Microglial peri-somatic abutments classify two novel types of GABAergic neuron in the inferior colliculus.

Samuel David Webb,Llwyd David Orton

doi:10.1111/ejn.15075

Abstract

Emerging evidence suggests functional roles for microglia in the healthy, mature nervous system. However, we know little of the cellular density and ramified morphology of microglia in sensory systems, and even less of their inter-relationship with inhibitory neurons. We therefore conducted fluorescent multi-channel immunohistochemistry and confocal microscopy in guinea pigs of both sexes for Iba1, GAD67, GFAP, calbindin, and calretinin. We explored these markers in the inferior colliculi (IC), which contain sub-regions specialized for different aspects of auditory processing. First, we found that while the density of Iba1+ somata is similar throughout the IC parenchyma, Iba1+ microglia in dorsal cortex are significantly more ramified than those in the central nucleus or lateral cortex. Conversely, Iba1+ ramifications in ventral central nucleus, a region with the highest density of GAD67+ (putative GABAergic) neurons in IC, are longer with fewer ramifications. Second, we observed extensive abutments of ramified Iba1+ processes onto GAD67+ somata throughout the whole IC and developed novel measures to quantify these. Cluster analyses revealed two novel sub-types of GAD67+ neuron that differ in the quantity of Iba1+ somatic abutments they receive. Unlike previous classification schemes for GAD67+ neurons in IC, these clusters are not related to GAD67+ soma size. Taken together, these data demonstrate that microglial ramifications vary between IC sub-regions in the healthy, adult IC, possibly related to the ongoing demands of their niche. Furthermore, Iba1+ abutments onto neuronal somata are a novel means by which GAD67+ neurons can be classified.

Highlights

Inhibition is an essential element of neural processing
Despite the essential role the inferior colliculi (IC) plays in auditory processing, little is known about microglia in the IC, nor how they may vary between sub-regions
Somata with neuronal morphology expressing cytoplasmic calbindin or calretinin were distributed in the outermost regions of the cortices of the IC, matching previous reports (Ouda et al, 2012; Zettel et al, 1997) and in close proximity to glial fibrillary acidic protein (GFAP)+ processes (Figure 1d). These findings demonstrate that many aspects of IC glial organization mirror those reported in other brain regions, with GFAP+ astrocytes forming the glia limitans externa and lining adjacent to blood vessels (Hafidi & Galifianakis, 2003), alongside ionized calcium binding adaptor molecule 1 (Iba1)+ microglia

Summary

| INTRODUCTION

Inhibition is an essential element of neural processing. GABAergic inhibition is prevalent in the auditory system, in the principal auditory midbrain nuclei, the inferior colliculi (IC). Despite the essential role the IC plays in auditory processing, little is known about microglia in the IC, nor how they may vary between sub-regions. Most investigations into sub-types of GABAergic neurons focus on the cells per se, including their morphology, electrophysiological firing characteristics, expression of cytoplasmic calcium binding proteins, and RNA transcriptome. Another approach is to characterize and classify GABAergic neurons based on differences in the afferent peri-somatic inputs they receive (Beebe et al, 2016; Ito et al, 2009). We employed multi-channel fluorescence immunohistochemistry and confocal microscopy and tested our hypotheses that (a) microglia exhibit sub-region-specific morphological adaptations to their niche, and (b) abutments from Iba1+ processes onto GAD67+ somata can be used to classify GAD67+ neuron sub-types in IC

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION

Findings

| CONCLUSION