Impaired Hearing and Altered Subplate Circuits During the First and Second Postnatal Weeks of Otoferlin-Deficient Mice

Abstract

Sensory deprivation from the periphery impacts cortical development. Otoferlin deficiency leads to impaired cochlear synaptic transmission and is associated with progressive hearing loss in adults. However, it remains elusive how sensory deprivation due to otoferlin deficiency impacts the early development of the auditory cortex (ACX) especially before the onset of low threshold hearing. To test that, we performed in vivo imaging of the ACX in awake mice lacking otoferlin (Otof-/-) during the first and second postnatal weeks and found that spontaneous and sound-driven cortical activity were progressively impaired. We then characterized the effects on developing auditory cortical circuits by performing in vitro recordings from subplate neurons (SPN), the first primary targets of thalamocortical inputs. We found that in Otof-/- pups, SPNs received exuberant connections from excitatory and inhibitory neurons. Moreover, as a population, SPNs showed higher similarity with respect to their circuit topology in the absence of otoferlin. Together, our results show that otoferlin deficiency results in impaired hearing and has a powerful influence on cortical connections and spontaneous activity in early development even before complete deafness. Therefore, peripheral activity has the potential to sculpt cortical structures from the earliest ages, even before hearing impairment is diagnosed.

Keywords: auditory cortex; development; otoferlin; plasticity; subplate.

Figure 1

Altered spontaneous activity in the ACX of Otof^−/− pups. (A) Schematic diagram showing the role of otoferlin in promoting exocytosis at the IHC ribbon synapses (Moser and Vogl 2016). (B) Left: Experimental setup for in vivo widefield imaging in awake mouse pups. Right: surface of the brain through intact and cleared skull in a representative pup. Bright regions indicate expression of GCaMP6s. (C) Open red circles show ROIs identified using dimensionality reduction technique. (D) Raw trace (left) and histogram (right) showing identification of L- and H-events in a representative pup. (E) Cumulative distribution functions (CDFs) showing peak amplitude of spontaneous H- and L-events in WT control (dashed line) and Otof^—/− (solid line) pups at both ages. The peak amplitude of the spontaneous H-events was higher and that of spontaneous L-events was lower in the Otof^—/− pups at P13–15. When Otof^—/− is compared across ages, the peak amplitude of both H- and L-events is higher at P7–P9 (gray line) than at P13–P15 (pink line). (F) CDFs showing interevent interval of spontaneous H- and L-events in control and Otof^—/− pups at both ages. The interevent interval of spontaneous H-events was shorter in Otof^—/− pups only at P13–P15, whereas that of spontaneous L-events was shorter in Otof^—/− pups at both ages. When compared across ages, the interevent interval of L-events was shorter at P13–P15 than at P7–P9.

Figure 2

Sound-evoked activity although present is impaired in the ACX of Otof^—/− pups. (A) Left: Filled areas denote active ROIs that showed significant increase in response within a 2-s window after tone onset. Colors indicate mean ΔF/F of responsive ROIs. Right: Fluorescence time-course of 2 representative responsive ROIs at P8 (top) and P14 (bottom) after an 8 kHz tone presentation. (B) Bar graphs showing number of responsive ROIs in control and Otof^−/− pups at both ages. (C) Left: CDFs showing mean ΔF/F of active ROIs in a 2-s window after tone onset is higher in Otof^−/− (solid line) than WT controls (dashed line) at P7–P9 but not at P13–P15. When Otof^−/− pups are compared across ages, the mean ΔF/F of active ROIs is higher at P7–P9 (gray line) than that at P13–P15 (pink line). Right: CDFs showing Fano factors of mean ΔF/F of active ROIs in a 2-s window after tone onset is lower in WT controls than Otof^−/− only at P13–P15. When Otof^−/− pups are compared across ages, the Fano factor is higher at P7–P9 (gray line) than at P13–P15 (pink line). (D) CDFs showing peak amplitude of sound-evoked H- and L-events across all repeats of sound stimuli for all ROIs in WT control (dashed line) and Otof^−/− pups (solid line) at both ages. The peak amplitude of evoked H-events was significantly higher in Otof^−/− only at P13–P15, whereas that of evoked L-events were significantly lower in Otof^−/− at both ages. When Otof^−/− pups are compared across ages, the amplitude of both H- and L-events was significantly higher at P7–P9. (E) Spatial correlation of both spontaneous and sound-evoked activity is higher in Otof^−/− than WT controls at P13–P15. When Otof^−/− is compared across ages, the spatial correlation of both spontaneous and sound-evoked activity is higher in P13–P15 (pink line) than P7–P9 (gray line). Solid lines denote mean and shaded areas denote confidence intervals.

