Galanin-immunoreactivity identifies a distinct population of inhibitory interneurons in laminae I-III of the rat spinal cord

Abstract

Background: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III.

Results: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted ~7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in ~6% of GABAergic boutons in laminae I-IIo, and ~1% of those in laminae IIi-III.

Conclusions: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina II.

Figure 1

Galanin immunoreactivity in laminae I-III. A confocal image taken from a transverse section of the L3 segment that had been reacted to reveal galanin. The solid line represents the outer limit of the grey matter, and the dashed lines show the borders between laminae I, II and III. There is a dense plexus of immunoreactive axons in lamina I and the outer part of lamina II, with some staining in deeper parts of the dorsal horn. Scattered immunoreactive cell bodies are visible, and three of these are marked with arrows. The insets show the two regions outlined by boxes in the main figure, and have been enlarged to show the immunoreactive cell bodies more clearly. The image is a projection of 6 optical sections at 1 μm z-spacing. Scale bar = 100 μm.

Figure 2

The distribution of cells that were galanin or parvalbumin immunoreactive in sections reacted to reveal galanin, parvalbumin and nNOS. The diagram shows the laminar location of all of the cells that were galanin (blue) or parvalbumin (red) immunoreactive in the 6 sections that were analysed (2 each from 3 rats). For the galanin cells, those that were also nNOS immunoreactive are shown as filled circles, while those that lacked nNOS are open circles. None of the parvalbumin cells contained nNOS, and there was no colocalisation of galanin and parvalbumin.

Figure 3

Lack of co-localisation of galanin, nNOS and parvalbumin in lamina II. a Confocal image showing part of lamina II that has been scanned to reveal galanin (Gal, red). Two galanin-containing cell bodies are visible (arrowheads). b, c The same field scanned for nNOS (green) and parvalbumin (PV, blue). In each case, immunoreactive cell bodies can be seen. d The merged image shows the lack of co-localisation of the three types of immunoreactivity in the cell bodies. The images were obtained from a single optical section. Scale bar = 20 μm.

Figure 4

Co-localisation of galanin and nNOS in a lamina III neuron. a Confocal image shows a lamina III neuron that is immunoreactive for nNOS (green). b The same field scanned for galanin (Gal, magenta) shows that the same cell is weakly immunoreactive. c A merged image. The images are from three optical sections at 2 μm z-spacing. Scale bar = 20 μm.

Figure 5

Lack of co-localisation of galanin and NPY. a, b, c show a confocal image from laminae I-IIo scanned for galanin (Gal, red), NPY (green) and the neuronal marker NeuN (blue), respectively. d A merged image. One of the NeuN-positive cell bodies is galanin-immuno-reactive (arrow) and another is NPY-immunoreactive (arrowhead). The image is a projection of 3 optical sections at 1 μm z-separation. Scale bar = 20 μm.

Figure 6

Lack of co-localisation of NPY and nNOS. A single confocal optical section through lamina II, scanned to reveal: a NPY (green) and b nNOS (magenta). A merged image is shown in c. This field contains cell bodies that are immunoreactive for NPY (arrowhead) or nNOS, but the two types of immunoreactivity are not co-localised. Scale bar = 20 μm.

Figure 7

Staining for galanin, NeuN and Sytox in a section used for stereological analysis. a A single confocal optical section through laminae I and II scanned to reveal galanin (Gal, red). b The same field scanned for NeuN (blue), and the nuclear stain Sytox (green). c A merged image. Neuronal nuclei can be recognised by the presence of both NeuN and Sytox staining, and therefore appear cyan, while non-neuronal nuclei are green. Two of the neurons (arrowheads) are immunoreactive for galanin. This appears in a ring surrounding the nucleus, corresponding to the perikaryal cytoplasm. Scale bar = 20 μm.

Figure 8

Co-localisation of galanin with CGRP and VGAT. a, b, c A confocal image through part of lamina II scanned for galanin (Gal, green), CGRP (red) and VGAT (blue), respectively. d A merged image. The small immunoreactive structures correspond to axonal boutons. Several Gal immunoreactive profiles can be seen and most of these are also labelled with the CGRP antibody (two indicated with arrowheads). These appear yellow in d. Two of the galanin-positive boutons (arrows) are also immunostained for VGAT. The image was obtained from 2 optical sections at 0.3 μm z-separation. Scale bar = 5 μm.