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. 2016 Aug 4;329:171-81.
doi: 10.1016/j.neuroscience.2016.05.009. Epub 2016 May 13.

Immunostaining for Homer Reveals the Majority of Excitatory Synapses in Laminae I-III of the Mouse Spinal Dorsal Horn

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Free PMC article

Immunostaining for Homer Reveals the Majority of Excitatory Synapses in Laminae I-III of the Mouse Spinal Dorsal Horn

Maria Gutierrez-Mecinas et al. Neuroscience. .
Free PMC article

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.

Keywords: confocal microscopy; excitatory interneuron; glutamatergic synapse; pain; primary afferent; spinal cord.

Figures

Fig. 1
Fig. 1
A low magnification view of Homer-immunoreactivity in a transverse section through the dorsal horn. Immunostaining is present throughout the gray matter, but is densest in lamina II. The inset is a higher magnification view of part of lamina II, and shows that the staining is in the form of small puncta that are scattered throughout the neuropil. Both images are from a single optical section. Scale bars: 100 μm (main image) and 5 μm (inset).
Fig. 2
Fig. 2
The ultrastructural appearance of Homer, seen with pre-embedding immunoperoxidase labeling. The DAB reaction product is confined to synapses, where it is always restricted to the postsynaptic aspect. (A) a bouton (b) makes two synapses (arrowheads), both of which show DAB labeling. (B) The central bouton of a type I glomerulus (CI) can be recognized because of its indented contour, lack of mitochondria and the presence of densely packed synaptic vesicles of highly variable size. Three of the synapses (arrowheads) formed by the central bouton are strongly labeled with DAB. (C) The central bouton of a type II glomerulus (CII) can be identified because of its large size and numerous mitochondria. It forms several synapses with adjacent peripheral profiles, and two of these (arrowheads) are clearly DAB-labeled. Scale bar = 0.5 μm.
Fig. 3
Fig. 3
A confocal scan (single optical section) through the middle part of lamina II in a section that had been pepsin-treated and then immunostained with antibodies against Homer and the AMPA receptor GluR2 subunit. (A, B) Homer- and GluR2-immunoreactive puncta are shown in magenta and green, respectively. (C) A merged image. Note that numerous puncta are present (some indicated with arrowheads) and that virtually all of these are stained with both antibodies, although the relative intensity of staining with the two antibodies varies considerably. Scale bar = 5 μm.
Fig. 4
Fig. 4
Confocal scan (single optical section) of a section reacted with antibodies against Homer, gephyrin, VGLUT2 and VGAT. (A) Numerous puncta immunoreactive for Homer (magenta) and gephyrin (green) are present in the neuropil of lamina II, but these are never co-localized. (B) The same field scanned to reveal VGLUT2 (blue) shows that some of the Homer puncta are in contact with VGLUT2-immunoreactive boutons. (C) The same field showing staining for VGAT (blue) reveals that most of the gephyrin puncta are adjacent to VGAT-immunoreactive boutons. Scale bar = 5 μm.
Fig. 5
Fig. 5
Confocal images showing the association between Homer puncta and various types of glutamatergic axon in laminae I–III of the dorsal horn. (A–D) part of lamina I from a section stained to reveal Homer (red), VGLUT2 (green) and CGRP (blue). This field contains a few CGRP-immunoreactive boutons (arrowheads), which are also weakly stained for VGLUT2, and numerous boutons that lack CGRP and show strong VGLUT2 immunoreactivity (some indicated with arrows). The merged image shows that the CGRP-immunoreactive boutons, and many of those with strong VGLUT2, are in contact with Homer puncta. Note that some of the boutons may be in contact with Homer puncta that are above or below the z-stack, and would therefore not be seen in this image. (E–H) A similar set of confocal images from lamina II in a section stained to reveal Homer (red), IB4 binding (green) and CGRP (blue). Although much of the IB4 is bound to intervaricose axons (as judged from the confocal z-stack), some of the labeled profiles are boutons (arrowheads), and each of these is surrounded by numerous Homer puncta. A single CGRP bouton is present (arrow) and this is also in contact with Homer puncta. (I–L) A similar set of confocal images from lamina III in a section stained to reveal Homer (red), VGLUT1 (green) and VGLUT2 (blue). Several large VGLUT1-immunoreactive boutons are visible (some indicated with arrowheads). These show weak VGLUT2 immunoreactivity, and each is associated with numerous Homer puncta. In addition, the field contains a large number of boutons that lack VGLUT1 but show strong VGLUT2 immunoreactivity, and many of these are in contact with Homer puncta. The images were generated from 5 (A–D), 4 (E–H) and 3 (I–L) confocal optical sections at 0.3 μm z-spacing. Scale bar = 5 μm.

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