imaging modalities, in the context of population activity (without single-cell resolution) in brain slices. GECI optical signals showed 8–20 times better SNR than GEVI signals, but GECI signals attenuated more with distance from the stimulation site. We show the exact temporal discrepancy between calcium and voltage imaging modalities, and discuss the misleading aspects of GECI imaging. For example, population voltage signals already repolarized to the baseline (~ disappeared), while the GECI signals were still near maximum. The region-to-region propagation latencies, easily captured by GEVI imaging, are blurred in GECI imaging. Temporal summation of GECI signals is highly exaggerated, causing uniform voltage events produced by neuronal populations to appear with highly variable amplitudes in GECI population traces. Relative signal amplitudes in GECI recordings are thus misleading. In simultaneous recordings from multiple sites, the compound EPSP signals in cortical neuropil (population signals) are less distorted by GEVIs than by GECIs.

Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro.

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

HIV and opiates dysregulate K+- Cl− cotransporter 2 (KCC2) to cause GABAergic dysfunction in primary human neurons and Tat-transgenic mice

Approximately half of people infected with HIV (PWH) exhibit HIV-associated neuropathology (neuroHIV), even when receiving combined antiretroviral therapy. Opiate use is widespread in PWH and exacerbates neuroHIV. While neurons themselves are not infected, they incur sublethal damage and GABAergic disruption is selectively vulnerable to viral and inflammatory factors released by infected/affected glia. Here, we demonstrate diminished K+-Cl− cotransporter 2 (KCC2) levels in primary human neurons after exposure to HIV-1 or HIV-1 proteins ± morphine. Resulting disruption of GABAAR-mediated hyperpolarization/inhibition was shown using genetically-encoded voltage (Archon1) and calcium (GCaMP6f) indicators. The HIV proteins Tat (acting through NMDA receptors) and R5-gp120 (acting via CCR5) but not X4-tropic gp120 (acting via CXCR4), and morphine (acting through μ-opioid receptors) all induced KCC2 loss. We demonstrate that modifying KCC2 levels or function, or antagonizing NMDAR, CCR5 or MOR rescues KCC2 and GABAAR-mediated hyperpolarization/inhibition in HIV, Tat, or gp120 ± morphine-exposed neurons. Using an inducible, Tat-transgenic mouse neuroHIV model, we found that chronic exposure to Tat also reduces KCC2. Our results identify KCC2 as a novel therapeutic target for ameliorating the pathobiology of neuroHIV, including PWH exposed to opiates.

Audio-visual experience strengthens multisensory assemblies in adult mouse visual cortex

We experience the world through multiple senses simultaneously. To better understand mechanisms of multisensory processing we ask whether inputs from two senses (auditory and visual) can interact and drive plasticity in neural-circuits of the primary visual cortex (V1). Using genetically-encoded voltage and calcium indicators, we find coincident audio-visual experience modifies both the supra and subthreshold response properties of neurons in L2/3 of mouse V1. Specifically, we find that after audio-visual pairing, a subset of multimodal neurons develops enhanced auditory responses to the paired auditory stimulus. This cross-modal plasticity persists over days and is reflected in the strengthening of small functional networks of L2/3 neurons. We find V1 processes coincident auditory and visual events by strengthening functional associations between feature specific assemblies of multimodal neurons during bouts of sensory driven co-activity, leaving a trace of multisensory experience in the cortical network.

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

De Novo Designed Proteins for Ultrafast Detection of Membrane Potential Changes

Neuronal networks communicate through changes in membrane potentials. Optically recording these changes on the sub-microsecond timescale provides a better understanding of interneuronal communication. While organic probes that have been developed detect signals with fast temporal resolution, these probes lack cell and membrane specificity. Genetically-encoded voltage indicators (GEVIs) remedy this problem; however, these protein-based optical voltage indicators are dimmer and slower than their organic probe counterparts. Here, we present our progress on the design, development, and characterization of novel GEVIs based on artificial 4-α-helical bundle proteins, called maquettes. We demonstrated that membrane maquettes are able to bind 3 hemes upon assembly in vesicles, each with different redox midpoint potentials. To sense changes in membrane potential, we are modifying our first prototype maquettes to develop two classes of maquette GEVIs. The first class of maquette GEVIs covalently binds a fluorescent cofactor, and we are using directed evolution to improve its fluorescent signal and quantum yield. The second class of maquette GEVIs is a fusion construct of a maquette and a fluorescent protein, whose fluorescence can be modulated by a chain of voltage-sensitive hemes embedded within the maquette. Energy transfer has been demonstrated between the fluorescent protein and the membrane maquette. These maquette GEVI prototypes have been expressed in E. coli and purified for in vitro characterization. Parallel maquettes are being developed for expression in hippocampal neuronsto validate the in vitro biophysical approach in mammalian cells.

