Voltage-sensitive Dye Imaging Research Articles

Surprising multifunctionality of a Tritonia swim CPG neuron: C2 drives the early phase of post-swim crawling despite being silent during the behavior.

In response to a suitably aversive skin stimulus, the marine mollusk Tritonia diomedea launches an escape swim followed by several minutes of high-speed crawling. The two escape behaviors are highly dissimilar: whereas the swim is a muscular behavior involving alternating ventral and dorsal whole-body flexions, the crawl is a non-rhythmic gliding behavior mediated by the beating of foot cilia. The serotonergic dorsal swim interneurons (DSIs) are members of the swim CPG and also strongly drive crawling. While the swim network is very well understood, the Tritonia crawling network to date comprises only three neurons: the DSIs, and pedal neurons 5 and 21 (Pd5 and Pd21). Since Tritonia's swim network has been suggested to have arisen from a pre-existing crawling network, we examined the possible role that another swim CPG neuron, C2, may play in crawling. Due to its complete silence in the post-swim crawling period, C2 had not previously been considered to play a role in driving crawling. However, semi-intact preparation experiments demonstrated that a brief C2 spike train surprisingly and strongly drives the foot cilia for ~30 s, something which cannot be explained by its synaptic connections to Pd5 and Pd21. Voltage-sensitive dye (VSD) imaging in the pedal ganglion identified many candidate crawling motor neurons that fire at an elevated rate following the swim, and also revealed several pedal neurons that are strongly excited by C2. It is intriguing that unlike the DSIs, which fire tonically after the swim to drive crawling, C2 does so despite its post-swim silence.

Neural correlates of perceptual similarity masking in primate V1.

Visual detection is a fundamental natural task. Detection becomes more challenging as the similarity between the target and the background in which it is embedded increases, a phenomenon termed 'similarity masking'. To test the hypothesis that V1 contributes to similarity masking, we used voltage sensitive dye imaging (VSDI) to measure V1 population responses while macaque monkeys performed a detection task under varying levels of target-background similarity. Paradoxically, we find that during an initial transient phase, V1 responses to the target are enhanced, rather than suppressed, by target-background similarity. This effect reverses in the second phase of the response, so that in this phase V1 signals are positively correlated with the behavioral effect of similarity. Finally, we show that a simple model with delayed divisive normalization can qualitatively account for our findings. Overall, our results support the hypothesis that a nonlinear gain control mechanism in V1 contributes to perceptual similarity masking.

Neural correlates of perceptual similarity masking in primate V1

Visual detection is a fundamental natural task. Detection becomes more challenging as the similarity between the target and the background in which it is embedded increases, a phenomenon termed ‘similarity masking’. To test the hypothesis that V1 contributes to similarity masking, we used voltage sensitive dye imaging (VSDI) to measure V1 population responses while macaque monkeys performed a detection task under varying levels of target-background similarity. Paradoxically, we find that during an initial transient phase, V1 responses to the target are enhanced, rather than suppressed, by target-background similarity. This effect reverses in the second phase of the response, so that in this phase V1 signals are positively correlated with the behavioral effect of similarity. Finally, we show that a simple model with delayed divisive normalization can qualitatively account for our findings. Overall, our results support the hypothesis that a nonlinear gain control mechanism in V1 contributes to perceptual similarity masking.

Encoding luminance surfaces in the visual cortex of mice and monkeys: difference in responses to edge and center.

Luminance and spatial contrast provide information on the surfaces and edges of objects. We investigated neural responses to black and white surfaces in the primary visual cortex (V1) of mice and monkeys. Unlike primates that use their fovea to inspect objects with high acuity, mice lack a fovea and have low visual acuity. It thus remains unclear whether monkeys and mice share similar neural mechanisms to process surfaces. The animals were presented with white or black surfaces and the population responses were measured at high spatial and temporal resolution using voltage-sensitive dye imaging. In mice, the population response to the surface was not edge-dominated with a tendency to center-dominance, whereas in monkeys the response was edge-dominated with a "hole" in the center of the surface. The population response to the surfaces in both species exhibited suppression relative to a grating stimulus. These results reveal the differences in spatial patterns to luminance surfaces in the V1 of mice and monkeys and provide evidence for a shared suppression process relative to grating.

