Neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNAs.

<p>Fluorescence micrographs (A1, B1) and their magnifications (A2, B2) showing neurons in the rostral ventromedial medulla (RVM) retrogradely labeled with fluorescent microspheres (red) from the lumbar (L4) dorsal horn (<b>A</b>) and the cervical (C5) spinal cord (<b>B</b>), and neurons labeled for GlyT2 and/or GAD67 mRNAs (Gly/GABA neurons, green) using fluorescent in situ hybridization (FISH). Arrowheads indicate Gly/GABA neurons that are also retrogradely labeled from the spinal cord. Note that the fluorescent microspheres and the labeling for Gly/GAD67 mRNA are located in the cytoplasm, outside of the neuronal nuclei. The intensity of the fluorescent labeling varied between the labeled neurons, several of which are also out of focus. Gi: gigantocellular reticular nucleus, alpha part (GiA); LPGi: lateral paragigantocellular reticular nucleus, alpha part (LPGiA), external part (LPGiE); Ml: medial lemniscus; RMg: raphe magnus nucleus. Scale bar: 50 µm.</p

The injection sites of fluorescent microspheres in the rat spinal cord.

<p>Drawings illustrating fluorescent microsphere injection sites in the dorsal and ventral cervical (C5–C6) spinal cord (<b>A</b>), in the dorsal horn (laminae I to VI) (<b>B</b>) and in the ventral horn (laminae VII-X) (<b>C</b>) of the lumbar (L4–L6) spinal cord. Note that all injections were confined to one side, and that in some cases fluorescent tracer was also present the adjacent white matter. Roman figures indicate spinal laminae.</p

Neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNAs.

<p>Fluorescence micrographs showing neurons in the ventromedial medulla retrogradely labeled with fluorescent microspheres (red) from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNA (green) using FISH. Arrowheads indicate GABAergic (GAD67 mRNA containing) neurons in the caudal ventromedial medulla (CVM) retrogradely labeled from the cervical spinal cord (<b>A</b>); glycinergic (GlyT2 mRNA containing) neurons in the rostral ventromedial medulla (RVM) retrogradely labeled from the cervical spinal cord (<b>B</b>); GlyT2 and/or GAD67 mRNA containing neurons (Gly/GABA) in the RVM retrogradely labeled from the lumbar dorsal horn (<b>C</b>); Gly/GABA neurons in the RVM retrogradely labeled from the lumbar ventral horn (<b>D</b>); Gly/GABA neurons in the CVM retrogradely labeled from the lumbar ventral horn (<b>E</b>). Scale bar = 50 µm.</p

Spinal neurons retrogradely labeled from the rostral ventromedial medulla, and spinal neurons labeled for GlyT2 and/or GAD67 mRNAs.

<p>Fluorescence micrographs showing spinal neurons (red) retrogradely labeled with fluorescent microspheres from the rostral ventromedial medulla (RVM) and spinal neurons (green) containing GlyT2 and/or GAD67 mRNA (glycinergic and/or GABAergic neurons) identified with FISH. Arrowheads indicate spinal neurons in the area around the central canal, which are retrogradely labeled from the RVM and are also labeled for GlyT2 mRNA in segment C5 (<b>A</b>) and for GlyT2 and/or GAD67 mRNAs in segments L5 (C) and L6 (<b>B and D</b>). Scale bar = 50 µm.</p

The distribution patterns of neurons in the ventromedial medulla that were retrogradely labeled from the lumbar dorsal or ventral horn.

<p>Drawings illustrating representative single brainstem sections of the rostral part of ventromedial medulla (RVM) and the caudal part of ventromedial medulla (CVM). Black filled circles represent neurons retrogradely labeled with fluorescent microspheres from the lumbar dorsal (<b>A</b>) or ventral (<b>B</b>) horn. Red filled triangles represent retrogradely labeled neurons that also contained GlyT2 and/or GAD67 mRNA (Gly/GABA). Note that the caudal part of the CVM contains fewer retrogradely labeled neurons than the rostral part of the CVM, and that most retrogradely labeled Gly/GABA neurons in both the CVM and RVM are located on the side ipsilateral to the injection. Gi: gigantocellular reticular nucleus, alpha part (GiA); IRt: intermediate reticular nucleus, alpha part (IRtA); LPGi: lateral paragigantocellular reticular nucleus, alpha part (LPGiA), external part (LPGiE); LRt: lateral reticular nucleus; Pn: pontine reticular nucleus, ventral part (PnV) and caudal part (PnC); Py: pyramid tract; Oi: olivary inferior nucleus; RMg: raphe magnus; Tz: nucleus trapezoid body; 7Nuc: nucleus of seventh nerve; 1: raphe pallidus nucleus; 2: raphe obscurus nucleus; 3: raphe interpositus nucleus.</p

