The simultaneous recording of many individual neurons is fundamental to understanding the integral functionality of neural systems. Imaging with voltage-sensitive dyes (VSDs) is a key approach to achieve this goal and a promising technique to supplement electrophysiological recordings. However, the lack of connectivity maps between imaged neurons and the requirement of averaging over repeated trials impede functional interpretations. Here we demonstrate fast, high resolution and single-sweep VSD imaging of identified and synaptically interacting neurons. We show for the first time the optical recording of individual action potentials and mutual inhibitory synaptic input of two key players in the well-characterized pyloric central pattern generator in the crab stomatogastric ganglion (STG). We also demonstrate the presence of individual synaptic potentials from other identified circuit neurons. We argue that imaging of neural networks with identifiable neurons with well-known connectivity, like in the STG, is crucial for the understanding of emergence of network functionality.

Voltage-sensitive dye (VSD) imaging and intrinsic optical signals (IOS) are widely used methods for monitoring spatiotemporal neural activity in extensive networks. In spite of that, identification of their major cellular and molecular components has not been concluded so far.

The aim of this paper is to present a dynamic causal modeling (DCM) framework for hippocampal activity measured via voltage-sensitive dye imaging (VSDI). We propose a DCM model of the hippocampus that summarizes interactions between the hilus, CA3 and CA1 regions. The activity of each region is governed via a neuronal mass model with two inhibitory and one/two excitatory neuronal populations, which can be linked to measurement VSDI by scaling neuronal activity. To optimize the model structure for the hippocampus, we propose two Bayesian schemes: Bayesian hyperparameter optimization to estimate the unknown electrophysiological properties necessary for constructing a mesoscopic hippocampus model; and Bayesian model reduction to determine the parameterization of neural properties, and to test and include potential connections (morphologically inferred without direct evidence yet) in the model by evaluating group-level model evidence. The proposed method was applied to model spatiotemporal patterns of accumulative responses to consecutive stimuli in separate groups of wild-type mice and epileptic aristaless-related homeobox gene (Arx) conditional knock-out mutant mice (Arx-/+;Dlx5/6CRE-IRES-GFP) in order to identify group differences in the effective connectivity within the hippocampus. The causal role of each group-differing connectivity in generating mutant-like responses was further tested. The group-level analysis identified altered intra- and inter-regional effective connectivity, some of which are crucial for explaining mutant-like responses. The modelling results for the hippocampal activity suggest the plausibility of the proposed mesoscopic hippocampus model and the usefulness of utilizing the Bayesian framework for model construction in the mesoscale modeling of neural interactions using DCM.

Voltage-sensitive dye imaging (VSDI) is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but with effective resolution limits that confound interpretation. To address this limitation, we developed an in silico model of VSDI in a biologically faithful digital reconstruction of rodent neocortical microcircuitry. Using this model, we extend previous experimental observations regarding the cellular origins of VSDI, finding that the signal is driven primarily by neurons in layers 2/3 and 5, and that VSDI measurements do not capture individual spikes. Furthermore, we test the capacity of VSD image sequences to discriminate between afferent thalamic inputs at various spatial locations to estimate a lower bound on the functional resolution of VSDI. Our approach underscores the power of a bottom-up computational approach for relating scales of cortical processing.

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.

Non-clinical toxicology is a major cause of drug candidate attrition during development. In particular, drug-induced seizures are the most common finding in central nervous system (CNS) toxicity. Current safety pharmacology tests for assessing CNS functions are often inadequate in detecting seizure-inducing compounds early in drug development, leading to significant delays. This paper presents an in vitro seizure liability assay using voltage-sensitive dye (VSD) imaging techniques in hippocampal brain slices, offering a powerful alternative to traditional electrophysiological methods. Hippocampal slices were isolated from mice, and VSD optical responses evoked by stimulating the Schaffer collateral pathway were recorded and analyzed in the stratum radiatum (SR) and stratum pyramidale (SP). VSDs allow for the comprehensive visualization of neuronal action potentials and postsynaptic potentials on a millisecond timescale. By employing this approach, we investigated the in vitro drug-induced seizure liability of representative pro-convulsant compounds. Picrotoxin (PiTX; 1-100 μM), gabazine (GZ; 0.1-10 μM), and 4-aminopyridine (4AP; 10-100 μM) exhibited seizure-like responses in the hippocampus, but pilocarpine hydrochloride (Pilo; 10-100 μM) did not. Our findings demonstrate the potential of VSD-based assays in identifying seizurogenic compounds during early drug discovery, thereby reducing delays in drug development and providing insights into the mechanisms underlying seizure induction and the associated risks of pro-convulsant compounds.

