Figure 2. Different subtypes of AN synapses converge onto individual CN neurons.

(A) Diagram of the CN and experimental setup. It depicts two endbulb of Held synapses (E1 and E2) from different AN fibers (AN1 and AN2) that innervate the target bushy neuron, which is recorded and filled with fluorescent dye (magenta). AN is activated by electric stimulation. CN: cochlear nucleus; AVCN: anteroventral CN; PVCN: posteroventral CN; DCN: dorsal CN.

(B) Example responses of a bushy neuron to current step injections (top) and trains of AN stimulation at 100 Hz (bottom). Ticks mark the stimulus onset. Scale: 10 mV and 20 ms.

(C) Single frame confocal images of the bushy neuron in (B) filled with Alexa Fluor 594 dye (magenta), with immunostained VGluT1 (green) and calretinin (CR; red). The bushy neuron receives a large type I_a endbulb of Held synapse (asterisk), which contains VGluT1-labeled puncta (arrow). Double-labeled puncta are shown in yellow in the merged panel (arrow). VGluT1-labeled puncta that do not overlap with CR staining are shown in green in the merged panel (arrowhead), which are from AN synapses that do not express calretinin (non-type I_a synapse). Scale: 10 μm.

(D) 3D reconstruction of the bushy neuron and AN synapses in (C). Red: CR-stained type I_a endbulb of Held synapse. Scale: 10 μm, also applies to E-H. See also Supplemental Video 1.

(E) same as in (D) except showing only the VGluT1-labeled puncta. Yellow (arrow): VGluT1-labeled puncta inside the calretinin-expressing type I_a synapses; green (arrowhead): puncta from non-calretinin-expressing (non-type I_a) synapses.

(F) View of all VGluT1-labeled puncta from (E) without revealing the postsynaptic bushy neuron. Note that this bushy neuron received mostly type I_a synaptic inputs from the AN in terms of VGluT1-labeled puncta volume.

(G) Morphology of the single type I_a endbulb of Held synapse from (D).

(H) Type I_a VGluT1-labeled puncta inside the single endbulb in (G).