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

Abstract

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.

Brain-Derived Neurotrophic Factor Is Involved in Activity-Dependent Tonotopic Refinement of MNTB Neurons

“…Several genetically modified mice lacking tonotopic gradients of neuronal structure display auditory function abnormalities. CXCR1 mutant mice, which have disrupted microglia-neuron communication, lacked the soma size tonotopic gradient observed in WT mice and had longer peak latencies of the auditory brainstem responses (ABRs) with the normal threshold of ABRs (Milinkeviciute et al, 2021). Ephrin-A3 mutants, which lack a key axon guidance signaling factor, have degraded frequency-responsiveness tonotopy within the cochlear nucleus.…”

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

“…The mature MNTB contains a cell size gradient, which increases from the most medial (high frequency) to the most lateral (low frequency) regions ( Weatherstone et al, 2017 ; Milinkeviciute et al, 2021a ). Fmr1 KO mice have a delay in the establishment of the cell size gradient across the MNTB mediolateral axis ( Rotschafer et al, 2015 ).…”

“…Fmr1 KO mice have a delay in the establishment of the cell size gradient across the MNTB mediolateral axis ( Rotschafer et al, 2015 ). Calyces also increase in size along the tonotopic axis ( Milinkeviciute et al, 2021a ). Membrane capacitance is correlated with larger synaptic input across the tonotopic axis and time constants are faster in the medial neurons compared to the lateral neurons ( Weatherstone et al, 2017 ).…”

“…Glycine transporter 2 (GLYT2) positive puncta can be detected in the MNTB prior to hearing onset and increase in expression across the mediolateral axis in the adult mouse ( Friauf et al, 1999 ; Altieri et al, 2014 ; Milinkeviciute et al, 2021a ). Loss of the microglial fractalkine receptor Cx3cr1 , an ASD and SZ-linked gene ( Ishizuka et al, 2017 ), disrupts the topographic distribution of GLYT2 in the MNTB, leads to a loss of MNTB neural size gradients and faster signal transmission, as measured by the auditory brainstem response (ABR) ( Ishizuka et al, 2017 ; Milinkeviciute et al, 2021a ). The MNTB in Fmr1 KO mice shows elevated levels of GABA/glycinergic marker vesicular GABA transporter (VGAT) ( Rotschafer et al, 2015 ; Ruby et al, 2015 ).…”

“…When microglia return to control levels following the cessation of treatment, GFAP expression is restored to that of age-matched control mice ( Milinkeviciute et al, 2021b ). Deletion of Cx3cr1, expressed primarily on microglia, leads to an increase in GFAP expression in the MNTB ( Figure 1B ; Milinkeviciute et al, 2021a ). Mice lacking Fmr1 have significantly more astrocytes in the VCN and LSO at P14, but there were no differences in the MNTB at this age, despite the abnormal cell numbers and sizes found in the MNTB at that age ( Rotschafer et al, 2015 ).…”