Fig 1.
Detection of ecto-ATPase activity in trigeminal nerves.
A: Diagram illustrating the trigeminal nerve system. B: Histochemical staining for ecto-ATPase activity in trigeminal ganglia. B1: Ecto-ATPase activity is detected in both trigeminal ganglia (TG) neurons (solid triangles) as well as in nerve fibers (arrows); B2: Ecto-ATPase activity is detected in both large (solid triangles) and small (open triangle) TG neurons. C: Histochemical staining for ecto-ATPase activity in trigeminal nerves trunks. Ecto-ATPase activity is detected in trigeminal nerve fibers projecting to the brainstem (arrows). Scale bars: 10 μm for B1, B2 and C respectively.
Fig 2.
Ecto-NTPDase3 is expressed in TG neurons.
A: Ecto-NTPDase3 is detected in TG neurons. Immunostaining for NTPDase3 (A1) is co-localized with the neuronal marker MAP2 (A2) in TG neurons (arrows in A1) and their axonal fibers (white triangles in merged image in A3. B: Ecto-NTPDase3 is detected in trigeminal nerve trunks projecting to the brainstem. Immunostaining for NTPDase3 (B1) is co-localized with the neuronal marker MAP2 (B2) in nerve fibers (white triangles in merged image in B3) as well as in a cluster of TG neurons (arrows in B1) within the nerve trunk projecting to the brainstem. C: Ecto-NTPDase3 is detected in TG nociceptive neurons. Immunostaining for NTPDase3 (C1) is co-localized with the nociceptive neuronal marker IB4 (C2) in TG nociceptive neurons (arrows in C1) and their axonal nerve fibers (white triangles in merged image in C3). Scale bars: 15, 15 and 15μm for A-C, respectively.
Fig 3.
Detection of ecto-ATPase activity in the trigeminal subnucleus caudalis of the brainstem.
A: Diagram illustrating the trigeminal subnucleus caudalis (TSNC) of the brainstem. B: Ecto-ATPase activity is detected in the trigeminal subnucleus caudalis corresponding to the region (a) marked in A. C: Ecto-ATPase activity in the trigeminal subnucleus caudalis corresponding to the region (b) marked in A. D: Ecto-ATPase activity in the corresponding region (c) marked in B. Note: For panels B-C, ecto-ATPase activity is mainly located at the outermost layer of the trigeminal subnucleus caudalis (black triangles). Ecto-ATPase activity is also detected in large blood vessels in the brainstem (white triangles). Scale bars: 50, 50 and 20μm for B-D respectively.
Fig 4.
Expression of ecto-NTPDase3 in trigeminal subnucleus caudalis.
A: Ecto-NTPDase3 is expressed in the outermost layer of the trigeminal subnucleus caudalis. A1: Immunostaining for ecto-NTPDase3 is detected in the brainstem at the lower level of the trigeminal subnucleus caudalis. A2: Co-staining with neuronal marker MAP2 shows the outline of the trigeminal subnucleus caudalis. A3: Merged image highlights that NTPDase3 is expressed within the outermost layer of the trigeminal subnucleus caudalis. Scale bar: 40μm. B: Ecto-NTPDase3 is expressed in the nociceptive lamina of the trigeminal subnucleus caudalis. B1: Immunostaining of ecto-NTPDase3 in the brainstem at the lower level of the trigeminal subnucleus caudalis. B2: Co-staining with the nociceptive neuronal marker IB4 in the brainstem demonstrates the location of the nociceptive lamina of the trigeminal subnucleus caudalis. B3: Merged image of ecto-NTPDase3 and IB4 staining shows that NTPDase3 is expressed in the nociceptive lamina of the trigeminal subnucleus caudalis. Scale bar: 50μm.
Fig 5.
Ecto-NTPDase3 mediates ATP degradation in the nociceptive lamina of the spinal cord dorsal horn and trigeminal subnucleus caudalis.
A: Enzymatic histochemistry detection of ecto-ATPase activity in the spinal cord. Positive staining for ecto-ATPase activity is detected in dorsal root nerve (arrows) and in the outermost layer (nociceptive lamina) of the dorsal horn. Ecto-ATPase activity is also detected in large blood vessels (open triangles) in the spinal cord. B: Ecto-ATPase activity is detected in dorsal root nerve fibers that project to the nociceptive lamina of the dorsal horn. Arrows indicate the positive ecto-ATPase activity staining in dorsal root nerve fibers that penetrate the surface of the spinal cord and project to the nociceptive lamina of the dorsal horn. C: Pre-incubation with specific anti-NTPDase3 serum reduces ecto-ATPase activity staining in the spinal cord dorsal horn. C1: Ecto-ATPase activity staining in spinal cord sections incubated with either control serum (left) or anti-NTPDase3 serum (right). The intensity of ecto-ATPase activity staining in the dorsal horn nociceptive lamina is reduced after incubation with anti-NTPDase3 serum. C2: Changes of normalized ecto-ATPase activity staining intensity in sections after incubation with either control or anti-NTPDase3 serum. The staining intensity for ecto-ATPase activity either in the dorsal horn nociceptive lamina or blood vessels was normalized by the background insensitivity. Unpaired student’s t-test, n = 4, **p<0.01. D: Pre-incubation with anti-NTPDase3 serum reduces the ecto-ATPase activity staining in nociceptive outermost layer of the trigeminal subnucleus caudalis. Enzymatic histochemical staining for ecto-ATPase activity in sections after incubation with either control serum (left) or anti-NTPDase3 serum (right). The intensity of ecto-ATPase activity staining in nociceptive outermost layer of the trigeminal subnucleus caudalis is reduced after incubation with anti-NTPDase3 serum. Note the intensity of ecto-ATPase activity staining in large blood vessels is not reduced, but is even enhanced after incubation with anti-NTPDase3 serum.