Figure 1.
Neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNAs.
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 (A) and the cervical (C5) spinal cord (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.
Figure 2.
The injection sites of fluorescent microspheres in the rat spinal cord.
Drawings illustrating fluorescent microsphere injection sites in the dorsal and ventral cervical (C5–C6) spinal cord (A), in the dorsal horn (laminae I to VI) (B) and in the ventral horn (laminae VII-X) (C) 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.
Figure 3.
The distribution patterns of neurons in the ventromedial medulla that were retrogradely labeled from the lumbar dorsal or ventral horn.
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 (A) or ventral (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.
Figure 4.
Neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and neurons labeled for GlyT2 and/or GAD67 mRNAs.
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 (A); glycinergic (GlyT2 mRNA containing) neurons in the rostral ventromedial medulla (RVM) retrogradely labeled from the cervical spinal cord (B); GlyT2 and/or GAD67 mRNA containing neurons (Gly/GABA) in the RVM retrogradely labeled from the lumbar dorsal horn (C); Gly/GABA neurons in the RVM retrogradely labeled from the lumbar ventral horn (D); Gly/GABA neurons in the CVM retrogradely labeled from the lumbar ventral horn (E). Scale bar = 50 µm.
Figure 5.
The percentages of neurons in the ventromedial medulla that were retrogradely labeled from the spinal cord and contained GlyT2 and/or GAD67 mRNAs.
Histograms showing the percentages of retrogradely labeled neurons in the ventromedial medulla that contained GAD67, GlyT2, and GlyT2 and/or GAD67 mRNAs after fluorescent microsphere injections in the cervical spinal cord (A), in the lumbar dorsal horn (B) and in the lumbar ventral horn (C). The percentages are shown separately for the caudal ventromedial medulla (CVM) and rostral ventromedial medulla (RVM) and were always significantly lower in the CVM than in the RVM (p<0.05). Differences between the various percentages within the CVM and the RVM were never significant (p>0.1).
Figure 6.
The distribution patterns of neurons in the spinal cord that were retrogradely labeled from the rostral ventromedial medulla.
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.
Figure 7.
Spinal neurons retrogradely labeled from the rostral ventromedial medulla, and spinal neurons labeled for GlyT2 and/or GAD67 mRNAs.
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 (A) and for GlyT2 and/or GAD67 mRNAs in segments L5 (C) and L6 (B and D). Scale bar = 50 µm.