Exogenous capture accounts for fundamental differences between prosaccade and antisaccade performance

Oculomotor circuits generate eye movements based on the physical salience of objects and current behavioral goals, exogenous and endogenous influences, respectively. However, the interactions between exogenous and endogenous mechanisms and their dynamic contributions to target selection have been difficult to resolve because they evolve extremely rapidly. In a recent study (Salinas et al., 2019), we achieved the necessary temporal precision using an urgent variant of the antisaccade task wherein motor plans are initiated early and choice accuracy depends sharply on when exactly the visual cue information becomes available. Empirical and modeling results indicated that the exogenous signal arrives ~80 ms after cue onset and rapidly accelerates the (incorrect) plan toward the cue, whereas the informed endogenous signal arrives ~25 ms later to favor the (correct) plan away from the cue. Here, we scrutinize a key mechanistic hypothesis about this dynamic, that the exogenous and endogenous signals act at different times and independently of each other. We test quantitative model predictions by comparing the performance of human participants instructed to look toward a visual cue versus away from it under high urgency. We find that, indeed, the exogenous response is largely impervious to task instructions; it simply flips its sign relative to the correct choice, and this largely explains the drastic differences in psychometric performance between the two tasks. Thus, saccadic choices are strongly dictated by the alignment between salience and behavioral goals.

Optogenetic Stimulation of Gi Signaling Enables Instantaneous Modulation of Cardiomyocyte Pacemaking

G-protein signaling pathways are central in the regulation of cardiac function in physiological and pathophysiological conditions. Their functional analysis through optogenetic techniques with selective expression of opsin proteins and activation by specific wavelengths allows high spatial and temporal precision. Here, we present the application of long wavelength-sensitive cone opsin (LWO) in cardiomyocytes for activation of the Gi signaling pathway by red light. Murine embryonic stem (ES) cells expressing LWO were generated and differentiated into beating cardiomyocytes in embryoid bodies (EBs). Illumination with red light (625 nm) led to an instantaneous decrease up to complete inhibition (84–99% effectivity) of spontaneous beating, but had no effect on control EBs. By using increasing light intensities with 10 s pulses, we determined a half maximal effective light intensity of 2.4 μW/mm2 and a maximum effect at 100 μW/mm2. Pre-incubation of LWO EBs with pertussis toxin completely inhibited the light effect proving the specificity for Gi signaling. Frequency reduction was mainly due to the activation of GIRK channels because the specific channel blocker tertiapin reduced the light effect by ~80%. Compared with pharmacological stimulation of M2 receptors with carbachol with slow kinetics (>30 s), illumination of LWO had an identical efficacy, but much faster kinetics (<1 s) in the activation and deactivation demonstrating the temporal advantage of optogenetic stimulation. Thus, LWO is an effective optogenetic tool for selective stimulation of the Gi signaling cascade in cardiomyocytes with red light, providing high temporal precision.

Imaging microvasculature network evolution and neurodegeneration with precise photothrombosis approach

In the cerebral cortex, the vasculature plays important homeostatic functions, especially at the highly connected complex capillary networks. The association of focal capillary ischemia with the neurodegenerative disease as well as the laminar vascular dynamics have prompted studies of vascular micro-occlusion via photothrombosis. However, technical challenges of this approach remain, including increased temporal precision of occlusion, increasing the depth of vascular occlusion, understanding how such micro-occlusion impacts local blood flow, and ultimately the neuronal effects of such changes. Here, we have developed a novel approach that employs ultra-fast multiphoton light to induce focal Rose Bengal-induced photothrombosis. We demonstrated induction of highly precise and fast occlusion of microvessels at various types and depths. The change of the microvascular architecture and hemodynamics after occlusion revealed the autoregulation and significant difference between upstream vs downstream in layer 2/3. Further, we found that micro-occlusion at two different layers within the same vascular arbor results in distinct effects on the acute flow redistribution mechanism. To examine neuronal effects of such micro-occlusion, we produced infarct of capillaries surrounding a labeled target neuron and found this induces dramatic and rapid lamina-specific degeneration in neuronal dendritic architecture. In sum, our technique enhanced the precision and power of the photothrombotic study of microvascular function. The current results pointed to the importance of laminar scale regulation within the microvascular network, a finding which may be relevant for models of neurovascular disease.

A deep learning framework for inference of single-trial neural population activity from calcium imaging with sub-frame temporal resolution

In many brain areas, neural populations act as a coordinated network whose state is tied to behavior on a moment-by-moment basis and millisecond timescale. Two-photon (2p) calcium imaging is a powerful tool to probe network-scale computation, as it can measure the activity of many individual neurons, monitor multiple layers simultaneously, and sample from identified cell types. However, estimating network states and dynamics from 2p measurements has proven challenging because of noise, inherent nonlinearities, and limitations on temporal resolution. Here we describe RADICaL, a deep learning method to overcome these limitations at the population level. RADICaL extends methods that exploit dynamics in spiking activity for application to deconvolved calcium signals, whose statistics and temporal dynamics are quite distinct from electrophysiologically-recorded spikes. It incorporates a novel network training strategy that exploits the timing of 2p sampling to recover network dynamics with high temporal precision. In synthetic tests, RADICaL infers network states more accurately than previous methods, particularly for high-frequency components. In real 2p recordings from sensorimotor areas in mice performing a "water grab" task, RADICaL infers network states with close correspondence to single-trial variations in behavior, and maintains high-quality inference even when neuronal populations are substantially reduced.