The distribution patterns of neurons in the spinal cord that were retrogradely labeled from the rostral ventromedial medulla.

<p>Schematic drawings showing the brainstems of three rats with fluorescent microsphere injections (blue, purple and red) and the corresponding spinal cords with the distribution of retrogradely labeled neurons (filled black circles), and retrogradely labeled spinal neurons also containing GlyT2 and/or GAD67 mRNA (red triangles). Each segmental drawing, i.e. cervical (C4–C8), thoracic (T1–T3 and T10–T12), lumbar (L1–L6) and sacral (S1–S4), shows the cumulative results obtained in 8-10 sections. Tracer injections were aimed at rostral ventromedial medulla (RVM) with injections confined to the midline (purple), to one side only (blue), or both sides of the RVM (red). Note that tracer injections limited to the midline yield fewer retrogradely labeled neurons in the spinal cord (purple), and injections limited to one side of the brainstem, yield more retrogradely labeled neurons on the contralateral side of the spinal cord (blue as compared to purple and red). Roman figures indicate spinal laminae. LSN: lateral spinal nucleus.</p

Mean intensity of the AFI signal (A) and area of excitation (B) following nociceptive electrical stimulation of the sciatic nerve, in naïve versus neuropathic rats (Seltzer model), on the ipsilateral (i) and contralateral (c) side of the nerve injury and nerve stimulation.

<p>Error bars indicate SEM; <i>n</i> = 20 naïve rats, <i>n</i> = 13 neuropathic rats.</p

Effect of 30 minutes SCS or sham stimulation on the intensity of the AFI signal and area of excitation in response to sciatic nerve electrical stimulation, in rats with partial ligation of the proximal sciatic nerve (Seltzer model).

<p>(A,D) Images of background fluorescence of the dorsal surface of the spinal cord at T13 of a sham (A) and SCS treated rat (D). (B,E) Area of excitation (yellow) on the ipsilateral side, directly after sham stimulation (B); after SCS, in this rat, there is no area exceeding the predefined ΔF/F level (E). (C) Time course of the intensity of the AFI signal after SCS or sham stimulation (T = 0 min), as a percentage of ΔF/F before treatment (T = -30 min), in 20×20 pixel square selections on the ipsilateral side at the L4-L6 spinal level. (F) Mean areas of excitation on the ipsilateral side directly after SCS or sham stimulation (T = 0 min), as a percentage of the areas before treatment (T = -30 min). Scale bar, 1 mm; Grayscale bar ranging from −0.75% (black) to +0.75% (white) of the 16-bit range; Error bars indicate SEM; *<i>p</i><0.05; paired <i>t</i>-tests; <i>n</i> = 7 SCS, <i>n</i> = 6 sham stimulation.</p

Behavioral data of rats that underwent partial ligation of the proximal sciatic nerve (Seltzer model).

<p>(A) Combined results of the von Frey withdrawal thresholds and hotplate latencies, at baseline and 10, 12 and 14 days after nerve ligation, from al 18 rats in the study, demonstrating tactile and thermal hyperalgesia. Error bars indicate SEM. <i>p</i><0.01; repeated measures ANOVAs, source of variation timepoint; ***<i>p</i><0.01; pairwise comparisons of day 0 versus day 10, 12 and 14, using Bonferroni correction. (B,C) Von Frey withdrawal thresholds (B) and hotplate latencies (C) from 7 neuropathic rats that subsequently underwent SCS and 6 neuropathic rats that subsequently underwent sham stimulation, demonstrating a similar degree of tactile and thermal hyperalgesia in both groups. Error bars indicate SEM. <i>p</i>>0.06; repeated measures ANOVAs, source of variation treatment.</p