Fig 2: Confocal analysis of MNTB neurons. A, Monoinnervated neuron from a three-week-old control mouse. Reconstructed RDA-labeled calyx of Held (magenta); MNTB neuron (blue); and VGLUT1/2 labeling (green). B, Monoinnervated neuron from a three-week-old BLZ945-treated mouse. C, D, Monoinnervated neuron from a four-week-old control and BLZ945-injected mouse, respectively. E, F, Polyinnervated neuron from a three-week-old control and BLZ945-injected mouse, respectively. Dashed line indicates the contour of the neuron while asterisks show the VGLUT1/2-positive terminal contacting the same neuron. G, H, Polyinnervated neuron from a four-week-old control and BLZ945-treated animal, respectively. An extra calyceal input was determined by a continuous VGLUT1/2 staining outside of the RDA-positive area (asterisks) surrounding the MNTB neuron. I, Comparison of calyx of Held surface area in control and BLZ945-treated mice at three and four weeks. There was no difference between the two animal groups at both ages tested. No age-related increase in calyceal area was observed. J, Comparison of polyinnervated MNTB neurons as a percentage of MNTB neurons analyzed revealed significantly more polyinnervated neurons in BLZ945-injected mice at three weeks when compared with controls. No difference between control and BLZ945 injected mice was found at four weeks. The age-related change in BLZ945-injected animals did not reach significance (p = 0.0574). Scale bars: 5 µm (A, B, E, F), 3 µm (C, G, H), and 4 µm (D).

Fig 3: Microglial association with VGluT1/2-positive puncta. (A–C) 3D reconstructions of EGFP-positive microglia (green) and VGluT1/2 immunolabeling (magenta) in CX3CR1+/EGFP mice at P8. White patches denote EGFP and VGluT1/2 co-localization areas. (A’–C’) Higher magnification images from panels (A–C). Asterisks point to overlapping areas at the surface of microglial processes or cell bodies. Arrows denote internalized VGluT1/2 particles. (D–F) 3D confocal z-stack reconstructions of microglial cells and VGluT1/2 labeling in CX3CR1+/EGFP mice at P13. (D’–F’) Higher magnification images of areas of co-localization between EGFP and VGluT1/2 (white). At both ages, association between EGFP and VGluT1/2 is clearly visible. Co-localization is more frequently observed at the glial surface than as internalized excitatory puncta. Scale bar in panel (A) = 200 µm and applies to panels (A–F). Scale bar in panel (A’) = 5 µm and applies to panels (A’–F’).

Fig 4: Calyx labeling, reconstruction in 3D, and analysis. (A) A schematic of the interaural intensity differences (IID) pathway and RDA injection site in the VAS to sparsely label calyces of Held in the MNTB. GBCs in the AVCN receive direct input from the AN. In turn, GBCs synapse on inhibitory neurons in the contralateral MNTB. MNTB neurons then innervate excitatory neurons in the LSO. LSO neurons also receive tonotopically matched excitatory terminals from SBCs in the ipsilateral AVCN. LSO neurons compute the balance of frequency-matched inhibition and excitation arising from both ears to determine the sound source location in the horizontal plane. (B) Timeline of injections. Two age cohorts were included in the study. The P8 mice group received vehicle or BLZ945 injections at P2, P4, and P6, and then were perfused at P8. Animals in the P13 age group were injected every 2 days from P2 until P10, and then perfused at P13. (C) A photograph of an RDA injection site (magenta) in the VAS and sparsely labeled calyces in the mouse brainstem. VGluT1/2 labeling is shown in green. (D) A higher magnification of the left MNTB with sparsely RDA-labeled calyces of Held. (E) Reconstructed calyx area used for size and volume measurements (magenta). MNTB neurons were classified into mono- or polyinnervated based on the presence of PV or VGluT1/2 labeling outside the RDA-labeled calyx. Shown is an example of a monoinnervated neuron. VGluT1/2 labeling is present and is co-localized to the RDA-labeled calyx. Dashed line indicates approximate boundary of the Nissl-stained neuron that is contacted by the RDA-filled calyx of Held. (E’) Schematic illustration of a monoinnervated MNTB neuron contacted by an RDA-labeled calyx. (F) An example of a polyinnervated MNTB neuron. It is contacted by an RDA-filled calyx and an additional VGluT1/2 positive calyceal input (indicated by white asterisks) around the remaining surface of the same neuron (dashed line). (F’) Schematic illustration of a polyinnervated MNTB neuron. Both the RDA-labeled calyx and one or more additional inputs terminate on the same MNTB neuron. Scale bar in panel (C) = 200 µm; scale bar in panel (D) = 100 µm; scale bar in panel (E) = 6 µm; scale bar in panel (F) = 5 µm. Abbreviations: AN – auditory nerve, AVCN – anterior ventral cochlear nucleus, GBC – globular bushy cell, LSO – lateral superior olive, MNTB – medial nucleus of the trapezoid body, SBC – spherical bushy cell, VAS – ventral acoustic stria. Panel (A) has been adapted from Joris and Yin (1998).

Fig 5: Effects of microglia depletion on synaptic protein expression in the MNTB. (A) Images of the MNTB (dashed line) from control mice at P8 and (B) at P13. Nissl-stained cell bodies are shown in magenta; VGluT1/2 immunolabel is shown in green. Panels (A’,B’) are higher magnifications of insets in panels (A,B), respectively. (C) VGluT1/2 labeling in the MNTB from BLZ945-treated mice at P8 and (D) at P13. Panels (C’,D’) are higher magnifications of insets in panels (C,D), respectively. VGluT1/2 labeling appears to be comparable throughout all panels. (E) Images of the MNTB (dashed line) from control mice at P8 and (F) at P13. Nissl-stained cell bodies are shown in magenta; VGAT labeling is shown in green. Panels (E’,F’) are higher magnifications of insets in panels (E,F), respectively. (G) VGAT labeling in the MNTB from BLZ945-treated mice at P8 and (H) at P13. Panels (G’,H’) are higher magnifications of insets in panels (G,H), respectively. VGAT immunolabel is similar throughout all panels. (I) Images of the MNTB (dashed line) from control mice at P8 and (J) at P13. Nissl-stained neurons are shown in magenta; Syn label is shown in green. Panels (I’,J’) are higher magnifications of insets in panels (I,J), respectively. (K) Syn labeling in the MNTB from BLZ945-treated mice at P8 and (L) at P13. Panels (K’,L’) are higher magnifications of insets in panels (K,L), respectively. Syn labeling is similar throughout all panels. (M) Areal coverage ratio of VGluT1/2 labeling in the MNTB. There was no significant difference between control and microglia-depleted animals before or after hearing onset as well as between different age groups. (N) Areal coverage ratio of VGAT in the MNTB. There was a significant increase in VGAT labeling with age in both control and microglia-depleted mice. (O) Areal coverage ratio of Syn labeling in the MNTB. There was a significant increase in Syn expression with age in control mice. No difference was observed between control and BLZ945-treated mice. Scale bar in panel (A) = 200 µm, applies to panels (A–L). Scale bar in panel (A’) = 20 µm, applies to panels (A’–L’).