Incorporating optical methods into implantable neural sensing devices is a challenging approach for brain-machine interfacing. Specifically, voltage-sensitive dye (VSD) imaging is a powerful tool enabling visualization of the network activity of thousands of neurons at high spatiotemporal resolution. However, VSD imaging usually requires removal of the dura mater for dye staining, and thereafter the exposed cortex needs to be protected using an optically transparent artificial dura. This is a major disadvantage that limits repeated VSD imaging over the long term. To address this issue, we propose to use an atelocollagen membrane as the dura substitute. We fabricated a small cranial chamber device, which is a tubular structure equipped with a collagen membrane at one end of the tube. We implanted the device into rats and monitored neural activity in the frontal cortex 1 week following surgery. The results indicate that the collagen membrane was chemically transparent, allowing VSD staining across the membrane material. The membrane was also optically transparent enough to pass light; forelimb-evoked neural activity was successfully visualized through the artificial dura. Because of its ideal chemical and optical manipulation capability, this collagen membrane may be widely applicable in various implantable neural sensors.

A neural field model is presented that captures the essential non-linear characteristics of activity dynamics across several millimeters of visual cortex in response to local flashed and moving stimuli. We account for physiological data obtained by voltage-sensitive dye (VSD) imaging which reports mesoscopic population activity at high spatio-temporal resolution. Stimulation included a single flashed square, a single flashed bar, the line-motion paradigm--for which psychophysical studies showed that flashing a square briefly before a bar produces sensation of illusory motion within the bar--and moving squares controls. We consider a two-layer neural field (NF) model describing an excitatory and an inhibitory layer of neurons as a coupled system of non-linear integro-differential equations. Under the assumption that the aggregated activity of both layers is reflected by VSD imaging, our phenomenological model quantitatively accounts for the observed spatio-temporal activity patterns. Moreover, the model generalizes to novel similar stimuli as it matches activity evoked by moving squares of different speeds. Our results indicate that feedback from higher brain areas is not required to produce motion patterns in the case of the illusory line-motion paradigm. Physiological interpretation of the model suggests that a considerable fraction of the VSD signal may be due to inhibitory activity, supporting the notion that balanced intra-layer cortical interactions between inhibitory and excitatory populations play a major role in shaping dynamic stimulus representations in the early visual cortex.

Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits.

Optical imaging using voltage-sensitive dyes (VSDs) is a promising technique for the simultaneous activity recording of many individual neurons. While such simultaneous recordings are critical for the understanding of the integral functionality of neural systems, functional interpretations on a single neuron level are difficult without knowledge of the connectivity of the underlying circuit. Central pattern generating circuits, such as the pyloric and gastric mill circuits in the stomatogastric ganglion (STG) of crustaceans, allow such investigations due to their well-known connectivities and have already contributed much to our understanding of general neuronal mechanisms. Here we present for the first time simultaneous optical recordings of the pattern generating neurons in the STG of two crustacean species using bulk loading of the VSD di-4-ANEPPS. We demonstrate the recording of firing activities and synaptic interactions of the circuit neurons as well as inter-circuit interactions in their functional context, i.e. without artificial stimulation. Neurons could be uniquely identified using simple event-triggered averaging. We tested this technique in two different species of crustaceans (lobsters and crabs), since several crustacean species are used for studying motor pattern generation. The signal-to-noise ratio of the optical signal was high enough in both species to derive phase-relationship between the network neurons, as well as action potentials and excitatory and inhibitory postsynaptic potentials. We argue that imaging of neural networks with identifiable neurons with well-known connectivity, like in the STG, is crucial for the understanding of emergence of network functionality.

How is contextual processing as demonstrated with simplified stimuli, cortically enacted in response to ecologically relevant complex and dynamic stimuli? Using voltage-sensitive dye imaging, we captured mesoscopic population dynamics across several square millimeters of cat primary visual cortex. By presenting natural movies locally through either one or two adjacent apertures, we show that simultaneous presentation leads to mutual facilitation of activity. These synergistic effects were most effective when both movie patches originated from the same natural movie, thus forming a coherent stimulus in which the inherent spatio-temporal structure of natural movies were preserved in accord with Gestalt principles of perceptual organization. These results suggest that natural sensory input triggers cooperative mechanisms that are imprinted into the cortical functional architecture as early as in primary visual cortex.