Genetically-encoded voltage indicators

A holy grail in neuroscience is to understand how brain functions arise from neural network-level electrical activities. Voltage imaging allows for the direct visualization of electrical signaling at high spatial and temporal resolutions across a large neuronal population. Central to this technique is a palette of genetically-encoded fluorescent probes with fast and sensitive voltage responses. In this review, we chronicle the development and applications of genetically-encoded voltage indicators (GEVIs) over the past two decades, with a primary focus on the structural design that harness the power of fluctuating transmembrane electric fields. We hope this article will inform chemical biologists and protein engineers of the GEVI history and inspire novel design ideas.

Exploring the Movement of S4 by Intermolecular FRET of Genetically-Encoded Voltage Indicators

Exploring the Movement of S4 by Intermolecular FRET of Genetically-Encoded Voltage Indicators

遺伝子にコードされた膜電位センサーによる神経活動計測の現状と展望

遺伝子にコードされた膜電位センサー（GEVIs）は、多くの神経細胞からの電気シグナルを同時計測可能であるため、神経科学の分野で注目されている。しかしながらGEVIsが現在まで数多く発表されているため、使用する側としてはどのGEVIsを実験に使用すべきかわからなくなることが多い。実際にそれぞれのGEVIsには利点と欠点があり、使用する際には注意が必要である。そこで本稿では初期のイオンチャネル型から2015年に発表された最新のロドプシン-蛍光タンパク質型まで、GEVIsの開発競争の流れを振り返り、それぞれのGEVIsの特徴や実験による使い分けについて概説する。

Expression and testing in plants of ArcLight, a genetically-encoded voltage indicator used in neuroscience research.

BackgroundIt is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues. An emerging method for non-invasive optical imaging of electrical activity at cell membranes uses genetically-encoded voltage indicators (GEVIs). Developed by neuroscientists to chart neuronal circuits in animals, GEVIs comprise a fluorescent protein that is fused to a voltage-sensing domain. One well-known GEVI, ArcLight, undergoes strong shifts in fluorescence intensity in response to voltage changes in mammalian cells. ArcLight consists of super-ecliptic (SE) pHluorin (pH-sensitive fluorescent protein) with an A227D substitution, which confers voltage sensitivity in neurons, fused to the voltage-sensing domain of the voltage-sensing phosphatase of Cionaintestinalis (Ci-VSD). In an ongoing effort to adapt tools of optical electrophysiology for plants, we describe here the expression and testing of ArcLight and various derivatives in different membranes of root cells in Arabidopsis thaliana.ResultsTransgenic constructs were designed to express ArcLight and various derivatives targeted to the plasma membrane and nuclear membranes of Arabidopsis root cells. In transgenic seedlings, changes in fluorescence intensity of these reporter proteins following extracellular ATP (eATP) application were monitored using a fluorescence microscope equipped with a high speed camera. Coordinate reductions in fluorescence intensity of ArcLight and Ci-VSD-containing derivatives were observed at both the plasma membrane and nuclear membranes following eATP treatments. However, similar responses were observed for derivatives lacking the Ci-VSD. The dispensability of the Ci-VSD suggests that in plants, where H+ ions contribute substantially to electrical activities, the voltage-sensing ability of ArcLight is subordinate to the pH sensitivity of its SEpHluorin base. The transient reduction of ArcLight fluorescence triggered by eATP most likely reflects changes in pH and not membrane voltage.ConclusionsThe pH sensitivity of ArcLight precludes its use as a direct sensor of membrane voltage in plants. Nevertheless, ArcLight and derivatives situated in the plasma membrane and nuclear membranes may offer robust, fluorescence intensity-based pH indicators for monitoring concurrent changes in pH at these discrete membrane systems. Such tools will assist analyses of pH as a signal and/or messenger at the cell surface and the nuclear periphery in living plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0633-z) contains supplementary material, which is available to authorized users.

