Differential distribution of diacylglycerol lipase-alpha and N-acylphosphatidylethanolamine-specific phospholipase d immunoreactivity in the superficial spinal dorsal horn of rats

Abstract

It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGL-α) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGL-α and NAPE-PLD showed a remarkably different distribution. DGL-α immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGL-α and NAPE-PLD. Glial processes immunostained for DGL-α were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGL-α, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release 2-AG and anandamide in the superficial spinal dorsal horn. The 2-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from nonsynaptic dendritic and glial compartments.

Figure 1

The distribution of DGLα and NAPE-PLD immunoreactivity in the spinal dorsal horn. a and b, Photomicrograph showing immunoreactivity for DGLα in the spinal dorsal horn of rats following immunoperoxidase (a) and immunofluorescence (b) staining. We observed an abundant punctuate immunoreactivity for DGLα. Lamina II of the superficial spinal dorsal horn appeared as a heavily stained band, whereas lamina I was more sparsely stained. Besides the characteristic punctuate labeling, larger immunoreactive spots resembling somata of neurons or glial cells were also scattered throughout both the gray and white matters. c and d, Photomicrograph showing immunoreactivity for NAPE-PLD in the dorsal horn of rat’s spinal cord following immunoperoxidase (c) and immunofluorescence (d) staining. Generally, we observed a homogeneous punctuate immunostaining for NAPE-PLD, but larger NAPE-PLD immunoreactive spots resembling somata of neurons or glial cells were also seen both in the gray and white matter. Note that the immunoperoxidase and immunofluorescent stainings show very similar patterns of immunoreactivity. Scale bar: 100 μm.

Figure 2

Co-localization of DGLα and NAPE-PLD with axonal markers. Micrographs of single 1μm thick laser scanning confocal optical sections assessing co-localization between immunolabeling for DGLα (red; a, c, e, g) or NAPE-PLD (red; b, d, f, h) and immunoreactivity for markers that are specific for axon terminals of specific populations of peptidergic (CGRP, green; a, b) and non-peptidergic (IB4-binding, green; c, d) nociceptive primary afferents, as well as for axon terminals of putative excitatory (VGLUT2, green; e, f) and inhibitory (GAD65/67, green; g, h) intrinsic neurons in the superficial spinal dorsal horn. Note that mixed colors (yellow) that may indicate double labeled structures are not observed on the superimposed images. Scale bar: 2 μm.

Figure 3

The degree of co-localization of DGLα and NAPE-PLD with axonal and glial markers. Histograms showing the degree of co-localization between immunoreactivity for DGLα or NAPE-PLD and selected axonal and glial markers in laminae I–II of the spinal dorsal horn. a) Percentage of profiles immunoreactive for the applied axonal and glial markers that were found to be labeled also for DGLα or NAPE-PLD. b) Percentage of profiles immunoreactive for DGLα or NAPE-PLD that were found to be labeled also for the applied axonal and glial markers. Data are shown as mean ± SEM.

Figure 4

Co-localization of DGLα and NAPE-PLD with glial markers. Micrographs of single 1.6 μm thick laser scanning confocal optical section (compressed images of three consecutive 1 μm thick optical sections with 0.3 μm separation in the Z axis) illustrating the co-localization between immunolabeling for DGLα (green; b) and immunoreactivity for a marker which is specific for astrocytes (GFAP, red; a); between immunolabeling for DGLα (green; e) and immunoreactivity for a marker which is specific for microglial cells (CD11b, red; d); between immunolabeling for NAPE-PLD (green; h) and immunoreactivity for a marker which is specific for astrocytes (GFAP, red; g); between immunolabeling for NAPE-PLD (green; k) and immunoreactivity for a marker which is specific for microglial cells (CD11b, red; j) in the superficial spinal dorsal horn. Mixed colors (yellow) on the superimposed image (c, f, i, l) indicate double labeled spots within the illustrated glial processes. Puncta immunoreactive for DGLα or NAPE-PLD which are also stained for the glial marker are marked with arrowheads. Scale bar: 2 μm.

Figure 5

Co-localization between NAPE-PLD and GFAP illustrated in X-Y, X-Z and Y-Z projections of confocal optical sections. Micrographs of a singe1.6 μm thick laser scanning confocal optical section, shown also in Figure 6i, illustrating X-Y, X-Z and Y-Z projections of the optical section double immunostained for GFAP (red) and NAPE-PLD (green). The points of co-localization between the two markers are at the crossing point of two lines indicating the planes through which orthogonal views of X-Z and Y-Z projections were drawn. The X-Z and Y-Z images of puncta 1, 2, 3 on insert a are identified by the serial numbers of the puncta beside and above the X-Z and Y-Z projections, respectively. Insert b shows a part of insert a (without the lines indicating the planes of the orthogonal views) at the site of the NAPE-PLD immunoreactive puncta. According to the orthogonal images, as it is indicated by the mixed color (yellow), NAPE-PLD immunostained puncta 1 and 2 are within the confines of the GFAP immunoreactive profile (see the mixed color (yellow) on all the three projections). However, in case of immunoreactive punctum 3, although there appears to be some overlap between the two markers indicated by the mixed color (yellow) on the X-Y and X-Z projections, the green color on the Y-Z projectional image clearly shows that the NAPE-PLD immunoreactive punctum is not within but adjacent to the GFAP immunoreactive profile.

Figure 6

Ultrastructural localization of DGLα immunoreactivity in neurons and glial cells. Electron micrographs of preembedding immunoperoxidase (a–e) and nanogold (f–h) stained sections showing the distribution of DGLα on postsynaptic dendrites (a–c, f–h) establishing synaptic contacts with axon terminals, in an axon terminal (d) and in glial profiles (a, b, e) in laminae I–II of the spinal dorsal horn. Immunoprecipitates and silver particles labeling DGLα in dendrites and axon terminal aligned along the perisynaptic surface membrane. Immunoprecipitates labeling DGLα in glial cells are also aligned along the surface membrane of glial processes. In some glial processes, the immunolabeled membrane compartments are in close vicinity to synapses (a, b). a: axon terminal, d: postsynaptic dendrite, asterisk: glial profile. Arrows point at immunoperoxidase deposits and silver intensified nanogold particles. Bars: 0.5 μm.

Figure 7

Ultrastructural localization of NAPE-PLD immunoreactivity in neurons and glial cells. Electron micrographs of preembedding immunoperoxidase stained sections showing the distribution of NAPE-PLD on dendrites (a, b, d), in an axon terminal (c) and in glial profiles (e, f) in laminae I–II of the spinal dorsal horn. Immunoprecipitates labeling NAPE-PLD in dendrites, axon terminal and glial processes are aligned along the surface membrane. a: axon terminal immunoreactive for NAPE-PLD, d: postsynaptic dendrite, asterisk: glial profile. Arrows point at immunoperoxidase deposits. Bars: 0.5 μm.