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

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.

Thrombospondins 1 and 2 are important for afferent synapse formation and function in the inner ear.

Thrombospondins (TSPs) constitute a family of secreted extracellular matrix proteins that have been shown to be involved in the formation of synapses in the central nervous system. In this study, we show that TSP1 and TSP2 are expressed in the cochlea, and offer the first description of their putative roles in afferent synapse development and function in the inner ear. We examined mice with deletions of TSP1, TSP2 and both (TSP1/TSP2) for inner ear development and function. Immunostaining for synaptic markers indicated a significant decrease in the number of formed afferent synapses in the cochleae of TSP2 and TSP1/TSP2 knockout (KO) mice at postnatal day (P)29. In functional studies, TSP2 and TSP1/TSP2 KO mice showed elevated auditory brainstem response (ABR) thresholds as compared with wild-type littermates, starting at P15, with the most severe phenotype being seen for TSP1/TSP2 KO mice. TSP1/TSP2 KO mice also showed reduced wave I amplitudes of ABRs and vestibular evoked potentials, suggesting synaptic dysfunction in both the auditory and vestibular systems. Whereas ABR thresholds in TSP1 KO mice were relatively unaffected at early ages, TSP1/TSP2 KO mice showed the most severe phenotype among all of the genotypes tested, suggesting functional redundancy between the two genes. On the basis of the above results, we propose that TSPs play an important role in afferent synapse development and function of the inner ear.

TSP1 and TSP2 Have Unique and Overlapping Roles in Protecting against Noise-Induced Auditory Synaptopathy.

Thrombospondins (TSPs) are cell adhesion molecules that play an important role in the maintenance of hearing and afferent synaptic connections. Based on their reported function in restoring synaptic connections after stroke, we tested a potential role for TSP1 and TSP2 genes in repairing cochlear synapses following noise injury. We observed a tonotopic gradient in the expression of TSP1 and TSP2 mRNA in control mouse cochleae and an upregulation of these genes following noise exposure. Examining the functional sequelae of these changes revealed that afferent synaptic counts and auditory brainstem responses (ABRs) in noise-exposed TSP1 and TSP2 knockout (-/-) mice exhibited a worst recovery when compared to controls. Consistent with their tonotopic expression, TSP1-/- mice showed greater susceptibility to noise-induced hearing loss (NIHL) at 8 kHz and 16 kHz frequencies, whereas NIHL in TSP2-/- mice occurred only at mid and high frequencies. Further analysis of the ABR waveforms indicated peripheral neuronal damage in TSP2-/- but not in TSP1-/- mice. Noise trauma affecting mid to high frequencies triggered severe seizures in the TSP2-/- mice. We found that decreased susceptibility to audiogenic seizures in TSP1-/- mice was correlated with increased TSP2 protein levels in their inner ears, suggesting that TSP2 might functionally compensate for the loss of TSP1 in these mice. Our data indicate that TSP1 and TSP2 are both involved in susceptibility to NIHL, with TSP2 playing a more prominent role.

A lack of immune system genes causes loss in high frequency hearing but does not disrupt cochlear synapse maturation in mice.

Early cochlear development is marked by an exuberant outgrowth of neurites that innervate multiple targets. The establishment of mature cochlear neural circuits is, however, dependent on the pruning of inappropriate axons and synaptic connections. Such refinement also occurs in the central nervous system (CNS), and recently, genes ordinarily associated with immune and inflammatory processes have been shown to play roles in synaptic pruning in the brain. These molecules include the major histocompatibility complex class I (MHCI) genes, H2-K(b) and H2-D(b), and the complement cascade gene, C1qa. Since the mechanisms involved in synaptic refinement in the cochlea are not well understood, we investigated whether these immune system genes may be involved in this process and whether they are required for normal hearing function. Here we report that these genes are not necessary for normal synapse formation and refinement in the mouse cochlea. We further demonstrate that C1qa expression is not necessary for normal hearing in mice but the lack of expression of H2-K(b) and H2-D(b) causes hearing impairment. These data underscore the importance of the highly polymorphic family of MHCI genes in hearing in mice and also suggest that factors and mechanisms regulating synaptic refinement in the cochlea may be distinct from those in the CNS.