In This Issue

HomeCirculation ResearchVol. 117, No. 5In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published14 Aug 2015https://doi.org/10.1161/RES.0000000000000070Circulation Research. 2015;117:389Optogenetic Voltage Sensing in Cardiac Myocytes (p 401)Download figureDownload PowerPointLiao et al measure electrical activity of heart cells and whole hearts with a fluorescent reporter.Monitoring electrical activity of the heart or cardiomyocytes in culture provides important information about normal and pathological cardiac physiology. But each recording method has limitations. Extracellular electrodes are non-invasive but have poor resolution. Intracellular recordings provide good resolution but are time-consuming, invasive and resource-intensive. Genetically-encoded voltage indicators (GEVI), however, may provide the best of both. They have been used to non-invasively measure electrical activity in brain cells of the mouse and fly, and now Liao and colleagues have tested one GEVI—called VSFP2.3—in whole mouse hearts and heart cells. Similar to VSFP2.3’s behavior in neurons, the protein’s fluorescent signal altered upon heart cell membrane depolarization: yellow emission increased and cyan decreased. This measurable change in fluorescence enabled the team to perform optical cardiograms on whole ex vivo mouse hearts, and on live mice—via optical fibers inserted into the animals’ chests. Lastly, the team used VSFP2.3 to monitor electrical activity of induced pluripotent stem cell-derived cardiomyocytes in vitro. Together the results provide a proof of principle that the use of GEVIs can provide high resolution monitoring of cardiovascular activity in vitro and in vivo.Methods to Study Cardiac Myocyte Epigenomics (p 413)Download figureDownload PowerPointPreissl et al investigate the epigenetic code and transcription profile of cardiomyocyte nuclei.The identity of a cell does not just come down to the proteins it expresses but to the genetic and epigenetic mechanisms that regulate protein expression. A thorough understanding of the epigenetic landscape of cardiomyocytes and how that landscape changes during development and disease could thus offer valuable insights into heart physiology. To gain such an understanding, Preissl and colleagues first devised a method for isolating highly pure cardiomyocyte nuclei, which relied on cold conditions and avoided enzymatic digestion to preserve the structural integrity of the nuclei and chromatin therein. The team separated cardiomyocyte nuclei from those of other heart cell types on the basis of expression of a specific marker protein—PCM1—and then analyzed nuclear mRNA expression, cellular mRNA expression and a number of epigenetic marks. Their study revealed that the pattern of epigenetic marks correlated more closely with nuclear mRNA expression than cellular mRNA, confirming that the former is a more accurate reflection of the transcription activity of cells. The procedure described in this paper serves as a springboard for further epigenetic analyses of cardiomyocytes from different stages of development or under different pathological conditions, say the authors.Role of miR-34a Post-MI (p 470)Download figureDownload PowerPointMicroRNA-34a impairs heart regeneration after myocardial infarction, report Yang et al.The hearts of newborn mice can almost entirely recover from injury, but by one week of age this regenerative capacity diminishes and remains lost throughout adulthood. If researchers could understand what makes regeneration possible in young mice they might be able to recapitulate regeneration in human hearts after myocardial infarctions. To this end, Yang et al have now investigated the role of microRNA miR34a in mouse heart regeneration. Mir34a is a suppressor of cell proliferation known to increase in expression in the heart with age. Furthermore, suppression of miR34a reduces age-associated cardiomyocyte death. The team showed that levels of miR34a are low in the newborn mouse heart and rise to adult levels within a week of birth. Over-expression of miR34a in the newborn mice prevented heart regeneration, whereas inhibition of miR34a in the adult heart improved cardiac function after infarction. Lastly, the team identified direct targets of miR34a repression—Sirt1, cyclinD1, and Bcl2—which are known to be involved in cell aging, proliferation and survival. Inhibition of miR34a in adult hearts led to the upregulation of these genes. Together the results suggest that inhibiting miR34a, or boosting its targets, may offer therapeutic routes to improve heart recovery after myocardial infarction. Previous Back to top Next FiguresReferencesRelatedDetails August 14, 2015Vol 117, Issue 5 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000070 Originally publishedAugust 14, 2015 PDF download Advertisement

Comparative performance of a genetically-encoded voltage indicator and a blue voltage sensitive dye for large scale cortical voltage imaging.

