Long-term microglia depletion impairs synapse elimination and auditory brainstem function.

Specialized sound localization circuit development requires synapse strengthening, refinement, and pruning. Many of these functions are carried out by microglia, immune cells that aid in regulating neurogenesis, synaptogenesis, apoptosis, and synaptic removal. We previously showed that postnatal treatment with BLZ945 (BLZ), an inhibitor of colony stimulating factor 1 receptor (CSF1R), eliminates microglia in the brainstem and disables calyceal pruning and maturation of astrocytes in the medial nucleus of the trapezoid body (MNTB). BLZ treatment results in elevated hearing thresholds and delayed signal propagation as measured by auditory brainstem responses (ABR). However, when microglia repopulate the brain following the cessation of BLZ, most of the deficits are repaired. It is unknown whether this recovery is achievable without the return of microglia. Here, we induced sustained microglial elimination with a two-drug approach using BLZ and PLX5622 (PLX). We found that BLZ/PLX treated mice had impaired calyceal pruning, diminished astrocytic GFAP in the lateral, low frequency, region of MNTB, and elevated glycine transporter 2 (GLYT2) levels. BLZ/PLX treated mice had elevated hearing thresholds, diminished peak amplitudes, and altered latencies and inter-peak latencies. These findings suggest that microglia are required to repopulate the brain in order to rectify deficits from their ablation.

Synapse Maturation and Developmental Impairment in the Medial Nucleus of the Trapezoid Body.

Sound localization requires rapid interpretation of signal speed, intensity, and frequency. Precise neurotransmission of auditory signals relies on specialized auditory brainstem synapses including the calyx of Held, the large encapsulating input to principal neurons in the medial nucleus of the trapezoid body (MNTB). During development, synapses in the MNTB are established, eliminated, and strengthened, thereby forming an excitatory/inhibitory (E/I) synapse profile. However, in neurodevelopmental disorders such as autism spectrum disorder (ASD), E/I neurotransmission is altered, and auditory phenotypes emerge anatomically, molecularly, and functionally. Here we review factors required for normal synapse development in this auditory brainstem pathway and discuss how it is affected by mutations in ASD-linked genes.

CX3CR1 mutation alters synaptic and astrocytic protein expression, topographic gradients, and response latencies in the auditory brainstem.

The precise and specialized circuitry in the auditory brainstem develops through adaptations of cellular and molecular signaling. We previously showed that elimination of microglia during development impairs synaptic pruning that leads to maturation of the calyx of Held, a large encapsulating synapse that terminates on neurons of the medial nucleus of the trapezoid body (MNTB). Microglia depletion also led to a decrease in glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. Here, we investigated the role of signaling through the fractalkine receptor (CX3CR1), which is expressed by microglia and mediates communication with neurons. CX3CR1-/- and wild-type mice were studied before and after hearing onset and at 9 weeks of age. Levels of GFAP were significantly increased in the MNTB in mutants at 9 weeks. Pruning was unaffected at the calyx of Held, but we found an increase in expression of glycinergic synaptic marker in mutant mice at P14, suggesting an effect on maturation of inhibitory inputs. We observed disrupted tonotopic gradients of neuron and calyx size in MNTB in mutant mice. Auditory brainstem recording (ABR) revealed that CX3CR1-/- mice had normal thresholds and amplitudes but decreased latencies and interpeak latencies, particularly for the highest frequencies. These results demonstrate that disruption of fractalkine signaling has a significant effect on auditory brainstem development. Our findings highlight the importance of neuron-microglia-astrocyte communication in pruning of inhibitory synapses and establishment of tonotopic gradients early in postnatal development.

Auditory Brainstem Deficits from Early Treatment with a CSF1R Inhibitor Largely Recover with Microglial Repopulation.

Signaling between neurons and glia is necessary for the formation of functional neural circuits. A role for microglia in the maturation of connections in the medial nucleus of the trapezoid body (MNTB) was previously demonstrated by postnatal microglial elimination using a colony stimulating factor 1 receptor (CSF1R). Defective pruning of calyces of Held and significant reduction of the mature astrocyte marker glial fibrillary acidic protein (GFAP) were observed after hearing onset. Here, we investigated the time course required for microglia to populate the mouse MNTB after cessation of CSF1R inhibitor treatment. We then examined whether defects seen after microglial depletion were rectified by microglial repopulation. We found that microglia returned to control levels at four weeks of age (18 d postcessation of treatment). Calyceal innervation of MNTB neurons was comparable to control levels at four weeks and GFAP expression recovered by seven weeks. We further investigated the effects of microglia elimination and repopulation on auditory function using auditory brainstem recordings (ABRs). Temporary microglial depletion significantly elevated auditory thresholds in response to 4. 8, and 12 kHz at four weeks. Treatment significantly affected latencies, interpeak latencies, and amplitudes of all the ABR peaks in response to many of the frequencies tested. These effects largely recovered by seven weeks. These findings highlight the functions of microglia in the formation of auditory neural circuits early in development. Further, the results suggest that microglia retain their developmental functions beyond the period of circuit refinement.

Microglia Regulate Pruning of Specialized Synapses in the Auditory Brainstem.

The assembly of uniquely organized sound localization circuits in the brainstem requires precise developmental mechanisms. Glial cells have been shown to shape synaptic connections in the retinogeniculate system during development, but their contributions to specialized auditory synapses have not been identified. Here we investigated the role of microglia in auditory brainstem circuit assembly, focusing on the formation and pruning of the calyx of Held in the medial nucleus of the trapezoid body (MNTB). Microglia were pharmacologically depleted in mice early in development using subcutaneous injections of an inhibitor of colony stimulating factor 1 receptor, which is essential for microglia survival. Brainstems were examined prior to and just after hearing onset, at postnatal days (P) 8 and P13, respectively. We found that at P13 there were significantly more polyinnervated MNTB neurons when microglia were depleted, consistent with a defect in pruning. Expression of glial fibrillary acidic protein (GFAP), a mature astrocyte marker that normally appears in the MNTB late in development, was significantly decreased in microglia-depleted mice at P13, suggesting a delay in astrocyte maturation. Our results demonstrate that monoinnervation of MNTB neurons by the calyx of Held is significantly disrupted or delayed in the absence of microglia. This finding may reflect a direct role for microglia in synaptic pruning. A secondary role for microglia may be in the maturation of astrocytes in MNTB. These findings highlight the significant function of glia in pruning during calyx of Held development.