Fast Voltage-sensitive Dye Imaging Research Articles

Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging.

Activity-dependent changes in the input-output (I-O) relationship of a neural circuit are central in the learning and memory function of the brain. To understand circuit-wide adjustments, optical imaging techniques to probe the membrane potential at every component of neurons, such as dendrites, axons and somas, in the circuit are essential. We have been developing fast voltage-sensitive dye (VSD) imaging methods for quantitative measurements, especially for single-photon wide-field optical imaging. The long-term continuous measurements needed to evaluate circuit-wide modifications require stable and quantitative long-term recordings. Here, we show that VSD imaging (VSDI) can be used to record changes in circuit activity in association with theta-burst stimulation (TBS)-induced long-term potentiation (LTP) of synaptic strength in the CA1 area. Our optics, together with the fast imaging system, enabled us to measure neuronal signals from the entire CA1 area at a maximum frame speed of 0.1 ms/frame every 60 s for over 12 h. We also introduced a method to evaluate circuit activity changes by mapping the variation in recordings from the CA1 area to coordinates defined by the morphology of CA1 pyramidal cells. The results clearly showed two types of spatial heterogeneity in LTP induction. The first heterogeneity is that LTP increased with distance from the stimulation site. The second heterogeneity is that LTP is higher in the stratum pyramidale (SP)-oriens region than in the stratum radiatum (SR). We also showed that the pattern of the heterogeneity changed according to the induction protocol, such as induction by TBS or high-frequency stimulation (HFS). We further demonstrated that part of the heterogeneity depends on the I-O response of the circuit elements. The results show the usefulness of VSDI in probing the function of hippocampal circuits.

Fast voltage-sensitive dye imaging of excitatory and inhibitory synaptic transmission in the rat granular retrosplenial cortex.

Rodent granular retrosplenial cortex (GRS) has dense connections between the anterior thalamic nuclei (ATN) and hippocampal formation. GRS superficial pyramidal neurons exhibit distinctive late spiking (LS) firing property and form patchy clusters with prominent apical dendritic bundles. The aim of this study was to investigate spatiotemporal dynamics of signal transduction in the GRS induced by ATN afferent stimulation by using fast voltage-sensitive dye imaging in rat brain slices. In coronal slices, layer 1a stimulation, which presumably activated thalamic fibers, evoked propagation of excitatory synaptic signals from layers 2-4 to layers 5-6 in a direction perpendicular to the layer axis, followed by transverse signal propagation within each layer. In the presence of ionotropic glutamate receptor antagonists, inhibitory responses were observed in superficial layers, induced by direct activation of inhibitory interneurons in layer 1. In horizontal slices, excitatory signals in deep layers propagated transversely mainly from posterior to anterior via superficial layers. Cortical inhibitory responses upon layer 1a stimulation in horizontal slices were weaker than those in the coronal slices. Observed differences between coronal and horizontal planes suggest anisotropy of the intracortical circuitry. In conclusion, ATN inputs are processed differently in coronal and horizontal planes of the GRS and then conveyed to other cortical areas. In both planes, GRS superficial layers play an important role in signal propagation, which suggests that superficial neuronal cascade is crucial in the integration of multiple information sources.NEW & NOTEWORTHY Superficial neurons in the rat granular retrosplenial cortex (GRS) show distinctive late-spiking (LS) firing property. However, little is known about spatiotemporal dynamics of signal transduction in the GRS. We demonstrated LS neuron network relaying thalamic inputs to deep layers and anisotropic distribution of inhibition between coronal and horizontal planes. Since deep layers of the GRS receive inputs from the subiculum, GRS circuits may work as an integrator of multiple sources such as sensory and memory information.

Functional optical probing of the hippocampal trisynaptic circuit in vitro: network dynamics, filter properties, and polysynaptic induction of CA1 LTP.

Decades of brain research have identified various parallel loops linking the hippocampus with neocortical areas, enabling the acquisition of spatial and episodic memories. Especially the hippocampal trisynaptic circuit [entorhinal cortex layer II → dentate gyrus (DG) → cornu ammonis (CA)-3 → CA1] was studied in great detail because of its seemingly simple connectivity and characteristic structures that are experimentally well accessible. While numerous researchers focused on functional aspects, obtained from a limited number of cells in distinct hippocampal subregions, little is known about the neuronal network dynamics which drive information across multiple synapses for subsequent long-term storage. Fast voltage-sensitive dye imaging in vitro allows real-time recording of activity patterns in large/meso-scale neuronal networks with high spatial resolution. In this way, we recently found that entorhinal theta-frequency input to the DG most effectively passes filter mechanisms of the trisynaptic circuit network, generating activity waves which propagate across the entire DG-CA axis. These “trisynaptic circuit waves” involve high-frequency firing of CA3 pyramidal neurons, leading to a rapid induction of classical NMDA receptor-dependent long-term potentiation (LTP) at CA3-CA1 synapses (CA1 LTP). CA1 LTP has been substantially evidenced to be essential for some forms of explicit learning in mammals. Here, we review data with particular reference to whole network-level approaches, illustrating how activity propagation can take place within the trisynaptic circuit to drive formation of CA1 LTP.

