(A and B) Side view of control (A) and Insm1 cKO third-row outer-compartment HCs at age P10 (A′, from basal turns) or P35 (A and A″, from middle turns). Parvalbumin labels HCs (oc-IHCs stronger than OHCs) and type II collaterals. At P10, OHCs and oc-IHCs (‡) receive at least one collateral projection (A′, arrows). At P35, OHCs receive type II afferent innervation, but the oc-IHC shown (‡) in the top panel does not (A″). In the lower panels, the oc-IHCs (‡) have only fragments of former collaterals (arrows). (B) oc-IHCs (not contacted by a type I afferent) are innervated by type II collaterals early in development (P10), but by maturity (P30 to P42), many oc-IHCs lack them (****P < 0.0001, binomial test; n ≥ 15; from three to five animals; error bars = SD). (C and D) 3D reconstructions of parvalbumin-positive type II fibers and collaterals showing examples of mature (P30 to P42) oc-IHCs (‡) that maintain collaterals (+collaterals) or have lost them (−collaterals). (D) Colabeling for vAChT reveals an oc-IHC innervated by both a type II afferent collateral and an efferent (terminating axosomatically; left), as well as an oc-IHC receiving neither type II afferent nor efferent innervation (right). (E) Cells receiving type II collateral projections receive large vAChT efferent terminals, whereas cells without type II collaterals lack efferent terminals [****P < 0.0001, Mann-Whitney test; n ≥ 11 from four to five animals (P18 to P35); error bars = SD].