Figure 3. AACs innervate the proximal part of PC axons via multiple synaptic contacts. A, Maximum z intensity projection of 3D confocal image of a representative pair. Biocytin in the AAC was visualized with streptavidin-conjugated Alexa-594 (magenta) and Alexa-488 in the PC (green). B, Neurolucida reconstruction of the postsynaptic PC (green) with the putative synaptic sites marked in magenta. C, High-power magnification of the maximum z intensity projection image of the region indicated in B, showing a part of the AIS of the postsynaptic PC (green), which receives multiple boutons (arrows) from the presynaptic AAC (magenta). D, 3D analysis of confocal images of three boutons enclosed in the box in C shows close appositions of the presynaptic and postsynaptic structures. E, Verification of the functional synapses at the light microscopic level using neuroligin-2 staining. F–I, Analysis of the presence of synaptic contacts between the recorded pairs using correlative light and electron microscopy. F, Neurolucida reconstruction of another AAC-PC pair. G, Light micrograph of the boxed region in F. H, I, Electron micrographs of two boutons of the presynaptic AAC indicated in G are shown, both of which form synaptic contacts with the AIS of the postsynaptic PC (black arrows). White arrow in H indicates an unlabeled bouton synapsing on the biocytin-labeled AIS. J, Distribution of the number of boutons obtained by light microscopy (201 putative synaptic contacts from 24 pairs). In 28 cases, the presence of a synapse was verified using electron microscopy. K, Relationship between the integral of the summed IPSPs evoked by 3 action potentials and the number of synapses made by the AAC. L, Correlation between the effect of AACs on PC firing tested with a sinusoidal protocol (Fig. 2A–C) and the number of axon-AIS synapses (Hill fit, p < 0.001). M, Distribution of the number of boutons from in vitro filled AACs on ankyrin G-labeled profiles (n = 6 AACs, 616 contact sites on 112 ankyrin G-immunostained profiles, black) and AAC-PC pairs. The analysis of the varicosities was restricted to the average length obtained from the ankyrin G staining (39.4 μm) (n = 24 pairs, 134 boutons, magenta). N, Cumulative distributions of bouton numbers on randomly sampled individual ankyrin G-immunostained profiles taken from 6 filled AACs (each thin black line indicates data from single AACs). The average of these distributions (thick black line) compared with the pooled data of the paired recordings shows no difference (Kolmogorov–Smirnov test, p = 0.76). O, Spatial distribution of the synapses along the axon obtained in paired recordings (n = 24 pairs, 201 boutons). The 0 μm marks the origin of the axon at the soma. Data are mean ± SEM. Scale bars: A, B, F, 30 μm; C, 5 μm; D, E, 1 μm; G, 10 μm; H, I, 0.25 μm.