Fig. 5: 3D morphodynamics and chemogenetically induced Ca²⁺ activity in hippocampal mossy fiber-hilar mossy cell synapses.

a, Single plane of an isotropically super-resolved LIONESS volume in the hilus of the DG in an organotypic hippocampal slice culture, where a subset of mossy fiber boutons expressed both the excitatory DREADD hM3Dq (together with cytosolic dTomato, confocal, purple, virally delivered) and the calcium indicator GCaMP6f (confocal, green, Prox1-cre::Ai95 mouse). LIONESS and dTomato images are identical replicates, placing the overlaid time-varying Ca²⁺ signals after stimulation with the DREADD ligand CNO into structural context, showing three example points from a time series. Note that confocal signals extend beyond the structures defined by LIONESS. In particular, they partially originate from structures above and below the plane displayed here. Inset shows GCaMP signal (averaged pixel value normalized to first frame) as a function of time. LIONESS image is a maximum intensity projection spanning 150 nm. Scale bar, 2 µm. b, 3D reconstructions of a hM3Dq-expressing mossy fiber bouton (purple) and its postsynaptic partner, a hilar mossy cell (gold) with complex spines at two time points (top, day 0 (0 h); bottom, day 1 (19.5 h)). V_0h and V_19.5h are bouton volumes at the respective time points. Green (i) and blue (ii) frames indicate the viewing angles from opposite directions for the magnified views on the right. The structures designated by the lettering in a and b refer to the same bouton and complex spine. The scale cube refers to the center of the rendering. Scale bars in the magnified views correspond to 1 µm in the center of the respective bouton renderings.