Figure 2. Running-evoked differential rises in PV-driven GCaMP Ca²⁺ signal around dPCs and sPCs.

(A) Schematic of in vivo 2-photon imaging experiments. (B) Left: Example time-averaged fluorescence images (2000 frames) from the superficial and deep sublayers of the CA1 stratum pyramidale in the dorsal (septal) CA1. Relative change in GCaMP Ca²⁺ fluorescence (ΔF/F) was first calculated over 2-4 polygonal regions of interest (ROIs) for superficial and deep imaging planes and then averaged within FOVs. Wide-field ROIs were selected to avoid somatic or dendritic profiles of PV⁺ interneurons in both imaging planes; scale bar: 40μm. Right: Example ΔF/F traces from the two ROIs in superficial and deep sublayers. Horizontal bars indicate periods of running. (C) Summary of mean relative GCaMP Ca²⁺ fluorescence change during running activity in FOVs. (D) Left: Example time-averaged fluorescence images from superficial and deep sublayers taken at high magnification (red, tdTomato; green, GCaMP). Bouton ROIs (yellow circles show five examples; arrow heads point to the areas of the circles) were selected based on the stationary tdTomato signal. Scale bar: 10μm. Right: Representative fluorescence traces (all boutons in FOV with a response between 0 and 200% ΔF/F) of GCaMP Ca²⁺ signals in superficial and deep sublayers. Horizontal bars indicate periods of running activity. (E) Summary of mean running-evoked GCaMP Ca²⁺ signal in perisomatic PV⁺ boutons compared across the two sublayers. (F) Summary of bouton density per 100μm² in the two sublayers. (G, H, I) Summary of morphological measurements of axons of PVBCs filled in vitro and in vivo in the septal CA1. n.s.: not significant (in this and subsequent figures).