A double-sided microscope to realize whole-ganglion imaging of membrane potential in the medicinal leech.

Studies of neuronal network emergence during sensory processing and motor control are greatly facilitated by technologies that allow us to simultaneously record the membrane potential dynamics of a large population of neurons in single cell resolution. To achieve whole-brain recording with the ability to detect both small synaptic potentials and action potentials, we developed a voltage-sensitive dye (VSD) imaging technique based on a double-sided microscope that can image two sides of a nervous system simultaneously. We applied this system to the segmental ganglia of the medicinal leech. Double-sided VSD imaging enabled simultaneous recording of membrane potential events from almost all of the identifiable neurons. Using data obtained from double-sided VSD imaging, we analyzed neuronal dynamics in both sensory processing and generation of behavior and constructed functional maps for identification of neurons contributing to these processes.

Effect of spatial resolution and filtering on mapping cardiac fibrillation.

BACKGROUND: Endocardial mapping tools use variable interelectrode resolution, whereas body surface mapping tools use narrow bandpass filtering (BPF) to map fibrillatory mechanisms established by high-resolution optical imaging. OBJECTIVE: The purpose of this study was to study the effect of resolution and BPF on the underlying mechanism being mapped. METHODS: Hearts from 14 healthy New Zealand white rabbits were Langendorff perfused. We studied the effect of spatial resolution and BPF on the location and characterization of rotors by comparing phase singularities detected by high-resolution unfiltered optical maps and of fibrillating myocardium with decimated and filtered maps with simulated electrode spacing of 2, 5, and 8 mm. RESULTS: As we decimated the maps with 2-mm, 5-mm, and 8-mm interelectrode spacing, the mean ( ± SD) number of rotors detected decreased from 10.2 ± 9.6, 1.6 ± 3.2, and 0.2 ± 0.5, respectively. Lowering the resolution led to synthesized pseudo-rotors that may be inappropriately identified. Applying a BPF led to fewer mean phase singularities detected (248 ± 207 vs 333 ± 130; P<.01), giving the appearance of pseudo-spatial stability measured as translation index (with BPF 3.6 ± 0.4 mm vs 4.0 ± 0.5 mm without BPF; P<.01) and pseudo-temporal stability with longer duration (70.0 ± 17.6 ms in BPF maps vs 44.1 ± 6.6 ms in unfiltered maps; P<.001) than true underlying fibrillating myocardium mapped. CONCLUSION: Electrode resolution and BPF of electrograms can result in distortion of the underlying electrophysiology of fibrillation. Newer mapping techniques need to demonstrate sensitivity analysis to quantify the degree of distortion before clinical use to avoid inaccurate electrophysiologic interpretation.

Milliseconds of Sensory Input Abruptly Modulate the Dynamics of Cortical States for Seconds.

Spontaneous internal activity plays a major role in higher brain functions. The question of how it modulates sensory evoked activity and behavior has been explored in anesthetized rodents, cats, monkeys and in behaving human subjects. However, the complementary question of how a brief sensory input modulates the internally generated activity in vivo remains unresolved, and high-resolution mapping of these bidirectional interactions was never performed. Integrating complementary methodologies, at population and single cells levels, we explored this question. Voltage-sensitive dye imaging of population activity in anesthetized rats' somatosensory cortex revealed that spontaneous up-states were largely diminished for ~2 s, even after a single weak whisker deflection. This effect was maximal at the stimulated barrel but spread across several cortical areas. A higher velocity whisker deflection evoked activity at ~15Hz. Two-photon calcium imaging activity and cell-attached recordings confirmed the VSD results and revealed that for several seconds most single cells decreased their firing, but a small number increased firing. Comparing single deflection with long train stimulation, we found a dominant effect of the first population spike. We suggest that, at the onset of a sensory input, some internal messages are silenced to prevent overloading of the processing of relevant incoming sensory information.

Optogenetic Visualization of Presynaptic Tonic Inhibition of Cerebellar Parallel Fibers.

