Histamine in murine narcolepsy: What do genetic and immune models tell us?

Abstract

An increased number of histaminergic neurons, identified by labeling histidine-decarboxylase (HDC) its synthesis enzyme, was unexpectedly found in patients with narcolepsy type 1 (NT1). In quest for enlightenment, we evaluate whether an increase in HDC cell number and expression level would be detected in mouse models of the disease, in order to provide proof of concepts reveling possible mechanisms of compensation for the loss of orexin neurons, and/or of induced expression as a consequence of local neuroinflammation, a state that likely accompanies NT1. To further explore the compensatory hypothesis, we also study the noradrenergic wake-promoting system. Immunohistochemistry for HDC, orexin, and melanin-concentrating hormone (MCH) was used to count neurons. Quantitative-PCR of HDC, orexin, MCH, and tyrosine-hydroxylase was performed to evaluate levels of mRNA expression in the hypothalamus or the dorsal pons. Both quantifications were achieved in genetic and neuroinflammatory models of narcolepsy with major orexin impairment, namely the orexin-deficient (Orex-KO) and orexin-hemagglutinin (Orex-HA) mice respectively. The number of HDC neurons and mRNA expression level were unchanged in Orex-KO mice compared to controls. Similarly, we found no change in tyrosine-hydroxylase mRNA expression in the dorsal pons between groups. Further, despite the presence of protracted local neuroinflammation as witnessed by the presence of reactive microglia, we found no change in the number of neurons nor the expression of HDC in Orex-HA mice compared to controls. Importantly, no correlation was found in all conditions between HDC and orexin. Our findings indicate that, in mice, the expression of histamine and noradrenalin, two wake-promoting systems, are not modulated by orexin level whether the lack of orexin is constitutive or induced at adult age, showing thus no compensation. They also show no recruitment of histamine by local neuroinflammation. Further studies will be needed to further define the role of histamine in the pathophysiology of NT1.

Keywords: hypocretin; hypothalamus; orexin; sleep.

FIGURE 1

Evaluation of ORX, MCH, HDC, and TH content in a genetic model of NT1, the Orex‐KO mice. Number of ORX (A), MCH (C), and HDC (E) neurons in C57BL/6J WT and Orex‐KO mice. mRNA expression level for ORX (B), MCH (D), HDC (F), and TH (H) in C57BL/6J WT and Orex‐KO mice. Data are shown as median (Q1–Q3), individual data values are represented as dots and the mean is represented as a cross. Mann–Whitney U test, *p < 0.05; **p < 0.01. ns, not significant. (G) Microphotograph illustrating HDC immunostaining in the tuberomammillary nucleus of a WT (left) and a HDC‐KO mouse (right). Scale bars: 110 µm

FIGURE 2

Evaluation of microglial activation in Orex‐KO and Orex‐HA mice. Double immunostaining of HDC (black) and Iba1 (brown) in the hypothalamus of WT (A and B), Orex‐KO (C and D), and Orex‐HA mice transferred twice with autoreactive CD8+ T cells at 60 days post‐injection (E and F). Red squares in A, C, E illustrate the enlarged areas shown in B, D, and F, respectively. Scale bars: 560µm (A, C, and E) or 50µm (B, D, and F). 3v, third ventricle; cp, cerebral peduncle; mt, mammillothalamic tract

FIGURE 3

Evaluation of ORX, MCH and HDC content in the immune‐mediated model of narcolepsy. Number of ORX (A), MCH (C), and HDC (E) neurons in WT and in Orex‐HA mice 60 days after T cell transfer. mRNA expression level for ORX (B), MCH (D), and HDC (F) in WT and in Orex‐HA mice 60 days after T cell transfer. Data are shown as median (Q1–Q3), individual data values are represented as dots and the mean is represented as a cross. Mann–Whitney (two groups) or Kruskal–Wallis (three groups) tests, *p < 0.05; **p < 0.01. ns, not significant