UC Irvine

Sound source localization recruits intricate neural circuitry that detects a sound’s pitch, loudness, and timing. From there, a listener can determine the sound’s location, distance, and significance and behave accordingly. The auditory brainstem is the first brain region that mediates sound processing and is comprised of specialized synapses that ensure rapid and precise signal transmission. One of the key connections that mediates this process is the calyx of Held, a highly myelinated contralateral projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid body (MNTB). Calyces are large single dominant inputs to MNTB cells, relay cells that provide inhibitory input to the ipsilateral lateral superior olive (LSO). The LSO simultaneously receives excitatory input from ipsilateral VCN. The ratio of excitation and inhibition allows the listener to infer where a sound is located. The development of this pathway is highly regulated and involves multipartite signaling with glial cells. Microglia, the brain’s immune cells, have recently been discovered to have neurodevelopmental roles where they mediate synapse formation, removal, plasticity, and protection. Microglia are multifarious, their functions are correlated to neural region, developmental time periods, and environmental factors. The mouse MNTB contains microglia in the first postnatal week, and microglia peak around hearing onset. Here, I began to determine the roles of microglia during the development of the precise sound localization neural circuit. Our recent work found that temporary microglia ablation through pharmacological inhibition of CSF1R, a receptor essential for microglial survival and proliferation, results in impaired calyceal pruning and delayed astrocyte maturation. We also found that temporary microglial ablation results in abnormal auditory function, as measured by auditory brainstem responses. Mice that temporarily lacked microglia required higher sound intensities for a response to be detected. Remarkably, following cessation of the CSF1R inhibitor, we found that calyceal pruning, astrocyte maturation, and auditory function largely recovered. This finding brought forth a new phenomenon: robust brainstem circuitry can mature outside of the normal developmental timeframe. This led us to the following questions: first, do microglia mediate circuit recovery? Second, what are the microglial attributes during neural circuit repair? To determine the role of microglia during circuit recovery, we permanently removed microglia in the period where recovery was observed. We developed a dual-drug pharmacological microglia depletion method where mice lacked microglia from early postnatal stages through adulthood. We found that long-term CSF1R inhibition prevented calyceal pruning, diminished astrocyte maturation and reactivity markers, impaired inhibitory synapse removal, and led to worsened auditory brainstem responses. Therefore, microglial presence appears to be key in circuit formation and maturation regardless of the developmental period. Next, we sought to characterize microglial characteristics during normal brainstem development and recovery. We examined mice in wildtype untreated postnatal mice as well as mice that temporarily received the CSF1R inhibitor. We found that as microglia colonize the brainstem, they display large cell bodies and stunted branches, characteristic morphology of young and reactive microglia. Returning microglia following CSF1R treatment recapitulated some of these morphological qualities and were distinct from those in age-matched controls during the circuit recovery period. Together, it is apparent that microglia take on some neurodevelopmental properties to ensure formation of auditory brainstem circuitry. Finally, we examined whether a microglial signaling pathway regulates calyx pruning and auditory function. Our recent work found that removal of the microglial fractalkine receptor CX3CR1 does not affect calyceal pruning. However, loss of CX3CR1 led to impairments in the formation of the cell size gradient along the MNTB tonotopic axis, increases in an astrocyte reactivity marker, and reduced or hyperactive peak latencies in the auditory brainstem response. Here, we investigated the effects of microglial C1q on auditory brainstem development, another pathway that in some regions and developmental periods regulates synapse pruning and excitatory/inhibitory ratios. We determined the anatomical qualities of C1q expression in the MNTB before and after hearing onset. We found that genetic ablation of C1q does not affect the establishment of the 1:1 calyx-synapse connection. However, C1q knockout mice showed some reduced peak latencies in the auditory brainstem response as well. Our findings here highlight the heterogeneity of microglia and their signaling pathways during neural circuit development.