UNLABELLED: Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABAA receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABAA receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABAA receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits. SIGNIFICANCE STATEMENT: This paper demonstrates that an unconventional form of signaling, known as tonic inhibition, is found in presynaptic terminals and affects conventional synaptic communication. Our results establish the basic characteristics and mechanisms of presynaptic tonic inhibition and show that it occurs in vivo as well as in isolated brain tissue.

In Vivo Voltage-Sensitive Dye Imaging of Subcortical Brain Function.

The whisker system of rodents is an excellent model to study peripherally evoked neural activity in the brain. Discrete neural modules represent each whisker in the somatosensory cortex ("barrels"), thalamus ("barreloids"), and brain stem ("barrelettes"). Stimulation of a single whisker evokes neural activity sequentially in its corresponding barrelette, barreloid, and barrel. Conventional optical imaging of functional activation in the brain is limited to surface structures such as the cerebral cortex. To access subcortical structures and image sensory-evoked neural activity, we designed a needle-based optical system using gradient-index (GRIN) rod lens. We performed voltage-sensitive dye imaging (VSDi) with GRIN rod lens to visualize neural activity evoked in the thalamic barreloids by deflection of whiskers in vivo. We stimulated several whiskers together to determine the sensitivity of our approach in differentiating between different barreloid responses. We also carried out stimulation of different whiskers at different times. Finally, we used muscimol in the barrel cortex to silence the corticothalamic inputs while imaging in the thalamus. Our results show that it is possible to obtain functional maps of the sensory periphery in deep brain structures such as the thalamic barreloids. Our approach can be broadly applicable to functional imaging of other core brain structures.

Cardiac applications of optogenetics.

In complex multicellular systems, such as the brain or the heart, the ability to selectively perturb and observe the response of individual components at the cellular level and with millisecond resolution in time, is essential for mechanistic understanding of function. Optogenetics uses genetic encoding of light sensitivity (by the expression of microbial opsins) to provide such capabilities for manipulation, recording, and control by light with cell specificity and high spatiotemporal resolution. As an optical approach, it is inherently scalable for remote and parallel interrogation of biological function at the tissue level; with implantable miniaturized devices, the technique is uniquely suitable for in vivo tracking of function, as illustrated by numerous applications in the brain. Its expansion into the cardiac area has been slow. Here, using examples from published research and original data, we focus on optogenetics applications to cardiac electrophysiology, specifically dealing with the ability to manipulate membrane voltage by light with implications for cardiac pacing, cardioversion, cell communication, and arrhythmia research, in general. We discuss gene and cell delivery methods of inscribing light sensitivity in cardiac tissue, functionality of the light-sensitive ion channels within different types of cardiac cells, utility in probing electrical coupling between different cell types, approaches and design solutions to all-optical electrophysiology by the combination of optogenetic sensors and actuators, and specific challenges in moving towards in vivo cardiac optogenetics.

Functional architecture of the inferior colliculus revealed with voltage-sensitive dyes.

We used optical imaging with voltage-sensitive dyes to investigate the spatio-temporal dynamics of synaptically evoked activity in brain slices of the inferior colliculus (IC). Responses in transverse slices which preserve cross-frequency connections and in modified sagittal slices that preserve connections within frequency laminae were evoked by activating the lateral lemniscal tract. Comparing activity between small and large populations of cells revealed response areas in the central nucleus of the IC that were similar in magnitude but graded temporally. In transverse sections, these response areas are summed to generate a topographic response profile. Activity through the commissure to the contralateral IC required an excitation threshold that was reached when GABAergic inhibition was blocked. Within laminae, module interaction created temporal homeostasis. Diffuse activity evoked by a single lemniscal shock re-organized into distinct spatial and temporal compartments when stimulus trains were used, and generated a directional activity profile within the lamina. Using different stimulus patterns to activate subsets of microcircuits in the central nucleus of the IC, we found that localized responses evoked by low-frequency stimulus trains spread extensively when train frequency was increased, suggesting recruitment of silent microcircuits. Long stimulus trains activated a circuit specific to post-inhibitory rebound neurons. Rebound microcircuits were defined by a focal point of initiation that spread to an annular ring that oscillated between inhibition and excitation. We propose that much of the computing power of the IC is derived from local circuits, some of which are cell-type specific. These circuits organize activity within and across frequency laminae, and are critical in determining the stimulus-selectivity of auditory coding.

