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.

Voltage-Sensitive Dye versus Intrinsic Signal Optical Imaging: Comparison of Tactile Responses in Primary and Secondary Somatosensory Cortices of Rats

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

Evaluation and Optimization of Methods for Generating High-Resolution Retinotopic Maps Using Visual Cortex Voltage-Sensitive Dye Imaging

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

In silico voltage-sensitive dye imaging reveals the emergent dynamics of cortical populations

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

Anatomy and activity patterns in a multifunctional motor neuron and its surrounding circuits

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced DE-3’s processes. Further, we traced back the processes of DE-3’s presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all the behaviors were widely distributed over DE-3’s branches, yet that functional clusters were different during (fictive) swimming vs. crawling.