Figure 3

Excitatory and inhibitory connections to SPNs at P6–P9 are altered in Otof^−/− pups. (A) Left: Cartoon showing auditory pathway from the periphery to the cortex. Right: Schematic of LSPS experiment. Whole-cell patch clamp recordings are made from SPNs. Neurons are activated by laser photolysis (355 nm) of caged glutamate leading to action potentials (upper trace). If activated cells are connected to SPN, eEPSCs or eIPSCs are seen (lower traces) depending on the holding voltage (V_m). Traces on right show example of eEPSCs and eIPSCs from different stimulus locations. (B) Example of LSPS map shows eEPSC (left V_m = −70 mV) and eIPSC (right V_m = 0 mV) charge in a representative SPN from particular stimulus locations. Slice is oriented so that the tonotopic (rostro-caudal) axis can be sampled. The borders between cortical layers are indicated by the white bars. Scale bar 200 μm. (C) Cartoon demonstrating the assembly of average spatial connection probability maps by aligning individual maps to the SPN somata (dashed line). (D) Spatial connection probability map for excitatory and inhibitory connections at P6–P9. Lines show marginal profiles. (E) Bar graphs show comparison of the source area, integration width, mean charge, and percentage of excitatory (top) and inhibitory (bottom) laminar inputs to SPNs along the rostro-caudal axis in WT (black) and Otof^−/− (red) pups at P6–P9.

Figure 4

Otof ^−/− pups show higher circuit similarity in both excitatory (top) and inhibitory (bottom) connections at P6–P9. (A) Scatter plots showing distribution of correlation for each pair of functional connection maps. Bar graphs (inset) show the mean. (B) Fano factor of maps and difference in Fano factor between WT and Otof^−/−show reduced variability in Otof^−/−. White color represents areas where the probability of the connections was <10% and a Fano factor was not calculated. (C) Bar graphs showing layer specific comparison of correlation of connection maps. Overall, Otof^−/− exhibited higher correlation originating from different cortical layers.

Figure 5

Excitatory and inhibitory connections to SPNs at P10–P15 are altered in Otof^−/− pups. (A) Schematic of LSPS experiment. (B) Spatial connection probability map for excitatory and inhibitory connections shows Otof^−/− has hyperconnectivity of excitatory connections at P10–P15. Lines show marginal profiles. (C) Bar graphs show comparison of the source area, integration width, mean charge, and percentage of excitatory (top) and inhibitory (bottom) laminar inputs to SPNs along the rostro-caudal axis in WT (black) and Otof^−/− (red) pups at P10–P15.

Figure 6

Otof ^−/− pups show higher circuit similarity in excitatory (top) but not in inhibitory (bottom) connections at P10–P15. (A) Scatter plots showing distribution of correlation for each pair of functional connection maps. Bar graphs (inset) show the mean. (B) Fano factor of maps and difference in Fano factor between WT and Otof^−/−. White color represents areas where the probability of the connections was <10% and a Fano factor was not calculated. (C) Bar graphs showing layer specific comparison of correlation of connection maps. Overall, Otof^−/− exhibited higher correlation for excitatory connections originating from different cortical layers.