Cardiac electrophysiological imaging systems scalable for high-throughput drug testing.

Multi-parametric electrophysiological measurements using optical methods have become a highly valued standard in cardiac research. Most published optical mapping systems are expensive and complex. Although some applications demand high-cost components and complex designs, many can be tackled with simpler solutions. Here, we describe (1) a camera-based voltage and calcium imaging system using a single 'economy' electron-multiplying charge-coupled device camera and demonstrate the possibility of using a consumer camera for imaging calcium transients of the heart, and (2) a photodiode-based voltage and calcium high temporal resolution measurement system using single-element photodiodes and an optical fibre. High-throughput drug testing represents an application where system scalability is particularly attractive. Therefore, we tested our systems on tissue exposed to a well-characterized and clinically relevant calcium channel blocker, nifedipine, which has been used to treat angina and hypertension. As experimental models, we used the Langendorff-perfused whole-heart and thin ventricular tissue slices, a preparation gaining renewed interest by the cardiac research community. Using our simplified systems, we were able to monitor simultaneously the marked changes in the voltage and calcium transients that are responsible for the negative inotropic effect of the compound.

Genetically engineered fluorescent voltage reporters.

Fluorescent membrane voltage indicators that enable optical imaging of neuronal circuit operations in the living mammalian brain are powerful tools for biology and particularly neuroscience. Classical voltage-sensitive dyes, typically low molecular-weight organic compounds, have been in widespread use for decades but are limited by issues related to optical noise, the lack of generally applicable procedures that enable staining of specific cell populations, and difficulties in performing imaging experiments over days and weeks. Genetically encoded voltage indicators (GEVIs) represent a newer alternative that overcomes several of the limitations inherent to classical voltage-sensitive dyes. We critically review the fundamental concepts of this approach, the variety of available probes and their state of development.

Columnar interactions determine horizontal propagation of recurrent network activity in neocortex.

The cortex is organized in vertical and horizontal circuits that determine the spatiotemporal properties of distributed cortical activity. Despite detailed knowledge of synaptic interactions among individual cells in the neocortex, little is known about the rules governing interactions among local populations. Here, we used self-sustained recurrent activity generated in cortex, also known as up-states, in rat thalamocortical slices in vitro to understand interactions among laminar and horizontal circuits. By means of intracellular recordings and fast optical imaging with voltage-sensitive dyes, we show that single thalamic inputs activate the cortical column in a preferential layer 4 (L4) → layer 2/3 (L2/3) → layer 5 (L5) sequence, followed by horizontal propagation with a leading front in supragranular and infragranular layers. To understand the laminar and columnar interactions, we used focal injections of TTX to block activity in small local populations, while preserving functional connectivity in the rest of the network. We show that L2/3 alone, without underlying L5, does not generate self-sustained activity and is inefficient propagating activity horizontally. In contrast, L5 sustains activity in the absence of L2/3 and is necessary and sufficient to propagate activity horizontally. However, loss of L2/3 delays horizontal propagation via L5. Finally, L5 amplifies activity in L2/3. Our results show for the first time that columnar interactions between supragranular and infragranular layers are required for the normal propagation of activity in the neocortex. Our data suggest that supragranular and infragranular circuits, with their specific and complex set of inputs and outputs, work in tandem to determine the patterns of cortical activation observed in vivo.

Voltage-sensitive dye imaging reveals improved topographic activation of cortex in response to manipulation of thalamic microstimulation parameters.

