Extended Data Figure 5. Immuno-histochemical verification of presynaptic neuron classification method.

(a) Coronal maximum projection from a z-stack acquired in vivo, showing a section through a presynaptic network labelled with RV-dsRed (red) in a CaMK2a-GCaMP6s transgenic animal (green). Scale bar 50 μm. (b) Coronal brain slice matching the section in a. We could match n=94 out of the n=105 neurons traced in vivo. (c) Inset from b (dashed) showing the immunostaining for the inhibitory neurons marker GAD (blue). (d) ROC curve illustrating the sensitivity and specificity of the neuron classification method based on somatic GCaMP6 fluorescence (Extended Data Figure 4) against the ground-truth measurements obtained by immunostaining against GAD, for a range of classification boundaries. The fitted classification boundary was optimal (blue dot), yielding a specificity of 94% and sensitivity of 91%. (e) Two example presynaptic neurons classified as excitatory (n=49) from the z-stack in a. (f) Slice immunostaining of the two neurons in e. Neurons where confirmed excitatory if they expressed GCaMP6 and were GAD-negative (n = 45). Scale bar 40 μm. (g,h) Same as e,f, for two example presynaptic neurons classified as inhibitory. Neurons where confirmed inhibitory if they did not express GCaMP6 and were GAD-positive (n = 42).