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. 2020 Feb 7;21(3):1107.
doi: 10.3390/ijms21031107.

Implication of 5-HT in the Dysregulation of Chloride Homeostasis in Prenatal Spinal Motoneurons from the G93A Mouse Model of Amyotrophic Lateral Sclerosis

Elodie Martin  1 , William Cazenave  1 , Anne-Emilie Allain  1 , Daniel Cattaert  1 , Pascal Branchereau  1
Affiliations

Implication of 5-HT in the Dysregulation of Chloride Homeostasis in Prenatal Spinal Motoneurons from the G93A Mouse Model of Amyotrophic Lateral Sclerosis

Elodie Martin et al. Int J Mol Sci. .

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. The early presymptomatic onset of abnormal processes is indicative of cumulative defects that ultimately lead to a late manifestation of clinical symptoms. It remains of paramount importance to identify the primary defects that underlie this condition and to determine how these deficits lead to a cycle of deterioration. We recently demonstrated that prenatal E17.5 lumbar spinal motoneurons (MNs) from SOD1G93A mice exhibit a KCC2-related alteration in chloride homeostasis, i.e., the EGABAAR is more depolarized than in WT littermates. Here, using immunohistochemistry, we found that the SOD1G93A lumbar spinal cord is less enriched with 5-HT descending fibres than the WT lumbar spinal cord. High-performance liquid chromatography confirmed the lower level of the monoamine 5-HT in the SOD1G93A spinal cord compared to the WT spinal cord. Using ex vivo perforated patch-clamp recordings of lumbar MNs coupled with pharmacology, we demonstrated that 5-HT strongly hyperpolarizes the EGABAAR by interacting with KCC2. Therefore, the deregulation of the interplay between 5-HT and KCC2 may explain the alteration in chloride homeostasis detected in prenatal SOD1G93A MNs. In conclusion, 5-HT and KCC2 are two likely key factors in the presymptomatic phase of ALS, particular in familial ALS involving the SOD1G93A mutation.

Keywords: 5-HT; ALS; GABA/glycine; SOD1G93A mouse; chloride homeostasis; development; motoneuron; perforated patch-clamp; spinal cord.

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Conflict of interest statement

The authors declare no conflicts of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Descending 5-HT fibres in coronal sections of E17.5 WT and SOD1G93A spinal cords (SCs). (A) Global 5-HT staining (dark labelling) in the three parts of the SC used for quantification (ventral horn, VH; intermediate area, IA; central grey matter, CGM). (B1–B3) 5-HT staining at the cervical level in WT mice. (B4–B6) 5-HT staining at the cervical level in SOD1G93A mice. (C1–C3) 5-HT staining at the lumbar level in WT mice. (C4–C6) 5-HT staining at the lumbar level in SOD1G93A mice. Note the 5-HT positive fibres projecting into the grey matter from the lateral funiculus (arrows) (D1–D2) Quantitative analysis of global 5-HT innervation in the VH, IA, and CGM at the cervical level (D1) and lumbar level (D2) in WT (black) and SOD1G93A (red) SCs. ns, not significant; ** p < 0.01; *** p < 0.001, Mann-Whitney test); cc, central canal.
Figure 2
Figure 2
High-performance liquid chromatography (HPLC) assay of the monoamine 5-HT in E17.5 WT (black bar) and SOD1G93A lumbar SCs (red bar). The schematic drawings on both sides of the graph depict the reduction in the density of descending 5-HT fibres from caudal raphe nuclei in the SC of SOD1G93A mice compared to the SC of WT littermates. * p < 0.05, Mann-Whitney test.
Figure 3
Figure 3
Modulation of the EGABAAR by exogenous 5-HT application. (A1) 5-HT-induced inward current and EGABAAR decrease in a representative E17.5 MN. (A2) EGABAAR values measured in the same WT MNs (left panel) and SOD1G93A MNs (right panel) under control conditions (black circles) and in the presence of 5-HT (blue circles). (B1) Involvement of the 5-HT2 receptor in the 5-HT-mediated modulation of the EGABAAR in a representative E17.5 MN. (B2) Quantitative analysis of the EGABAAR in the same WT MNs (left panel) and SOD1G93A MNs (right panel) under control conditions (black circles), in the presence of 5-HT (blue circles) and in the presence of 5-HT + methysergide and ketanserine (grey circles). (C1) 5-HT hyperpolarized the EGABAAR by acting on KCC2, as depicted in a representative E17.5 MN. (C2) Quantitative analysis of the EGABAAR in the same WT MNs (left panel) and SOD1G93A MNs (right panel) under control conditions (black circles), in the presence of 5-HT (blue circles) and in the presence of 5-HT + VU0240551 (green circles). Drugs were applied at 10 µM. * p < 0.05; *** p < 0.001, Wilcoxon matched-pairs signed rank test. Experiments in B–C are from the set of experiments used in A.
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