Lysosome-targeted lipid probes reveal sterol transporters NPC1 and LIMP2 as sphingosine transporters

Lysosomes are central catabolic organelles involved in lipid homeostasis and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions as well as their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photo-crosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and LIMP-2/SCARB2, also directly bind to sphingosine. In addition, we showed that absence of either protein leads to lysosomal sphingosine accumulation which suggests a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

A positively tuned voltage indicator reveals electrical correlates of calcium activity in the brain

Neuronal activity is routinely recorded in vivo using genetically encoded calcium indicators (GECIs) and 2-photon microscopy, but calcium imaging is poorly sensitive for single voltage spikes under typical population imaging conditions, lacks temporal precision, and does not report subthreshold voltage changes. Genetically encoded voltage indicators (GEVIs) offer better temporal resolution and subthreshold sensitivity, but 2-photon detection of single spikes in vivo using GEVIs has required specialized imaging equipment. Here, we report ASAP4b and ASAP4e, two GEVIs that brighten in response to membrane depolarization, inverting the fluorescence-voltage relationship of previous ASAP-family GEVIs. ASAP4b and ASAP4e feature 180% and 210% fluorescence increases to 100-mV depolarizations, respectively, as well as modestly prolonged deactivation and high photostability. We demonstrate single-trial detection of spikes and oscillations in vivo with standard 1 and 2-photon imaging systems, and confirm improved temporal resolution in comparison to calcium imaging on the same equipment. Thus, ASAP4b and ASAP4e GEVIs extend the uses of existing imaging equipment to include multi-unit voltage imaging in vivo.

Tagging active neurons by soma-targeted Cal-Light

Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we developed the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio (SNR) of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also targeted kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppressed seizure symptoms, suggesting its applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in (KI) mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

Neural responses to shot changes by cut in cinematographic editing: An EEG (ERD/ERS) study

In order to analyze and detect neural activations and inhibitions in film spectators to shot changes by cut in films, we developed a methodology based on comparisons of recorded EEG signals and analyzed the event-related desynchronization/synchronization (ERD/ERS). The aim of the research is isolating these neuronal responses from other visual and auditory features that covary with film editing. This system of comparing pairs of signals using permutation tests, the Spearman correlation, and slope analysis is implemented in an automated way through sliding windows, analyzing all the registered electrodes signals at all the frequency bands defined. Through this methodology, we are able to locate, identify, and quantify the variations in neuronal rhythms in specific cortical areas and frequency ranges with temporal precision. Our results detected that after a cut there is a synchronization in theta rhythms during the first 188 ms with left lateralization, and also a desynchronization between 250 ms and 750 ms in the delta frequency band. The cortical area where most of these neuronal responses are detected in both cases is the parietal area.

Accuracy of Tempo Judgments in Disk Jockeys Compared to Musicians and Untrained Individuals

Professional disk jockeys (DJs) are an under-studied population whose performance involves creating new musical experiences by combining existing musical materials with a high level of temporal precision. In contemporary electronic dance music, these materials have a stable tempo and are composed with the expectation for further transformation during performance by a DJ for the audience of dancers. Thus, a fundamental aspect of DJ performance is synchronizing the tempo and phase of multiple pieces of music, so that over seconds or even minutes, they may be layered and transitioned without disrupting the rhythmic pulse. This has been accomplished traditionally by manipulating the speed of individual music pieces “by ear,” without additional technological synchronization aids. However, the cumulative effect of this repeated practice on auditory tempo perception has not yet been evaluated. Well-known phenomena of experience-dependent plasticity in other populations, such as musicians, prompts the question of whether such effects exist in DJs in their domain of expertise. This pilot study examined auditory judgments of tempo in 10 professional DJs with experience mixing by ear, compared to 7 percussionists, 12 melodic instrumental musicians, and 11 untrained controls. Participants heard metronome sequences between 80 and 160 beats per minute (BPM) and estimated the tempo. In their most-trained tempo range, 120–139 BPM, DJs were more accurate (lower absolute percent error) than untrained participants. Within the DJ group, 120–139 BPM exhibited greater accuracy than slower tempos of 80–99 or 100–119 BPM. DJs did not differ in accuracy compared to percussionists or melodic musicians on any BPM range. Percussionists were more accurate than controls for 100–119 and 120–139 BPM. The results affirm the experience-dependent skill of professional DJs in temporal perception, with comparable performance to conventionally trained percussionists and instrumental musicians. Additionally, the pattern of results suggests a tempo-specific aspect to this training effect that may be more pronounced in DJs than percussionists and musicians. As one of the first demonstrations of enhanced auditory perception in this unorthodox music expert population, this work opens the way to testing whether DJs also have enhanced rhythmic production abilities, and investigating the neural substrates of this skill compared to conventional musicians.

Selective electrical stimulation with currentfield modulation

Selective electrical stimulation using a multielectrode array is a promising technique that can potentially bring electrical stimulation treatment modalities a step forward. A microcontroller-controlled electrical stimulator system delivering a single pulse was designed, suitable for current-field modulation. The goal is to make electrical stimulation with surface electrodes more specific. A graphical user interface (GUI) was developed to control stimulation parameters and current-field within a multi-electrode array wirelessly. The stimulator generates arbitrary biphasic waveforms with a 5-bit resolution and high temporal precision (<10 μs) and was demonstrated to stimulate posterior lumbar root fibers in transcutaneous spinal cord stimulation (tSCS) treatment selectively. Current-field modulation throughout a sixteen-electrode array was achieved. The system has the goal to improve control of stimulation conditions in electrophysiological studies and time-dependent and site-specific stimulation patterns for neuromodulation applications. A novel feature is the current-field modulation ability of the stimulator for surface electrode arrays.