Bilateral Whisker Representations in the Primary Somatosensory Cortex in Robo3cKO Mice Are Reflected in the Primary Motor Cortex

In Robo3cKO mice, midline crossing defects of the trigeminothalamic projections from the trigeminal principal sensory nucleus result in bilateral whisker maps in the somatosensory thalamus and consequently in the face representation area of the primary somatosensory (S1) cortex (Renier et al., 2017; Tsytsarev et al., 2017). We investigated whether this bilateral sensory representation in the whisker-barrel cortex is also reflected in the downstream projections from the S1 to the primary motor (M1) cortex. To label these projections, we injected anterograde viral axonal tracer in S1 cortex. Corticocortical projections from the S1 distribute to similar areas across the ipsilateral hemisphere in control and Robo3cKO mice. Namely, in both genotypes they extend to the M1, premotor/prefrontal cortex (PMPF), secondary somatosensory (S2) cortex. Next, we performed voltage-sensitive dye imaging (VSDi) in the left hemisphere following ipsilateral and contralateral single whisker stimulation. While controls showed only activation in the contralateral whisker barrel cortex and M1 cortex, the Robo3cKO mouse left hemisphere was activated bilaterally in both the barrel cortex and the M1 cortex. We conclude that the midline crossing defect of the trigeminothalamic projections leads to bilateral whisker representations not only in the thalamus and the S1 cortex but also downstream from the S1, in the M1 cortex.

Alleviation of peripheral sensitization by quadriceps insertion of cog polydioxanone filaments in knee osteoarthritis rats

A recently established therapeutic strategy, involving the insertion of biodegradable cog polydioxanone filaments into the quadriceps muscles using the Muscle Enhancement and Support Therapy (MEST) device, has demonstrated significant efficacy in alleviating knee osteoarthritis (OA) pain. This study investigated changes in peripheral sensitization as the potential mechanism underlying MEST-induced pain relief in monoiodoacetate (MIA) induced OA rats. The results revealed that MEST treatment potently reduces MIA-induced sensitization of L3/L4 dorsal root ganglion (DRG) neurons, the primary nociceptor pathway for the knee joint. This reduction in DRG sensitization, as elucidated by voltage-sensitive dye imaging, is accompanied by a diminished overexpression of TRPA1 and NaV1.7, key nociceptor receptors involved in mechanical pain perception. Importantly, these observed alterations strongly correlate with a decrease in mechanically-evoked pain behaviors, providing compelling neurophysiological evidence that MEST treatment alleviates OA pain by suppressing peripheral sensitization.

Functional Dissection of Ipsilateral and Contralateral Neural Activity Propagation Using Voltage-Sensitive Dye Imaging in Mouse Prefrontal Cortex.

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.

Quantitative assessment of hippocampal network dynamics by combining Voltage Sensitive Dye Imaging and Optimal Transportation Theory

Quantitative assessment of hippocampal network dynamics by combining Voltage Sensitive Dye Imaging and Optimal Transportation Theory

Antagonism of a key peptide 'T14' driving neurodegeneration: Evaluation of a next generation therapeutic

T14, a 14mer peptide derived from the C-terminus of acetylcholinesterase (AChE) is a signalling molecule that could drive neurodegeneration via the alpha 7 nicotinic acetylcholine receptor. Its levels increase as Alzheimer’s pathology progresses; however, a cyclic variant of the compound, NBP14, can block the effects of the endogenous linear counterpart in-vitro, ex vivo, and in vivo. Here, we explore the antagonistic potential of two 6mer peptides, NBP6A and NBP6B. These are smaller linear versions of NBP14, designed to be more effective by modifying the amino acid residues to enhance receptor blockade alongside other relevant solubility parameters. The peptides were tested in-vitro in PC12 cells on three parameters, calcium influx, cell viability, and AChE release, and ex vivo using voltage sensitive dye imaging (VSDI) in rat brain slices. Neither NBP6A nor NBP6B applied alone had any effect. In PC12 cells, NBP6B was identified as the more potent molecule since it demonstrated more effective blockade of T14 action on calcium influx, cell viability, and AChE release. NBP6B was then further evaluated using VSDI, where it proved twice as potent as NBP14 in blocking the action of T14. The improved effect of NBP6B in blocking the actions of T14, combined with its smaller size suggests that this variant could have even greater therapeutic potential than its original cyclic compound, for treating neurodegenerative disorders.

Assessing seizure liability in vitro with voltage-sensitive dye imaging in mouse hippocampal slices.

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.

