Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses

Abstract

Functional maturation of afferent synaptic connections to inner hair cells (IHCs) involves pruning of excess synapses formed during development, as well as the strengthening and survival of the retained synapses. These events take place during the thyroid hormone (TH)-critical period of cochlear development, which is in the perinatal period for mice and in the third trimester for humans. Here, we used the hypothyroid Snell dwarf mouse (Pit1(dw)) as a model to study the role of TH in afferent type I synaptic refinement and functional maturation. We observed defects in afferent synaptic pruning and delays in calcium channel clustering in the IHCs of Pit1(dw) mice. Nevertheless, calcium currents and capacitance reached near normal levels in Pit1(dw) IHCs by the age of onset of hearing, despite the excess number of retained synapses. We restored normal synaptic pruning in Pit1(dw) IHCs by supplementing with TH from postnatal day (P)3 to P8, establishing this window as being critical for TH action on this process. Afferent terminals of older Pit1(dw) IHCs showed evidence of excitotoxic damage accompanied by a concomitant reduction in the levels of the glial glutamate transporter, GLAST. Our results indicate that a lack of TH during a critical period of inner ear development causes defects in pruning and long-term homeostatic maintenance of afferent synapses.

Keywords: auditory system; calcium current; capacitance measurements; glutamate transporter.

Fig. 1

Synaptic pruning is disrupted in Pit1^dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with afferent presynaptic (RIBEYE; green) and postsynaptic (SHANK1; red) markers at P5 in WT mice (a) and Pit1^dw mice (b). (c and d) The same markers at P14 in WT mice (c) and Pit1^dw mice (d). (e–i) Box plots of the quantification of RIBEYE, SHANK1 and RIBEYE–SHANK1 puncta from the mid-turn of the cochlea in WT and Pit1^dw mice. (e and f) RIBEYE, SHANK1 and RIBEYE–SHANK1 counts at P5, P9 and P14 in WT mice (e) and Pit1^dw mice (f). (g–i) Comparison of RIBEYE (g), SHANK1 (h) and RIBEYE–SHANK1 (i) counts between WT and Pit1^dw mice. significant comparisons (P < 0.05) are indicated with asterisks. The dots indicate outliers in the data.

Fig. 2

Abnormal CaV1.3 puncta clustering at the synapses of Pit1^dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with the afferent presynaptic marker RIBEYE (green) and the calcium channel CaV1.3 (red) at P7 in WT mice (a) and Pit1^dw mice (b). (c–f) The same markers at P14 for WT (c) and Pit1^dw mice (d), and at P24 for WT (e) and Pit1^dw mice (f). (g–i) Box plots of the quantification of RIBEYE (g), CaV1.3 (h) and RIBEYE–CaV1.3 (i) puncta from the mid-turn of the cochlea in WT and Pit1^dw mice. significant comparisons (P < 0.05) between genotypes are indicated with asterisks. To maintain clarity of the figure, P-values for significant comparisons across different ages within the same genotype are not indicated on the graph, but are mentioned in the relevant Results section. The dots indicate outliers in the data.

Fig. 3

(a and b) Calcium current measurements from WT (a) and Pit1^dw (b) IHCs. The stimulus is shown at the top. Measurements were made at the ages indicated on the right. Hair cells were held at −84 mV and depolarized in 10-mV steps from −120 mV to +20 mV. The data shown include 20-mV steps from −80 mV to −60 mV. (c) Plots of the calcium current amplitudes at different postnatal days for WT (black) and Pit1^dw (red) IHCs. The number of WT and Pit1^dw IHCs recorded were, respectively, P2 (12 and 5), P4 (4 and 0), P7 (13 and 4), P14 (11 and 20), and P24 (9 and 12).

Fig. 4

Two sine capacitance measurements from WT (a) and Pit1^dw (b) IHCs at different postnatal ages, as indicated. Capacitance was measured in response to a 3-s depolarization to −34 mV from a holding potential of −84 mV. The first component was measured at 200 ms into the step depolarization, and is summarized in (c), and full release is summarized in (d). The number of measurements were as follows for WT and Pit1^dw IHCs, respectively: P2 (6 and 4), P4 (2 and 0), P7 (4 and 3), P14 (6 and 5), and P24 (5 and 5).

Fig. 5

TH treatment from P3 to P8 restores normal synaptic counts in Pit1^dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with RIBEYE (green) and SHANK1 (red) markers at P14 in saline-treated WT mice (a) and Pit1^dw mice (b) treated with TH from P3 to P8. Scale bar: 10 lm. (c–g) Box plots of the quantification of RIBEYE, SHANK1 and RIBEYE–SHANK1 puncta from the mid-turn of the cochlea in saline-treated WT and Pit1^dw mice, and in TH-treated Pit1^dw mice, for the following treatment windows: P4 to P8 (c), P5 to P8 (d), P3 to P8 (e), P3 to P4 (f), and P3 to P6 (g). significant comparisons (P < 0.05) are indicated with asterisks. The dots indicate outliers in the data. (h) Percentage of presynaptic (green) and postsynaptic (red) pruning for the different TH treatment groups with respect to WT controls. The duration of each treatment period is indicated with respect to the entire period examined from P3 to P8.

Fig. 6

Reduced number of afferent postsynaptic counts in young adult Pit1^dw IHCs. (a and b) Projection of confocal sections obtained from the mid-turn of cochlear whole mounts stained with RIBEYE (green) and SHANK1 (red) markers in WT mice (a) and Pit1^dw mice (b) at P42. Scale bar: 10 lm. (c) Box plot of the quantification of RIBEYE, SHANK1 and RIBEYE–SHANK1 puncta from the mid-turn of WT and Pit1^dw cochlea at P42. significant comparisons (P < 0.05) are indicated with asterisks. The dots indicate outliers in the data.

Fig. 7

Swollen afferent type I terminals and reduced GLAST expression in young adult Pit1^dw mice. (a and b) Representative TEM images from the mid-turn of Pit1^dw and WT cochleas at P42. IHC and afferent boutons (aff) are indicated. Scale bar: 2 lm. (c and d) Projections of confocal sections obtained from the mid-turn of cochlear whole mounts co-stained with anti-GLAST (red) and anti-myosin VIIa (green) antibodies in WT and Pit1^dw mice at P14. (e and f) GLAST expression (red) alone in WT and Pit1^dw mice at P14 before TH treatment. (g and h) GLAST expression (red) at P14 in saline-treated WT mice and in TH-treated Pit1^dw mice from P3 to P8. Scale bar: 10 lm.

Fig. 8

A cartoon representing normal and hypothyroid adult IHC synapses. (a) WT IHC. (b) Hypothyroid IHC. (c) Magnification of the box in b. A hypothyroid IHC is characterized by an excess of afferent synapses caused by the abnormal retention of afferent neurite branches and terminals, calcium channels being more widely distributed around the hair cell, and higher glutamate buildup at the synaptic cleft, owing to lower GLAST levels (b and c). In comparison, a normal IHC shows fewer but well-organized afferent terminals and a more clustered pattern of CaV1.3 calcium channel expression at the basal region of the hair cell. a, afferent fibre; e, efferent fibre; r, ribbon synapse.