Voltage-sensitive dye imaging was used to quantify in vivo, network level spatiotemporal cortical activation in response to electrical microstimulation of the thalamus in the rat vibrissa pathway. Thalamic microstimulation evoked a distinctly different cortical response than natural sensory stimulation, with response to microstimulation spreading over a larger area of cortex and being topographically misaligned with the cortical column to which the stimulated thalamic region projects. Electrical stimulation with cathode-leading asymmetric waveforms reduced this topographic misalignment while simultaneously increasing the spatial specificity of the cortical activation. Systematically increasing the asymmetry of the microstimulation pulses revealed a continuum between symmetric and asymmetric stimulation that gradually reduced the topographic bias. These data strongly support the hypothesis that manipulation of the electrical stimulation waveform can be used to selectively activate specific neural elements. Specifically, our results are consistent with the prediction that cathode-leading asymmetric waveforms preferentially stimulate cell bodies over axons, while symmetric waveforms preferentially activate axons over cell bodies. The findings here provide some initial steps toward the design and optimization of microstimulation of neural circuitry, and open the door to more sophisticated engineering tools, such as nonlinear system identification techniques, to develop technologies for more effective control of activity in the nervous system.

Simultaneous measurement of membrane potential changes in multiple pattern generating neurons using voltage sensitive dye imaging.

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.

Anatomic localization and autonomic modulation of atrioventricular junctional rhythm in failing human hearts.

BACKGROUND: The structure-function relationship in the atrioventricular junction (AVJ) of various animal species has been investigated in detail; however, less is known about the human AVJ. In this study, we performed high-resolution optical mapping of the human AVJ (n = 6) to define its pacemaker properties and response to autonomic stimulation. METHODS AND RESULTS: Isolated, coronary-perfused AVJ preparations from failing human hearts (n = 6, 53 ± 6 years) were optically mapped using the near-infrared, voltage-sensitive dye, di-4-ANBDQBS, with isoproterenol (1 µmol/L) and acetylcholine (1 µmol/L). An algorithm detecting multiple components of optical action potentials was used to reconstruct multilayered intramural AVJ activation and to identify specialized slow and fast conduction pathways (SP and FP). The anatomic origin and propagation of pacemaker activity was verified by histology. Spontaneous AVJ rhythms of 29 ± 11 bpm (n = 6) originated in the nodal-His region (n = 3) and/or the proximal His bundle (n = 4). Isoproterenol accelerated the AVJ rhythm to 69 ± 12 bpm (n = 5); shifted the leading pacemaker to the transitional cell regions near the FP and SP (n = 4) and/or coronary sinus (n = 2); and triggered reentrant arrhythmias (n = 2). Acetylcholine (n = 4) decreased the AVJ rhythm to 18 ± 4 bpm; slowed FP/SP conduction leading to block between the AVJ and atrium; and shifted the pacemaker to either the transitional cell region or the nodal-His region (bifocal activation). CONCLUSIONS: We have demonstrated that the AVJ pacemaker in failing human hearts is located in the nodal-His region or His bundle regions and can be modified with autonomic stimulation. Moreover, we found that both the FP and SP are involved in anterograde and retrograde conduction.

Targeted mini-strokes produce changes in interhemispheric sensory signal processing that are indicative of disinhibition within minutes.

Most processing of sensation involves the cortical hemisphere opposite (contralateral) to the stimulated limb. Stroke patients can exhibit changes in the interhemispheric balance of sensory signal processing. It is unclear whether these changes are the result of poststroke rewiring and experience, or whether they could result from the immediate effect of circuit loss. We evaluated the effect of mini-strokes over short timescales (<2 h) where cortical rewiring is unlikely by monitoring sensory-evoked activity throughout much of both cortical hemispheres using voltage-sensitive dye imaging. Blockade of a single pial arteriole within the C57BL6J mouse forelimb somatosensory cortex reduced the response evoked by stimulation of the limb contralateral to the stroke. However, after stroke, the ipsilateral (uncrossed) forelimb response within the unaffected hemisphere was spared and became independent of the contralateral forelimb cortex. Within the unaffected hemisphere, mini-strokes in the opposite hemisphere significantly enhanced sensory responses produced by stimulation of either contralateral or ipsilateral pathways within 30-50 min of stroke onset. Stroke-induced enhancement of responses within the spared hemisphere was not reproduced by inhibition of either cortex or thalamus using pharmacological agents in nonischemic animals. I/LnJ acallosal mice showed similar rapid interhemispheric redistribution of sensory processing after stroke, suggesting that subcortical connections and not transcallosal projections were mediating the novel activation patterns. Thalamic inactivation before stroke prevented the bilateral rearrangement of sensory responses. These findings suggest that acute stroke, and not merely loss of activity, activates unique pathways that can rapidly redistribute function within the spared cortical hemisphere.