Spatio-temporal activation patterns of neuronal population evoked by optostimulation and the comparison to electrical microstimulation

Optostimulation and electrical microstimulation are well-established techniques that enable to artificially stimulate the brain. While the activation patterns evoked by microstimulation in cortical network are well characterized, much less is known for optostimulation. Specifically, the activation maps of neuronal population at the membrane potential level and direct measurements of these maps were barely reported. In addition, only a few studies compared the activation patterns evoked by microstimulation and optostimulation. In this study we addressed these issues by applying optostimulation in the barrel cortex of anesthetized rats after a short (ShortExp) or a long (LongExp) opsin expression time and compared it to microstimulation. We measured the membrane potential of neuronal populations at high spatial (meso-scale) and temporal resolution using voltage-sensitive dye imaging. Longer optostimulation pulses evoked higher neural responses spreading over larger region relative to short pulses. Interestingly, similar optostimulation pulses evoked stronger and more prolonged population response in the LongExp vs. the ShortExp condition. Finally, the spatial activation patterns evoked in the LongExp condition showed an intermediate state, with higher resemblance to the microstimulation at the stimulation site. Therefore, short microstimulation and optostimulation can induce wide spread activation, however the effects of optostimulation depend on the opsin expression time.

Combining voltage-sensitive dye, carbon fiber array, and extracellular nerve electrodes using a 3-D printed recording chamber and manipulators

BackgroundThe analyses of neuronal circuits require high-throughput technologies for stimulating and recording many neurons simultaneously with single-neuron precision. Voltage-sensitive dyes (VSDs) have enabled the monitoring of membrane potentials of many (10–100 s) neurons simultaneously. Carbon fiber electrode (CFE) arrays allow for stimulation and recording of many neurons simultaneously, including intracellularly. New methodCombining CFE with VSD leverages the advantages of both technologies, allowing for stimulation of single neurons while recording the activity of the entire network. 3-D printing technology was used to develop a chamber to simultaneously perform VSD imaging, CFE array recording, and extracellular recording from individual glass electrodes. ResultsAplysia buccal ganglia were stained with VSD and imaged while also recording using a CFE array and extracellular nerve electrodes. Coincident spiking activity was recorded by VSD, CFE, and extracellular nerve electrodes. Current injection with CFE electrodes could activate and inhibit individual neurons as detected by VSD and nerve recordings. Comparison to existing methodsThe large size of traditional manipulators limits the number of electrodes used and the number of neurons recorded during an experiment. Here we present a method to build a 3-D printed recording chamber that includes a 3-axis micromanipulator to position a CFE array and eight 2-axis manipulators to position eight extracellular electrodes. Conclusions3-D printing technology can be used to build a custom recording chamber and micromanipulators. Combining these technologies allows for the direct modulation of the activity of neurons while recording the activity of 100 s of neurons simultaneously.

Assessment of Tissue Viability by Functional Imaging of Membrane Potential.

Electrical activity plays a key role in physiology, in particular for signaling and coordination. Cellular electrophysiology is often studied with micropipette-based techniques such as patch clamp and sharp electrodes, but for measurements at the tissue or organ scale, more integrated approaches are needed. Epifluorescence imaging of voltage-sensitive dyes ("optical mapping") is a tissue non-destructive approach to obtain insight into electrophysiology with high spatiotemporal resolution. Optical mapping has primarily been applied to excitable organs, especially the heart and brain. Action potential durations, conduction patterns, and conduction velocities can be determined from the recordings, providing information about electrophysiological mechanisms, including factors such as effects of pharmacological interventions, ion channel mutations, or tissue remodeling. Here, we describe the process for optical mapping of Langendorff-perfused mouse hearts, highlighting potential issues and key considerations.

Amyloid Deposition and Dendritic Complexity of Corticocortical Projection Cells in Five Familial Alzheimer's Disease Mouse