In general, signal amplitude in optical imaging is normalized using the well-established DeltaF/F method, where functional activity is divided by the total fluorescent light flux. This measure is used both directly, as a measure of population activity, and indirectly, to quantify spatial and spatiotemporal activity patterns. Despite its ubiquitous use, the stability and accuracy of this measure has not been validated for voltage-sensitive dye imaging of mammalian neocortex in vivo. In this report, we find that this normalization can introduce dynamic biases. In particular, the DeltaF/F is influenced by dye staining quality, and the ratio is also unstable over the course of experiments. As methods to record and analyze optical imaging signals become more precise, such biases can have an increasingly pernicious impact on the accuracy of findings, especially in the comparison of cytoarchitechtonic areas, in area-of-activation measurements, and in plasticity or developmental experiments. These dynamic biases of the DeltaF/F method may, to an extent, be mitigated by a novel method of normalization, DeltaF/DeltaF(epileptiform). This normalization uses as a reference the measured activity of epileptiform spikes elicited by global disinhibition with bicuculline methiodide. Since this normalization is based on a functional measure, i.e. the signal amplitude of "hypersynchronized" bursts of activity in the cortical network, it is less influenced by staining of non-functional elements. We demonstrate that such a functional measure can better represent the amplitude of population mass action, and discuss alternative functional normalizations based on the amplitude of synchronized spontaneous sleep-like activity. These findings demonstrate that the traditional DeltaF/F normalization of voltage-sensitive dye signals can introduce pernicious inaccuracies in the quantification of neural population activity. They further suggest that normalization-independent metrics such as waveform propagation patterns, oscillations in single detectors, and phase relationships between detector pairs may better capture the biological information which is obtained by high-sensitivity imaging.

Understanding the spatiotemporal organization of long-term synaptic plasticity in neuronal networks demands techniques capable of monitoring changes in synaptic responsiveness over extended multineuronal structures. Among these techniques, voltage-sensitive dye imaging (VSD imaging) is of particular interest due to its good spatial resolution. However, improvements of the technique are needed in order to overcome limits imposed by its low signal-to-noise ratio. Here, we show that VSD imaging can detect long-term potentiation (LTP) and long-term depression (LTD) in acute cerebellar slices. Combined VSD imaging and patch-clamp recordings revealed that the most excited regions were predominantly associated with granule cells (GrCs) generating EPSP-spike complexes, while poorly responding regions were associated with GrCs generating EPSPs only. The correspondence with cellular changes occurring during LTP and LTD was highlighted by a vector representation obtained by combining amplitude with time-to-peak of VSD signals. This showed that LTP occurred in the most excited regions lying in the core of activated areas and increased the number of EPSP-spike complexes, while LTD occurred in the less excited regions lying in the surround. VSD imaging appears to be an efficient tool for investigating how synaptic plasticity contributes to the reorganization of multineuronal activity in neuronal circuits.

The localization and measurement of neuronal activity magnitude at high spatial and temporal resolution are essential for mapping and better understanding neuronal systems and mechanisms. One such example is the generation of retinotopic maps, which correlates localized retinal stimulation with the corresponding specific visual cortex responses. Here we evaluated and compared seven different methods for extracting and localizing cortical responses from voltage-sensitive dye imaging recordings, elicited by visual stimuli projected directly on the rat retina by a customized projection system. The performance of these methods was evaluated both qualitatively and quantitatively by means of two cluster separation metrics, namely, the (adjusted) Silhouette Index (SI) and the (adjusted) Davies-Bouldin Index (DBI). These metrics were validated using simulated data, which showed that Temporally Structured Component Analysis (TSCA) outperformed all other analysis methods for localizing cortical responses and generating high-resolution retinotopic maps. The analysis methods, as well as the use of cluster separation metrics proposed here, can facilitate future research aiming to localize specific activity at high resolution in the visual cortex or other brain areas.

Prefrontal cortex (PFC) intrahemispheric activity and the interhemispheric connection have a significant impact on neuropsychiatric disorder pathology. This study aimed to generate a functional map of FC intrahemispheric and interhemispheric connections. Functional dissection of mouse PFCs was performed using the voltage-sensitive dye (VSD) imaging method with high speed (1 ms/frame), high resolution (256 × 256 pixels), and a large field of view (∼10 mm). Acute serial 350 μm slices were prepared from the bregma covering the PFC and numbered 1-5 based on their distance from the bregma (i.e., 1.70, 1.34, 0.98, 0.62, and 0.26 mm) with reference to the Mouse Brain Atlas (Paxinos and Franklin, 2008). The neural response to electrical stimulation was measured at nine sites and then averaged, and a functional map of the propagation patterns was created. Intracortical propagation was observed in slices 3-5, encompassing the anterior cingulate cortex (ACC) and corpus callosum (CC). The activity reached area 33 of the ACC. Direct white matter stimulation activated area 33 in both hemispheres. Similar findings were obtained via DiI staining of the CC. Imaging analysis revealed directional biases in neural signals traveling within the ACC, whereby the signal transmission speed and probability varied based on the signal direction. Specifically, the spread of neural signals from cg2 to cg1 was stronger than that from cingulate cortex area 1(cg1) to cingulate cortex area 2(cg2), which has implications for interhemispheric functional connections. These findings highlight the importance of understanding the PFC functional anatomy in evaluating neuromodulators like serotonin and dopamine, as well as other factors related to neuropsychiatric diseases.

