A Toolbox for Spatiotemporal Analysis of Voltage-Sensitive Dye Imaging Data in Brain Slices

Abstract

Voltage-sensitive dye imaging (VSDI) can simultaneously monitor the spatiotemporal electrical dynamics of thousands of neurons and is often used to identify functional differences in models of neurological disease. While the chief advantage of VSDI is the ability to record spatiotemporal activity, there are no tools available to visualize and statistically compare activity across the full spatiotemporal range of the VSDI dataset. Investigators commonly analyze only a subset of the data, and a majority of the dataset is routinely excluded from analysis. We have developed a software toolbox that simplifies visual inspection of VSDI data, and permits unaided statistical comparison across spatial and temporal dimensions. First, the three-dimensional VSDI dataset (x,y,time) is geometrically transformed into a two-dimensional spatiotemporal map of activity. Second, statistical comparison between groups is performed using a non-parametric permutation test. The result is a 2D map of all significant differences in both space and time. Here, we used the toolbox to identify functional differences in activity in VSDI data from acute hippocampal slices obtained from epileptic Arx conditional knock-out and control mice. Maps of spatiotemporal activity were produced and analyzed to identify differences in the activity evoked by stimulation of each of two axonal inputs to the hippocampus: the perforant pathway and the temporoammonic pathway. In mutant hippocampal slices, the toolbox identified a widespread decrease in spatiotemporal activity evoked by the temporoammonic pathway. No significant differences were observed in the activity evoked by the perforant pathway. The VSDI toolbox permitted us to visualize and statistically compare activity across the spatiotemporal scope of the VSDI dataset. Sampling error was minimized because the representation of the data is standardized by the toolbox. Statistical comparisons were conducted quickly, across the spatiotemporal scope of the data, without a priori knowledge of the character of the responses or the likely differences between them.

Diminished amygdala activation and behavioral threat response following traumatic brain injury

“…Raster plot construction, analysis, and statistics were performed using the MATLAB VSD analysis toolbox provided by and described in (Bourgeois et al, 2014). To begin raster construction, regions of interest (anatomical regions) were defined for segmentation.…”

“…Faul et al, 2007) based on variability from similar previous experiments. All statistical tests for significance were conducted using Mann-Whitney U-tests, two-way repeated-measures ANOVA with Sidak's multiple comparison test in order to test for injury effect and stimulation intensity effect or the permutation test described in (Bourgeois et al, 2014), where applicable. Statistical significance was P <0.05 ( P <0.05*, P <0.01**, P <0.001***).…”

The Features and Functions of Neuronal Assemblies: Possible Dependency on Mechanisms beyond Synaptic Transmission

“…However, some anomalies become immediately evident when comparing assemblies generated in two different, well-established in vitro preparations: first, coronal brain slices where assemblies are evoked using direct electrical stimulation to the cortex (Yuste et al, 1997; Zochowski et al, 2000; Petersen and Sakmann, 2001; Bourgeois et al, 2014), and secondly, a thalamocortical section (Agmon and Connors, 1991; Takashima et al, 2001; Llinas et al, 2002; Hill and Greenfield, 2014) that enables indirect, remote activation of the cortex region via neuronal innervation resulting from thalamic stimulation. Other studies have also investigated the downstream cortico-cortical connectivity elicited by exogenous activation of the lemniscal pathway, leading for example to active communication between primary somatosensory cortex and motor cortex (Ferezou et al, 2007) or for the purposes of general brain mapping in vivo (Lim et al, 2012).…”

“…Single experiment data sets (as in iv ) can then be combined to produce overall experiment averaged space-time maps ( v , n = 7). Image processing via toolbox of Bourgeois et al (2014). Red box in B ii represents the imaging area of focus in A i – iii , while the time-span highlighted in red in B iv represents the epoch graphed out as a time-series in A .…”

Genetically encoded voltage indicators for large scale cortical imaging come of age

“…GEVIs with response dynamics that are compatible with recordings of fast action potentials in cultured cells are already available [20,21 ,23 ,26], but robust resolution of individual action potentials from multiple neurons in living rodents has not yet been demonstrated with any GEVI. While proof-ofprinciple experiments reaching this milestone can be expected in the very near future, further challenges will include the necessary advances in optical instrumentation and analysis methods of activity-map data [37,38]. For cellular resolution functional imaging, novel two-photon microscopes are required that provide activity maps updated at approximately each millisecond with large field-of-views ()1 mm 2 ).…”