In Alzheimer’s disease and related dementias, amyloid beta (Aβ) and amyloid plaques can disrupt long-term synaptic plasticity, learning and memory and cognitive function. Plaque accumulation can disrupt corticocortical circuitry leading to abnormalities in sensory, motor, and cognitive processing. In this study, using 5xFAD (five Familial Alzheimer's Disease – FAD – mutations) mice, we evaluated amyloid plaque formation in different cortical areas, and whether differential amyloid accumulation across cortical fields correlates with changes in dendritic complexity of layer 3 corticocortical projection neurons and functional responses in the primary somatosensory cortex following whisker stimulation. We focused on three cortical areas: the primary somatosensory cortex (S1), the primary motor cortex (M1), and the prefrontal cortex (PFC including the anterior cingulate, prelimbic, and infralimbic subdivisions). We found that Aβ and amyloid plaque accumulation is not uniform across 5xFAD cortical areas, while there is no expression in littermate controls. We also found that there are differential layer 3 pyramidal cell dendritic complexity changes across the three areas in 5xFAD mice, compared to same age controls, with no apparent relation to differential amyloid accumulation. We used voltage-sensitive dye imaging (VSDi) to visualize neural activity in S1, M1 and PFC following whisker activation. Control mice show normal physiological responses in all three cortical areas, whereas 5xFAD mice only display physiological responses in S1. Taken together our results show that 5xFAD mutation affects the overall dendritic morphology of layer 3 pyramidal cells across sensory-motor and association cortex irrespective of the density and distribution of the Aβ amyloid proteins. Corticocortical circuitry between the sensory and motor/association areas is most likely disrupted in 5xFAD mice as cortical responses to whisker stimulation are altered.

Microstimulation in the primary visual cortex: activity patterns and their relation to visual responses and evoked saccades.

Intracortical microstimulation (ICMS) in the primary visual cortex (V1) can generate the visual perception of a small point of light, termed phosphene, and evoke saccades directed to the receptive field of the stimulated neurons. Although ICMS is widely used, a direct measurement of the spatio-temporal patterns of neural activity evoked by ICMS and their relation to the neural responses evoked by visual stimuli or how they relate to ICMS-evoked saccades are still missing. To investigate this, we combined ICMS with voltage-sensitive dye imaging in V1 of behaving monkeys and measured neural activity at a high spatial (meso-scale) and temporal resolution. We then compared the population response evoked by small visual stimuli to those evoked by microstimulation. Both stimulation types evoked population activity that spread over few millimeters in V1 and propagated to extrastriate areas. However, the population responses evoked by ICMS have shown faster dynamics for the activation transients and the horizontal propagation of activity revealed a wave-like propagation. Finally, neural activity in the ICMS condition was higher for trials with evoked saccades as compared with trials without saccades. Our results uncover the spatio-temporal patterns evoked by ICMS and their relation to visual processing and saccade generation.

Computational modeling of color perception with biologically plausible spiking neural networks.

Biologically plausible computational modeling of visual perception has the potential to link high-level visual experiences to their underlying neurons' spiking dynamic. In this work, we propose a neuromorphic (brain-inspired) Spiking Neural Network (SNN)-driven model for the reconstruction of colorful images from retinal inputs. We compared our results to experimentally obtained V1 neuronal activity maps in a macaque monkey using voltage-sensitive dye imaging and used the model to demonstrate and critically explore color constancy, color assimilation, and ambiguous color perception. Our parametric implementation allows critical evaluation of visual phenomena in a single biologically plausible computational framework. It uses a parametrized combination of high and low pass image filtering and SNN-based filling-in Poisson processes to provide adequate color image perception while accounting for differences in individual perception.

Spatiotemporal patterns of population response in the visual cortex under isoflurane: from wakefulness to loss of consciousness.

Anesthetic drugs are widely used in medicine and research to mediate loss of consciousness (LOC). Isoflurane is a commonly used anesthetic drug; however, its effects on cortical sensory processing, in particular around LOC, are not well understood. Using voltage-sensitive dye imaging, we measured visually evoked neuronal population response from the visual cortex in awake and anesthetized mice at 3 increasing concentrations of isoflurane, thus controlling the level of anesthesia from wakefulness to deep anesthesia. At low concentration of isoflurane, the effects on neuronal measures were minor relative to the awake condition. These effects augmented with increasing isoflurane concentration, while around LOC point, they showed abrupt and nonlinear changes. At the network level, we found that isoflurane decreased the stimulus-evoked intra-areal spatial spread of local neural activation, previously reported to be mediated by horizontal connections, and also reduced intra-areal synchronization of neuronal population. The synchronization between different visual areas decreased with higher isoflurane levels. Isoflurane reduced the population response amplitude and prolonged their latencies while higher visual areas showed increased vulnerability to isoflurane concentration. Our results uncover the changes in neural activity and synchronization at isoflurane concentrations leading to LOC and suggest reverse hierarchical shutdown of cortical areas.

Inhalational Anesthetics Do Not Deteriorate Amyloid-β-Derived Pathophysiology in Alzheimer's Disease: Investigations on the Molecular, Neuronal, and Behavioral Level.