Voltage-sensitive dye imaging during functional development of the embryonic nervous system: a brief review with special thanks to Professor Larry Cohen.

Investigating the developmental organization of the embryonic nervous system is one of the major challenges in the field of neuroscience. Despite their significance, functional studies on the vertebrate embryonic central nervous system (CNS) have been hampered by the technical limitations associated with conventional electrophysiological methods. The advent of optical techniques using voltage-sensitive dyes, which were developed by Dr. Cohen and his colleagues, has enabled electrical activity in living cells to be monitored noninvasively and also facilitated the simultaneous recording of neural responses from multiple regions. Using optical recording techniques, it is now possible to follow the functional organization of the embryonic CNS and image the spatiotemporal dynamics involved in the formation of this neural network. We herein briefly reviewed optical studies on the embryonic CNS with a special emphasis on methodological considerations and the study of neuronal circuit formation, which demonstrates the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the embryonic CNS.

Monitoring Population Membrane Potential Signals During Development of the Vertebrate Nervous System.

The functional organization of the vertebrate central nervous system (CNS) during the early phase of development has long been unclear, because conventional electrophysiological means have several technical limitations. First, early embryonic neurons are small and fragile, and the application of microelectrodes is often difficult. Second, the simultaneous recording of electrical activity from multiple sites is limited, and as a consequence, response patterns of neural networks cannot be assessed. Optical recording techniques with voltage-sensitive dyes have overcome these obstacles and provided a new approach to the analysis of the functional development/organization of the CNS. In this review, we provide detailed information concerning the recording of optical signals in the embryonic nervous system. After outlining methodological considerations, we present examples of recent progress in optical studies on the embryonic nervous system with special emphasis on two topics. The first is the study of how synapse networks form in specific neuronal circuits. The second is the study of non-specific correlated wave activity, which is considered to play a fundamental role in neural development. These studies clearly demonstrate the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the vertebrate embryonic CNS.

Bidirectional global spontaneous network activity precedes the canonical unidirectional circuit organization in the developing hippocampus

Spontaneous network activity is believed to sculpt developing neural circuits. Spontaneous giant depolarizing potentials (GDPs) were first identified with single-cell recordings from rat CA3 pyramidal neurons, but here we identify and characterize a large-scale spontaneous network activity we term global network activation (GNA) in the developing mouse hippocampal slices, which is measured macroscopically by fast voltage-sensitive dye imaging. The initiation and propagation of GNA in the mouse is largely GABA-independent and dominated by glutamatergic transmission via AMPA receptors. Despite the fact that signal propagation in the adult hippocampus is strongly unidirectional through the canonical trisynaptic circuit (dentate gyrus [DG] to CA3 to CA1), spontaneous GNA in the developing hippocampus originates in distal CA3 and propagates both forward to CA1 and backward to DG. Photostimulation-evoked GNA also shows prominent backward propagation in the developing hippocampus from CA3 to DG. Mouse GNA is strongly correlated to electrophysiological recordings of highly localized single-cell and local field potential events. Photostimulation mapping of neural circuitry demonstrates that the enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA and reveals the underlying pathways accounting for GNA backward propagation from CA3 to DG. The disappearance of GNA coincides with a transition to the adult-like unidirectional circuit organization at about 2 weeks of age. Taken together, our findings strongly suggest a critical link between GNA activity and maturation of functional circuit connections in the developing hippocampus.

Low molecular weight Neutral Boron Dipyrromethene (Bodipy) dyads for fluorescence-based neural imaging

The neutral low molecular weight julolidine-based borondipyrromethene (Bodipy) dyads JULBD and MJULBD were used for fast voltage-sensitive dye imaging of neurons in the crab stomatogastric ganglion. The fluorescence modulation of the dyads mirrors alterations in the membrane potential of the imaged neurons. The toxicity of the dyes towards the neurons is related to their structure in that methyl groups at the 3,5 positions results in reduced toxic effects.

