Loss of the Reelin-signaling pathway differentially disrupts heat, mechanical and chemical nociceptive processing

Abstract

The Reelin-signaling pathway regulates neuronal positioning during embryonic development. Reelin, the extracellular matrix protein missing in reeler mutants, is secreted by neurons in laminae I, II and V, binds to Vldl and Apoer2 receptors on nearby neurons, and tyrosine phosphorylates the adaptor protein Disabled-1 (Dab1), which activates downstream signaling. We previously reported that reeler and dab1 mutants had significantly reduced mechanical and increased heat nociception. Here we extend our analysis to chemical, visceral, and cold pain and importantly, used Fos expression to relate positioning errors in mutant mouse dorsal horn to changes in neuronal activity. We found that noxious mechanical stimulation-induced Fos expression is reduced in reeler and dab1 laminae I-II, compared to wild-type mice. Additionally, mutants had fewer Fos-immunoreactive neurons in the lateral-reticulated area of the deep dorsal horn than wild-type mice, a finding that correlates with a 50% reduction and subsequent mispositioning of the large Dab1-positive cells in the mutant lateral-reticulated area. Furthermore, several of these Dab1 cells expressed Fos in wild-type mice but rarely in reeler mutants. By contrast, paralleling the behavioral observations, noxious heat stimulation evoked significantly greater Fos expression in laminae I-II of reeler and dab1 mutants. We then used the formalin test to show that chemical nociception is reduced in reeler and dab1 mutants and that there is a corresponding decrease in formalin-induced Fos expression. Finally, neither visceral pain nor cold-pain sensitivity differed between wild-type and mutant mice. As differences in the nociceptor distribution within reeler and dab1 mutant dorsal horn were not detected, these differential effects observed on distinct pain modalities suggest that dorsal horn circuits are organized along modality-specific lines.

Fig. 1

Schematic of dorsal horn lamination and the area of the lateral spinal nucleus (LSN) used for Fos analysis. DLF, dorsolateral funiculus; DF, dorsal funiculus.

Fig. 2

Fos expression in the dorsal horn following mechanical (A–D) and thermal (heat, E–H) stimulation. A, B: Compared to wild type controls, the number of Fos-immunoreactive cells stimulated by mechanical pinch is significantly decreased in laminae I–II, III–IV, and the LSN in reeler (A) and in laminae I–II and the LSN of dab1 mutant mice (B). C, D: Following mechanical stimulation, more Fos-expressing cells are present in wild type laminae I–II and the lateral reticulated area (C, arrow) than in reeler mutants (D, arrow). E, F: Following heat stimulation, reeler mutants (E) showed a significantly elevated Fos response in laminae I–II and dab1 mutants (F) displayed a pronounced increase in Fos throughout the dorsal horn compared to wild type mice. G, H: Heat stimulation elevates Fos-immunoreactive cells in reeler laminae I–II and the lateral reticulated area (at arrows) versus wild type mice. Significant differences between wild type and mutants at *p<0.05; **p<0.01; ***p<0.001. Error bars reflect SEM. Scale bar C, D, G, H = 200 μm.

Fig. 3

Fos and Dab1 expression in adult lumbar dorsal horn after noxious mechanical (A–D) or heat (E–F) stimulation. A, B: More Fos-labeled nuclei are found in between the dark gray axon bundles of the lateral reticulated area (arrows) in wild type (A) than in reeler mutant (B) mice. C, D: Large Dab1-labeled cells are more common in the lateral reticulated area (C, arrow) of wild type than in reeler mutants (D). Two Dab1 neurons express mechanically-induced Fos (C, enlarged in inset at arrowheads) in the wild type lateral reticulated area, but not in reeler mutants (D, arrow). E, F: After noxious heat stimulation several Dab-labeled neurons express Fos (E, enlarged in inset at arrowheads) in the wild type lateral reticulated area. An ectopic Dab1-labeled cell (F, arrowhead) is outside of the lamina I border in the dorsal funiculus of a reeler mutant and expresses Fos (enlarged in inset). Scale bar A–F = 100 μm.

Fig. 4

Formalin-evoked nocifensive behavior and Fos expression in reeler (A–D) and dab1 (E–H) mice. Formalin was injected into the hindpaw and the duration of licking/biting the injected paw was measured in 5-min intervals for 1 hr. A, E: Compared to wild type mice, reeler mutants had reduced nocifensive behavior in phases I and II (A), whereas in dab1 mutants, a decrease was seen only in phase II (E). B, F: Fewer Fos immunoreactive cells were found in reeler (B) and dab1 (F) mutant dorsal horn laminae than in wild type mice. Fos expressing neurons were decreased in the reeler lateral spinal nucleus (LSN), but not dab1 mutants. C, D, G, H: Representative sections illustrate distribution of Fos immunoreactive neurons in wild type (C, G), reeler (D) and dab1 mutant (H) mice. Significant differences between wild type and mutant mice, *p<0.05; **p<0.01; ***p<0.001. Scale bar C, D, G, H = 200 μm.

Fig. 5

Cold and visceral pain responsiveness is unchanged in reeler and dab1 mutants. A, B: Regardless of temperature, wild type, reeler and dab1 mutants spent equivalent time on the coldest plate (compared to the adjacent plate). C: Number of abdominal stretches induced by i.p. acetic acid did not differ between wild type, reeler, or dab1 mutant mice. D: The number of Fos immunoreactive cells induced by acetic acid in the thoracic superficial dorsal horn did not differ between wild type and dab1 mutant mice. p>0.05 for all comparisons; error bars reflect SEM.