Immunostaining for Homer reveals the majority of excitatory synapses in laminae I–III of the mouse spinal dorsal horn

Maria Gutierrez-Mecinas,Emily D Kuehn,Victoria E Abraira,Erika Polgár,Masahiko Watanabe,Andrew J Todd

doi:10.1016/j.neuroscience.2016.05.009

Maria Gutierrez-Mecinas, Emily D Kuehn + Show 4 more

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https://doi.org/10.1016/j.neuroscience.2016.05.009

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Abstract

The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I–III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states.

Highlights

The dorsal horn of the spinal cord is innervated by primary afferents that terminate in a highly ordered lamina-specific pattern (Todd, 2010; Abraira and Ginty, 2013; Braz et al, 2014)
Immunostaining with the Homer antibody appeared as small puncta of varying size and intensity. These were present throughout the spinal gray matter, but were densest in lamina II (Fig 1). This distribution resembled that seen with antibodies against the GluR2 subunit of the AMPAr or PSD-95 following antigen retrieval with pepsin (Nagy et al, 2004a; Polgar et al, 2008), Homer could be readily detected without pepsin treatment
The GluR2 subunit of the AMPA receptor is thought to be present at virtually all excitatory synapses in laminae I–III of the dorsal horn. This assumption is based on studies of rat dorsal horn involving antigen retrieval with pepsin, in which we found that 99% of puncta that were labeled with an antibody that recognizes all 4 subunits of the AMPA receptor were GluR2immunoreactive, and that 98% of puncta labeled with antibody against the major postsynaptic density protein PSD-95 were pan-AMPAr-immunoreactive (Polgar et al, 2008)

Summary

Introduction

The dorsal horn of the spinal cord is innervated by primary afferents that terminate in a highly ordered lamina-specific pattern (Todd, 2010; Abraira and Ginty, 2013; Braz et al, 2014). These contribute to complex synaptic circuits that involve spinal projection neurons, local interneurons and axons that descend from the brain. All primary afferents and some descending axons use glutamate as their principal fast transmitter, and glutamatergic synapses in the dorsal horn can originate from a variety of sources

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