Dual excitation wavelength epifluorescence imaging of transmural electrophysiological properties in intact hearts.

BACKGROUND: Epifluorescence imaging using voltage-sensitive dyes has provided unique insights into cardiac electrical activity and arrhythmias. However, conventional dyes use blue-green excitation light, which has limited depth penetration. OBJECTIVE: The aim of this study was to demonstrate that combining a short and a long excitation wavelength using near-infrared (NIR) dyes allows for epifluorescence imaging of transmural electrophysiological properties in intact hearts. METHODS: Epifluorescence imaging was performed in rat hearts (N = 11) using DI-4-ANEPPS and the NIR dye DI-4-ANBDQBS. Activation and action potential duration (APD) patterns were investigated at 2 excitation wavelengths (530 and 660 nm) after epicardial stimulation at various cycle lengths (160 to 70 ms). RESULTS: Optical action potential upstrokes acquired with 660-nm excitation of DI-4-ANBDQBS were significantly longer than upstrokes obtained with 530-nm excitation of DI-4-ANEPPS (P < .001). Comparison of activation maps showed counterclockwise rotation of isochrones consistent with a transmural rotation of myofibers. Pronounced APD modulation by the activation sequence was observed at both excitation wavelengths. Significantly prolonged APDs (P = .016) and steeper APD restitution curves were found with DI-4-ANBDQBS (660-nm excitation) when compared with DI-4-ANEPPS (530-nm excitation). Dual excitation wavelength experiments using solely DI-4-ANBDQBS yielded similar results. Monophasic action potential recordings showed prolonged APD and steeper APD restitution curves in the endocardium, indicating that 660-nm excitation provides a significant endocardial contribution to the signal. Three-dimensional computer simulations confirmed our findings. CONCLUSION: Dual excitation wavelength epifluorescence allows detecting transmural heterogeneity in intact hearts. It therefore has the potential to become an important tool in experimental cardiac electrophysiology.

Inhomogeneous distribution of action potential characteristics in the rabbit sino-atrial node revealed by voltage imaging.

The sino-atrial node (SAN) is the natural pacemaker of the heart. Mechanisms of the leading pacemaker site generation and dynamic pacemaker shifts in the SAN have been so far studied with an electrophysiological technique, but the detailed spatial distribution of action potential characteristics in the SAN has not been analyzed due to the limited number of simultaneously recorded sites in microelectrode recording. To elucidate the mechanism of leading pacemaker site generation in the SAN, we applied a voltage imaging technique and analyzed the spatial distribution of action potential characteristics in the rabbit SAN. Action potential parameters, i.e., action potential duration at 50% repolarization level, the slope of upstroke, and the slope of the linearly depolarizing early phase of pacemaker activity (phase-4), were calculated from optical signals. Action potential parameter values derived from intracellular recording with a microelectrode and those from optical recording were significantly correlated. The leading pacemaker site occurred in the region of either globally or locally maximum phase-4 slope in 7 of 12 preparations, however, it did not coincide with the region of the early maximum phase-4 slope in the other 5 preparations. Carbenoxolone, a gap junction blocker, changed action potential properties and caused pacemaker shifts. Model simulation, assuming an inhomogeneous distribution of intrinsic properties of SAN cells, reproduced the experimental results. We conclude that the functional structure of the SAN is more inhomogeneous than that dictated by previous models. Besides intrinsic cellular properties, cell-to-cell interaction through gap junctions influences action potential characteristics and leading pacemaker site generation.