Studies suggest that general anesthetics like isoflurane and sevoflurane may aggravate Alzheimer's disease (AD) neuropathogenesis, e.g., increased amyloid-β (Aβ) protein aggregation resulting in synaptotoxicity and cognitive dysfunction. Other studies showed neuroprotective effects, e.g., with xenon. In the present study, we want to detail the interactions of inhalational anesthetics with Aβ-derived pathology. We hypothesize xenon-mediated beneficial mechanisms regarding Aβ oligomerization and Aβ-mediated neurotoxicity on processes related to cognition. Oligomerization of Aβ1-42 in the presence of anesthetics has been analyzed by means of TR-FRET and silver staining. For monitoring changes in neuronal plasticity due to anesthetics and Aβ1-42, Aβ1-40, pyroglutamate-modified amyloid-(AβpE3), and nitrated Aβ (3NTyrAβ), we quantified long-term potentiation (LTP) and spine density. We analyzed network activity in the hippocampus via voltage-sensitive dye imaging (VSDI) and cognitive performance and Aβ plaque burden in transgenic AD mice (ArcAβ) after anesthesia. Whereas isoflurane and sevoflurane did not affect Aβ1-42 aggregation, xenon alleviated the propensity for aggregation and partially reversed AβpE3 induced synaptotoxic effects on LTP. Xenon and sevoflurane reversed Aβ1-42-induced spine density attenuation. In the presence of Aβ1-40 and AβpE3, anesthetic-induced depression of VSDI-monitored signaling recovered after xenon, but not isoflurane and sevoflurane removal. In slices pretreated with Aβ1-42 or 3NTyrAβ, activity did not recover after washout. Cognitive performance and plaque burden were unaffected after anesthetizing WT and ArcAβ mice. None of the anesthetics aggravated Aβ-derived AD pathology in vivo. However, Aβ and anesthetics affected neuronal activity in vitro, whereby xenon showed beneficial effects on Aβ1-42 aggregation, LTP, and spine density.

Region-dependent Millisecond Time-scale Sensitivity in Spectrotemporal Integrations in Guinea Pig Primary Auditory Cortex

Spectrotemporal integration is a key function of our auditory system for discriminating spectrotemporally complex sounds, such as words. Response latency in the auditory cortex is known to change with the millisecond time-scale depending on acoustic parameters, such as sound frequency and intensity. The functional significance of the millisecond-range latency difference in the integration remains unclear. Actually, whether the auditory cortex has a sensitivity to the millisecond-range difference has not been systematically examined. Herein, we examined the sensitivity in the primary auditory cortex (A1) using voltage-sensitive dye imaging techniques in guinea pigs. Bandpass noise bursts in two different bands (band-noises), centered at 1 and 16 kHz, respectively, were used for the examination. Onset times of individual band-noises (spectral onset-times) were varied to virtually cancel or magnify the latency difference observed with the band-noises. Conventionally defined nonlinear effects in integration were analyzed at A1 with varying sound intensities (or response latencies) and/or spectral onset-times of the two band-noises. The nonlinear effect measured in the high-frequency region of the A1 linearly changed depending on the millisecond difference of the response onset-times, which were estimated from the spatially-local response latencies and spectral onset-times. In contrast, the low-frequency region of the A1 had no significant sensitivity to the millisecond difference. The millisecond-range latency difference may have functional significance in the spectrotemporal integration with the millisecond time-scale sensitivity at the high-frequency region of A1 but not at the low-frequency region.

Optimization of Near-Infrared Fluorescence Voltage-Sensitive Dye Imaging for Neuronal Activity Monitoring in the Rodent Brain.

Many currently employed clinical brain functional imaging technologies rely on indirect measures of activity such as hemodynamics resulting in low temporal and spatial resolutions. To improve upon this, optical systems were developed in conjunction with methods to deliver near-IR voltage-sensitive dye (VSD) to provide activity-dependent optical contrast to establish a clinical tool to facilitate direct monitoring of neuron depolarization through the intact skull. Following the previously developed VSD delivery protocol through the blood-brain barrier, IR-780 perchlorate VSD concentrations in the brain were varied and stimulus-evoked responses were observed. In this paper, a range of optimal VSD tissue concentrations was established that maximized fluorescence fractional change for detection of membrane potential responses to external stimuli through a series of phantom, in vitro, ex vivo, and in vivo experiments in mouse models.