The ability to monitor changes in membrane potential is a useful tool for studying neuronal function, but there are only limited options available at present. Here, we have investigated the potential of a commercially available FLIPR membrane potential (FMP) dye, developed originally for high throughput screening using a plate reader, for imaging the membrane potential of cultured cells using an epifluorescence-based single cell imaging system. We found that the properties of the FMP dye make it highly suitable for such imaging since 1) its fluorescence displayed a high signal-to-noise ratio, 2) robust signals meant only minimal exposure times of around 5 ms were necessary, and 3) bidirectional changes in fluorescence were detectable resulting from hyper- or depolarising conditions, reaching equilibrium with a time constant of 4-8 s. Measurements were possible independently of whether membrane potential changes were induced by voltage clamping, or manipulating the ionic distribution of either Na(+) or K(+). Since FMP behaves as a charged molecule which accumulates in the cytosol, equations based on the Boltzmann distribution were developed determining that the apparent charge of FMP which represents a measure of the voltage sensitivity of the dye, is between -0.62 and -0.72. Finally, we demonstrated that FMP is suitable for use in a variety of neuronal cell types and detects membrane potential changes arising from spontaneous firing of action potentials and through stimulation with a variety of excitatory and inhibitory neurotransmitters.

Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.

Despite current progress achieved in the surgical technique of radical prostatectomy, post-operative complications such as erectile dysfunction and urinary incontinence persist at high incidence rates. In this paper, we present a methodology for functional intra-operative localization of the cavernous nerve (CN) network for nerve-sparing radical prostatectomy using near-infrared cyanine voltage-sensitive dye (VSD) imaging, which visualizes membrane potential variations in the CN and its branches (CNB) in real time. As a proof-of-concept experiment, we demonstrate a functioning complex nerve network in response to electrical stimulation of the CN, which was clearly differentiated from surrounding tissues in an in vivo rat prostate model. Stimulation of an erection was confirmed by correlative intracavernosal pressure (ICP) monitoring. Within 10 minutes, we performed trans-fascial staining of the CN by direct VSD administration. Our findings suggest the applicability of VSD imaging for real-time, functional imaging guidance during nerve-sparing radical prostatectomy.

Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, the olfactory and the trigeminal system. Within the early olfactory system, spatiotemporal responses of the olfactory bulb to various odorants have been mapped in great detail. In contrast, far less is known about the representation of volatile chemical stimuli at an early stage in the trigeminal system, the trigeminal ganglion (TG), which contains neurons directly projecting to the nasal cavity. We have established an in vivo preparation that allows high-resolution imaging of neuronal population activity from a large region of the rat TG using voltage-sensitive dyes (VSDs). Application of different chemical stimuli to the nasal cavity elicited distinct, stimulus-category specific, spatiotemporal activation patterns that comprised activated as well as suppressed areas. Thus, our results provide the first direct insights into the spatial representation of nasal chemosensory information within the trigeminal ganglion imaged at high temporal resolution.

Studies using functional magnetic resonance imaging assume that hemodynamic responses have roughly linear relationships with underlying neural activity. However, to accurately investigate the neurovascular transfer function and compare its variability across brain regions, it is necessary to obtain full-field imaging of both electrophysiological and hemodynamic responses under various stimulus conditions with superior spatiotemporal resolution. Optical imaging combined with voltage-sensitive dye (VSD) and intrinsic signals (IS) is a powerful tool to address this issue. We performed VSD and IS imaging in the primary (S1) and secondary (S2) somatosensory cortices of rats to obtain optical maps of whisker-evoked responses. There were characteristic differences in sensory responses between the S1 and S2 cortices: VSD imaging revealed more localized excitatory and stronger inhibitory neural activity in S1 than in S2. IS imaging revealed stronger metabolic responses in S1 than in S2. We calculated the degree of response to compare the sensory responses between cortical regions and found that the ratio of the degree of response of S2 to S1 was similar, irrespective of whether the ratio was determined by VSD or IS imaging. These results suggest that neurovascular coupling does not vary between the S1 and S2 cortices.