Article Figures & Data
Figures
- Figure 1.
Anterograde tracing of C2 DRG peripheral axons to the posterior dura (extracranial component). A, Illustration of the rat's skull view from behind showing intra-ganglionar injection of rAAV-GFP viral vector into C2 DRG. B, Transversal view of a C2 DRG section showing GFP expression in successfully transduced cells (green, left), some of which were also immunoreactive to CGRP (red, middle). Superimposition of these images and DAPI (blue) is displayed at the right with white arrowheads indicating double-labeled cells (yellow). C, Cross-section of the whole neck at the level of C1 vertebra showing the distribution and trajectories of GFP-labeled nerves and fibers traveling along blood vessels (BV), fascia, and muscle from C2 DRG to the cranium. The image shows the left dorsolateral quadrant of the neck and was created by stitching 268 high-resolution images into a single composite. Numbers and frames indicate the areas where the images displayed at the right and bottom were taken. The only not-numbered image was taken from another cross-section of the neck in the same animal. D, Cervical nerves and bundles of fibers expressing GFP were observed crossing neck muscles, traveling along blood vessels and fascia before entering the cranium in the occipital region through (E) a canal between the occipital bone (occ) and the periotic capsule (pcap) (1), emissary canals near the occipital condyle (2), the hypoglossal canal (3), foramen magnum (fmag) (4), and jugular foramen (5). Interparietal bone (ipar), parietal bone (par). Scale bars, 100 μm.
- Figure 2.
Anterograde tracing of C2 DRG peripheral axons in the posterior dura (intracranial component). A, Flat mount of the dura overlying the cerebellum showing GFP-labeled axons from C2 DRG spreading within the dura immediately after entering the cranium. These GFP-labeled dural axons were immunoreactive to CGRP (red) as evidenced in the superimposition of images at the bottom, where white arrowheads indicate double labeling of these axons (yellow) in the DAPI (blue) counterstained dura. B, Representative examples of GFP-labeled axons spreading extensively within the posterior dura overlying the cerebellum (left), large intra-cranial blood vessels (right), and (C) the dura covering the upper cervical segments of the spinal cord. D, Reconstruction of the GFP-labeled axons shown in A–C. Inset, Illustration of the rat brain showing the region of interest. Scale bars, 100 μm.
- Figure 3.
Retrograde labeling in C2 DRG from the posterior dura. A, Illustration of the rat's skull view from behind showing the area of FG application (green) on the posterior dura. B, Transversal view of a C2 DRG section showing retrogradely-labeled neurons filled with FG near the anterior edge of the left ganglion. C, A proportion of FG-labeled neurons (green) in C2 DRG was also immunoreactive to CGRP, TRPV1, or IB4 (red). Images in the left column were created by superimposition of the images in the right column. Filled arrowheads point to FG-positive cells. Open arrowheads indicate double-labeled cells with FG and with each of the three markers of sensory neurons used (yellow). Scale bars, 100 μm.
- Figure 4.
Electrophysiological characterization of posterior dura-sensitive neurons in C2–C4 dorsal horn. A, Illustration of the rat's skull, exposed posterior dura, neck muscles, and skin showing the mapping of all RFs obtained from a single dura-sensitive neuron at baseline. B, Identification of C2–C4 neurons responding to electrical and mechanical stimulation of the posterior dura, neck muscle, and skin.
- Figure 5.
Schematic representation of C2 to C4 spinal cord segments showing the recording sites marked by electrolytic lesions made at the end of each experiment. Red dots represent recording sites where neurons displaying sensitized responses were found. An example of these lesions in C2 spinal cord segment is shown in the dark-field image at the right. Scale bar, 500 μm.
- Figure 6.
RFs and neuronal responses to mechanical stimulation of the posterior dura. A, Illustration of the rat's skull showing the area of exposition of the posterior dura (gray) and dural RFs of C2–C4 spinal cord dura-sensitive neurons measured during mechanical stimulation with VFH at baseline (dark blue) and 2 h after application of IS (light blue). B, C, Responses of individual neurons to mechanical stimulation of the posterior dura before (B) and after IS (C). Neurons were classified as sensitized (red) if their response magnitude increased by at least 50% after IS. Numbers in parenthesis represent firing rate in mean spikes/s during baseline (10 s) and stimulation (10 s). Shaded areas indicate the period of mechanical stimulation.
- Figure 7.
RFs and neuronal responses to mechanical stimulation of neck muscles. A, Illustration of superficial and deep group of neck muscles showing the most sensitive area (blue) to mechanical stimulation with VFH. B, C, Responses of individual neurons to mechanical stimulation of muscle RFs before (B) and after IS (C). Neurons were classified as sensitized (red) if their response magnitude increased by at least 50% after IS. Numbers in parenthesis represent firing rate in mean spikes/s during baseline (10 s) and stimulation (10 s). Shaded areas indicate the period of mechanical stimulation.
- Figure 8.
RFs and neuronal responses to innocuous and noxious mechanical stimulation of the skin. A, Illustration of the rat's head and neck showing the cutaneous RFs of C2–C4 spinal cord dura-sensitive neurons measured during mechanical stimulation at baseline (dark blue) and 2 h after application of IS (light blue). B, Responses of individual neurons to mechanical stimulation (brush, pressure, and pinch) of the skin before (left columns) and after IS (right columns). Neurons were classified as sensitized (red) if their response magnitude increased by at least 50% after IS. Numbers in parenthesis represent firing rate in mean spikes/s during baseline (10 s) and stimulation (10 s). Shaded areas indicate the period of mechanical stimulation.
- Figure 9.
Neuronal responses of sensitized neurons to mechanical stimulation of (A) the posterior dura, (B) neck muscle, and (C) skin (brush, pressure, and pinch) before (green) and 2 h after application of IS (red). Thick lines and shadowed areas represent mean and SE, respectively.
