Microglia Regulate Pruning of Specialized Synapses in the Auditory Brainstem

Abstract

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.

RiboTag-Seq Reveals a Compensatory cAMP Responsive Gene Network in Striatal Microglia Induced by Morphine Withdrawal

“…Microglia are the innate immune cells of the brain but are also involved in intercellular interactions that are not directly related to immune defenses. The processes and filopodia of "resting" microglia constantly engage in surveillance of the parenchyma and are critical for cell-cell interaction, sensing of chemical cues, and are involved in synapse formation and elimination 8,9,30,31 . Microglia have been described as quiescent at baseline, but their interactions with nearby neurons and astrocytes are now recognized to play important roles in the maintenance of normal brain function 7,9,32 .…”

“…Little is known about the interface of opioids, microglia, and neuroinflammation, but it is likely that there are both direct and indirect effects of opioid receptor activation on microglia function. Resting microglia reside in the brain parenchyma, engage in synaptic pruning [7][8][9] and in chemical and mechanical surveillance for the presence of challenges such as infection or neuronal damage. Once engaged, microglia may release a wide range of effector molecules and enzymes, phagocytose debris, remodel nearby synapses, etc.…”

“…However, the broad range of these changes suggests indirect impacts of opioid withdrawal as well. Microglia interact intimately with neuronal synapses and are essential for healthy synaptic formation, pruning, and plasticity 8,30,31 . Morphine administration and withdrawal are known to inversely affect long term depression 33 and have myriad other effects on synaptic activity [34][35][36] .…”

“…Morphine tolerance tended to downregulate synaptic organization genes, while withdrawal upregulated them. While many of these gene sets associated with synaptic processes relate to neurons, microglia play an essential role in synapse formation, maintenance, and elimination 8,30,31 and they also express genes commonly associated with synapse differentiation such as Syndig1 40 . GSEA is explicitly built upon previously interpreted gene associations and many gene sets were originally annotated from transcriptomic studies that used whole tissue homogenates containing glial cells along with neurons, but they may have been interpreted as emanating from neurons.…”

Microglial trogocytosis and the complement system regulate axonal pruningin vivo

“…Constantly surveilling the brain (Nimmerjahn et al, 2005;Wake et al, 2009), these highly phagocytic cells are thought to contribute to developmental synaptic remodeling by phagocytosing inappropriate or supernumerary synapses, a hypothesis that has derived considerable support from histological and immunohistochemical evidence identifying synaptic components within endocytic compartments in microglia (Paolicelli et al, 2011;Schafer et al, 2012;Stevens et al, 2007;Tremblay et al, 2010). This hypothesis is further supported by numerous studies demonstrating that microglia depletion leads to exuberant axonal outgrowth (Pont-Lezica et al, 2014;Squarzoni et al, 2014), impaired pruning of excess synapses (Ji et al, 2013;Milinkeviciute et al, 2019) and increased spine density (Wallace et al, 2020) during development.…”