Traditional small molecule voltage sensitive dye indicators have been a powerful tool for monitoring large scale dynamics of neuronal activities but have several limitations including the lack of cell class specific targeting, invasiveness and difficulties in conducting longitudinal studies. Recent advances in the development of genetically-encoded voltage indicators have successfully overcome these limitations. Genetically-encoded voltage indicators (GEVIs) provide sufficient sensitivity to map cortical representations of sensory information and spontaneous network activities across cortical areas and different brain states. In this study, we directly compared the performance of a prototypic GEVI, VSFP2.3, with that of a widely used small molecule voltage sensitive dye (VSD), RH1691, in terms of their ability to resolve mesoscopic scale cortical population responses. We used three synchronized CCD cameras to simultaneously record the dual emission ratiometric fluorescence signal from VSFP2.3 and RH1691 fluorescence. The results show that VSFP2.3 offers more stable and less invasive recording conditions, while the signal-to-noise level and the response dynamics to sensory inputs are comparable to RH1691 recordings.

The Influence of Voltage Sensor Activity on Arclight Dynamics

Many genetically-encoded voltage indicators have been developed based on voltage-sensitive phosphatases. One of the most promising of these indicators is Arclight, largely due to its substantial signal in response to voltage changes. Although it is evident that the fluorophore is fundamentally following the voltage sensor movement, the fluorescence change is comprised of multiple components; how these fluorescence components reflect voltage sensor movement remains poorly understood. Using simultaneous electrophysiological and optical recordings of oocytes expressing wild-type and mutant Arclight constructs, we investigated the influence of altered voltage sensor behavior on Arclight fluorescence. We found that the latency before fluorescence change onset correlated with gating charge movement, and that the fluorescence change following prolonged depolarizations showed alterations in kinetics and thermodynamics consistent with voltage sensor relaxation. Other features of fluorophore movement, however, were less obviously coupled to voltage sensor dynamics. These results may help guide future attempts to optimize the properties of Arclight and related fluorescent proteins. Support: NIH GM030376.

Voltage Imaging in the Study of Hippocampal Circuit Function and Plasticity.

Synaptic plasticity has the capacity to alter the function of neural circuits, and long-term potentiation (LTP) of synaptic transmission induced by high frequency electrical activity has the capacity to store information in neural circuits. The cellular and molecular mechanisms of LTP have been studied intensively for many years and much progress has been made on this front. By contrast, how synaptic plasticity alters circuit function has received much less attention and remains poorly understood. Voltage imaging provides a powerful general technique for the study of neural circuitry, and studies of synaptic plasticity with voltage imaging are beginning to reveal important aspects of how the function of a neural circuit can change when the strength of its synapses has been modified. The hippocampus has an important role in learning and memory and the plasticity of its synapses has received much attention. Voltage imaging with voltage sensitive dye in the CA1 region of a hippocampal slice has shown that spatial patterns of enhancement following LTP induction can diverge from the spatial patterns elicited by electrical stimulation, suggesting that LTP exhibits a distinct organizational structure. LTP can alter the throughput of electrical activity in the dentate gyrus of a hippocampal slice, to gate transmission on to the CA3 region. The spatial patterns evoked by complex electrical stimulation can be stored within the CA3 region in a hippocampal slice, allowing patterns to be reconstructed with simpler electrical stimulation. Thus, voltage imaging has demonstrated that the CA3 circuit has the capacity for pattern completion. These studies with voltage sensitive dye illustrate a range of interesting and novel questions that can be addressed at the population level. It is hoped that future imaging experiments with single-cell resolution using genetically-encoded voltage sensors will provide a more detailed picture of how synaptic plasticity modifies the information processing capabilities of neural circuits.