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.

Introduction

The reeler gene is a naturally occurring autosomal recessive mutation identified and cloned by D’Arcangelo et al. (1995). The protein missing in these mice, Reelin, is a large secreted extracellular matrix-type molecule that is highly expressed during embryonic development and at lower levels in postnatal and adult mice (Rice et al., 1998, D’Arcangelo et al., 1999, Niu et al., 2004). Reelin binding to the Apolipoprotein E receptor 2 (Apoer2) and the Very low-density lipoprotein receptor (Vldlr) leads to tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1; D’Arcangelo et al., 1999, Hiesberger et al., 1999) by the Src family of non-receptor tyrosine kinases (Arnaud et al., 2003, Bock and Herz, 2003). Downstream signaling events activated by Dab1 phosphorylation influence embryonic neuronal positioning, postnatal dendritic development and synaptic plasticity in the adult (Howell et al., 1997, Niu et al., 2004, Matsuki et al., 2008). As mice with mutations in Reelin, both Apoer2 and Vldlr, or Dab1 are phenotypically indistinguishable in their ‘reeling’ gait and neuronal migration defects, these mutants provide strong genetic evidence that the Reelin-signaling pathway is required for correct neuronal positioning (Trommsdorff et al., 1999, Rice and Curran, 2001).

The classic migratory defects in reeler mutants occur in laminated structures of the cerebral and cerebellar cortices and in the hippocampal formation (Goffinet, 1984). Reelin signaling also affects less-laminated CNS structures, including the spinal cord, where it functions in a cell-specific manner (Phelps et al., 2002). That is, many neurons in reeler spinal cord are located correctly but others are aberrantly positioned. Prominent migratory errors in reeler spinal cord occur among sympathetic and parasympathetic preganglionic neurons (Yip et al., 2000, Phelps et al., 2002). Incorrectly positioned neurons are identified by their high Dab1 protein expression in reeler dorsal horn, specifically in laminae I, II and V and in the lateral spinal nucleus (LSN; Villeda et al., 2006), areas involved in the transmission of ‘pain’ signals (Menétrey et al., 1980, Burstein et al., 1990b, Craig, 2003, Olave and Maxwell, 2004, Basbaum et al., 2009, Pinto et al., 2010). Importantly, there is a behavioral correlate of these positioning errors. Thus, reeler mice exhibit a significant reduction in mechanical sensitivity and an increased thermal sensitivity/heat hyperalgesia (Villeda et al., 2006). As the same alterations of pain processing occur in dab1 mutants (Akopians et al., 2008), we concluded that the Reelin-signaling pathway is an essential contributor to the normal development of ‘pain’ circuits. The fact that there is differential processing of heat and mechanical pain messages in these mutants is of particular interest, and emphasizes the specificity of the circuits affected by these positioning defects.

To delineate further the modality specificity of the alterations in acute pain processing in reeler and dab1 mutant mice, we extended the analysis to chemical, noxious cold, and visceral pain, using behavioral and Fos expression studies. We report that despite presenting with an unusual combination of increased thermal (heat) sensitivity and reduced mechanical and chemical responsiveness, Reelin-signaling pathway mutants behave normally in tests of responsiveness to noxious cold and visceral stimulation.