Purinergic control of hippocampal circuit hyperexcitability in Dravet syndrome

Severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome is one of the most devastating childhood epilepsies. Children with SMEI have febrile and afebrile seizures (FS and aFS), ataxia, and social and cognitive dysfunctions. SMEI is pharmacologically intractable and can be fatal in 10-20% of patients. It remains to be elucidated how channelopathies that cause SMEI impact synaptic activities in key neural circuits, and there is an ongoing critical need for alternative methods of controlling seizures in SMEI. Using the SCN1A gene knock-in mouse model of SMEI (mSMEI), we studied hippocampal cell and circuit excitability, particularly during hyperthermia, and tested whether an adenosine A1 receptor (A1R) agonist can reliably control hippocampal circuit hyperexcitability. Using a combination of electrophysiology (extracellular and whole-cell voltage clamp) and fast voltage-sensitive dye imaging (VSDI), we quantified synaptic excitation and inhibition, spatiotemporal characteristics of neural circuit activity, and hyperthermia-induced febrile seizure-like events (FSLEs) in juvenile mouse hippocampal slices. We used hyperthermia to elicit FSLEs in hippocampal slices, while making use of adenosine A1R agonist N6-cyclopentyladenosine (CPA) to control abnormally widespread neural activity and FSLEs. We discovered a significant excitation/inhibition (E/I) imbalance in mSMEI hippocampi, in which inhibition was decreased and excitation increased. This imbalance was associated with an increased spatial extent of evoked neural circuit activation and a lowered FSLE threshold. We found that a low concentration (50nm) of CPA blocked FSLEs and reduced the spatial extent of abnormal neural activity spread while preserving basal levels of excitatory synaptic transmission. Our study reveals significant hippocampal synapse and circuit dysfunctions in mSMEI and demonstrates that the A1R agonist CPA can reliably control hippocampal hyperexcitability and FSLEs in vitro. These findings may warrant further investigations of purinergic agonists as part of the development of new therapeutic approaches for Dravet syndrome.

Inhibitory Neuron and Hippocampal Circuit Dysfunction in an Aged Mouse Model of Alzheimer's Disease

In Alzheimer's disease (AD), a decline in explicit memory is one of the earliest signs of disease and is associated with hippocampal dysfunction. Amyloid protein exerts a disruptive impact on neuronal function, but the specific effects on hippocampal network activity are not well known. In this study, fast voltage-sensitive dye imaging and extracellular and whole-cell electrophysiology were used on entorhinal cortical-hippocampal slice preparations to characterize hippocampal network activity in 12–16 month old female APPswe/PSEN1DeltaE9 (APdE9 mice) mice. Aged APdE9 mice exhibited profound disruptions in dentate gyrus circuit activation. High frequency stimulation of the perforant pathway in the dentate gyrus (DG) area of APdE9 mouse tissue evoked abnormally large field potential responses corresponding to the wider neural activation maps. Whole-cell patch clamp recordings of the identified inhibitory interneurons in the molecular layer of DG revealed that they fail to reliably fire action potentials. Taken together, abnormal DG excitability and an inhibitory neuron failure to generate action potentials are suggested to be important contributors to the underlying cellular mechanisms of early-stage Alzheimer's disease pathophysiology.

Optical stimulation and imaging of functional brain circuitry in a segmented laminar flow chamber

Microfluidic technology is emerging as a useful tool for the study of brain slices, offering precise delivery of chemical factors along with robust oxygen and nutrient transport. However, continued reliance upon electrode-based physiological recording poses inherent limitations in terms of physical access, as well as the number of sites that can be sampled simultaneously. In the present study, we combine a microfluidic laminar flow chamber with fast voltage-sensitive dye imaging and laser photostimulation via caged glutamate to map neural network activity across large cortical regions in living brain slices. We find that the closed microfluidic chamber results in greatly improved signal-to-noise performance for optical measurements of neural signaling. These optical tools are also leveraged to characterize laminar flow interfaces within the device, demonstrating a functional boundary width of less than 100 μm. Finally, we utilize this integrated platform to investigate the mechanism of signal propagation for spontaneous neural activity in the developing mouse hippocampus. Through the use of localized Ca(2+) depletion, we provide evidence for Ca(2+)-dependent synaptic transmission.

