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.

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 all of DE-3’s processes. Further, we traced back the processes of all 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 of the behaviors were widely distributed over DE-3’s branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

Voltage-sensitive dye imaging reveals inhibitory modulation of ongoing cortical activity

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. In particular, it is unclear how VSDI signals relate to population firing rates. 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. We proceed by exploring experimentally inaccessible circuit properties to show that during periods of spontaneous activity, membrane potential fluctuations are anticorrelated with population firing rates. Furthermore, we manipulate network connections to show that this effect depends on recurrent connectivity and is modulated by external input. We conclude that VSDI primarily reflects inhibitory responses to ongoing excitatory dynamics.

Spatial organization of activity evoked by focal stimulation within the rat spinal dorsal horn as visualized by voltage-sensitive dye imaging in the slice

In a prior study using laser scanning photostimulation, we found a pronounced cell type-specific mediolateral asymmetry in the local synaptic connectivity in the superficial laminae of the spinal dorsal horn (Kosugi M, Kato G, Lukashov S, Pendse G, Puskar Z, Kozsurek M, Strassman AM. J Physiol 591: 1935–1949, 2013). To obtain information on dorsal horn organization that might complement findings from microelectrode studies, voltage-sensitive dye imaging was used in the present study to examine patterns of activity evoked by focal electrical stimulation, in the presence and absence of synaptic blocking agents, at different positions in transverse, parasagittal, and horizontal slices of the dorsal horn of 2- to 3-wk -old male rats. A pronounced difference in responsiveness was found between medial and lateral dorsal horn, in that medial sites in the superficial dorsal horn showed much larger synaptic responses to focal stimulation than lateral sites. This difference appeared to be a result of a difference in the intrinsic elements of the dorsal horn, rather than a difference in the inputs from the white matter, because the stimulus intensities were subthreshold for evoking synaptic responses from stimulation at sites in the white matter, although it is also possible that the greater responsiveness is due, at least in part, to activation of Aβ primary afferent fibers that pass through the medial dorsal horn. The results raise the possibility of differences between medial and dorsal horn that need to be taken into account in the interpretation of studies of dorsal horn organization. NEW & NOTEWORTHY We used voltage-sensitive dye imaging to obtain information on spatial aspects of dorsal horn organization that are difficult to examine with single-cell approaches because of the limitations of microelectrode sampling. The most noteworthy finding was a previously unreported, extreme difference between medial and lateral dorsal horn in responsiveness to focal stimulation that appears to result, at least in part, from a greater degree of excitability or local connectivity in medial dorsal horn.