Figure 1. Dorsal periaqueductal gray (dPAG) neuronal ensembles encode arm type in the elevated plus maze (EPM).

(A) Gradient refractive index (GRIN) lens implantation, virus expression strategy, and example Ca²⁺ signals of neurons in the dorsal periaqueductal gray (dPAG). (B) Example mouse exploration path recorded in the EPM. Mice spent significantly more time in the closed arms compared to the open arms (data are represented as mean ± SEM; W = 0, p=0.012, Wilcoxon signed rank test, n = 8 mice). (C) Mean percentage of time in which mice engaged in head dips during the whole EPM session (n = 8 mice). (D) dPAG dF/F traces from the same mouse that display preferential activity in the closed (upper trace) and open (lower trace) arms of the EPM (open and closed arm-preferring cells). Epochs corresponding to exploration of the closed and open arms are shown respectively as green and blue shaded areas. (E) Activity heatmaps for corresponding example neurons shown in (D). (F) The open arm preference score was calculated for each neuron (orange bars; see Materials and methods), as was the distribution of open arm preference score for shuffled data (red line). Bars show the distribution of open arm preference score for open, closed, and neither cells (n = 857 cells). (G) Scatterplots showing correlations between neural activity across the two open arms (top) and between open and closed arms of the EPM (bottom). Each point represents one cell (n = 857 cells, r = Pearson’s correlation coefficient). (H) Pie chart shows the percent of all recorded neurons that were classified as open, closed, or neither cells (n = 857 cells). (I) For each subplot, each row depicts the mean normalized activity of an open, closed, or neither arm-preferring cell during behavior-aligned arm transitions (n = 857 cells).

Figure 1—figure supplement 1. Deep brain imaging of dorsal periaqueductal gray (dPAG) neurons and distribution of elevated plus maze (EPM) scores.

(A) Illustration and sequence of EPM and rat exposure assays. (B) (Left) Coronal section of the periaqueductal gray showing GCaMP6s expression and representative gradient refractive index (GRIN) lens placement in the dPAG. Position of the section relative to bregma is indicated in the lower left corner. Aq: aqueduct (Sylvius). Scale bar: 500 μm. (Right) Maximum projection of the dPAG field of view in an example mouse. (Top right) Anatomical scheme of GRIN lens front location of animals expressing GCaMP6s under the control of the Syn promoter in large populations of dPAG neurons (n = 8 mice). (C) Behavioral tests. (D) The EPM score (blue) differs significantly from distribution expected by chance (red line) (n = 857 cells; U = 15.62, p<0.001, Wilcoxon rank sum test). The EPM score is near +1 for cells that fire similarly in arms of the same type. For example, a cell that had high activity in both open arms and very low rate on both closed arms would have a score near 1. A cell with high activity in both closed arms and low activity in both open arms also would have a score near 1. Thus, both open and closed arm-preferring cells would have positive scores. A cell active uniformly in the environment would have a score near 0. A cell that fired differently in arms of the same type would have negative score, such as a cell that has high activity in one open arm and lower than mean activity in the other open arm. Note that this is a different metric from the open arm preference score metric as shown in Figure 1F.