β-Adrenergic modulation of spontaneous spatiotemporal activity patterns and synchrony in hyperexcitable hippocampal circuits

A description of healthy and pathological brain dynamics requires an understanding of spatiotemporal patterns of neural activity and characteristics of its propagation between interconnected circuits. However, the structure and modulation of the neural activation maps underlying these patterns and their propagation remain elusive. We investigated effects of β-adrenergic receptor (β-AR) stimulation on the spatiotemporal characteristics of emergent activity in rat hippocampal circuits. Synchronized epileptiform-like activity, such as interictal bursts (IBs) and ictal-like events (ILEs), were evoked by 4-aminopyridine (4-AP), and their dynamics were studied using a combination of electrophysiology and fast voltage-sensitive dye imaging. Dynamic characterization of the spontaneous IBs showed that they originated in dentate gyrus/CA3 border and propagated toward CA1. To determine how β-AR modulates spatiotemporal characteristics of the emergent IBs, we used the β-AR agonist isoproterenol (ISO). ISO significantly reduced the spatiotemporal extent and propagation velocity of the IBs and significantly altered network activity in the 1- to 20-Hz range. Dual whole cell recordings of the IBs in CA3/CA1 pyramidal cells and optical analysis of those regions showed that ISO application reduced interpyramidal and interregional synchrony during the IBs. In addition, ISO significantly reduced duration not only of the shorter duration IBs but also the prolonged ILEs in 4-AP. To test whether the decrease in ILE duration was model dependent, we used a different hyperexcitability model, zero magnesium (0 Mg(2+)). Prolonged ILEs were readily formed in 0 Mg(2+), and addition of ISO significantly reduced their durations. Taken together, these novel results provide evidence that β-AR activation dynamically reshapes the spatiotemporal activity patterns in hyperexcitable circuits by altering network rhythmogenesis, propagation velocity, and intercellular/regional synchronization.

Validation of independent component analysis for rapid spike sorting of optical recording data.

Independent component analysis (ICA) is a technique that can be used to extract the source signals from sets of signal mixtures where the sources themselves are unknown. The analysis of optical recordings of invertebrate neuronal networks with fast voltage-sensitive dyes could benefit greatly from ICA. These experiments can generate hundreds of voltage traces containing both redundant and mixed recordings of action potentials originating from unknown numbers of neurons. ICA can be used as a method for converting such complex data sets into single-neuron traces, but its accuracy for doing so has never been empirically evaluated. Here, we tested the accuracy of ICA for such blind source separation by simultaneously performing sharp electrode intracellular recording and fast voltage-sensitive dye imaging of neurons located in the central ganglia of Tritonia diomedea and Aplysia californica, using a 464-element photodiode array. After running ICA on the optical data sets, we found that in 34 of 34 cases the intracellularly recorded action potentials corresponded 100% to the spiking activity of one of the independent components returned by ICA. We also show that ICA can accurately sort action potentials into single neuron traces from a series of optical data files obtained at different times from the same preparation, allowing one to monitor the network participation of large numbers of individually identifiable neurons over several recording episodes. Our validation of the accuracy of ICA for extracting the neural activity of many individual neurons from noisy, mixed, and redundant optical recording data sets should enable the use of this powerful large-scale imaging approach for studies of invertebrate and suitable vertebrate neuronal networks.

Multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the myenteric plexus of the guinea pig ileum

An important feature of the enteric nervous system (ENS) is its capability to respond to mechanical stimulation which, as currently suggested for the guinea-pig ileum, is encoded by specialized intrinsic primary afferent neurons (IPANs). We used von Frey hairs or intraganglionic volume injections to mimic ganglion deformation as observed in freely contracting preparations. Using fast voltage-sensitive dye imaging we identified rapidly adapting mechanosensitive enteric neurons (RAMEN, 25% of all neurons) in the myenteric plexus of the guinea pig ileum. RAMEN responded with phasic spike discharge to dynamic changes during ganglion deformation. This response was reproducible and increased with increasing forces. Deformation-evoked spike discharge was not changed by synaptic blockade with hexamethonium, omega-conotoxin or low Ca(2+)/high Mg(2+), defunctionalization of extrinsic afferents with capsaicin or muscle paralysis with nifedipine, suggesting direct activation of RAMEN. All RAMEN received hexamethonium-sensitive fast EPSPs, which were blocked by omega-conotoxin and low Ca(2+)/high Mg(2+). Seventy-two per cent of RAMEN were cholinergic, 22% nitrergic, and 44% were calbindin and NeuN negative, markers used to identify IPANs. Mechanosensitivity was observed in 31% and 47% of retrogradely traced interneurons and motor neurons, respectively. RAMEN belong to a new population of mechanosensitive neurons which differ from IPANs. We provided evidence for multifunctionality of RAMEN which may fulfil sensory, integrative and motor functions. In light of previously identified mechanosensitive neuron populations, mechanosensitivity appears to be a property of many more enteric neurons than generally assumed. The findings call for a revision of current concepts on sensory transmission within the ENS.

In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V(m) can accurately represent population V(m) changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.