Orexin/Hypocretin and Histamine Cross-Talk on Hypothalamic Neuron Counts in Mice

Abstract

The loss of hypothalamic neurons that produce wake-promoting orexin (hypocretin) neuropeptides is responsible for narcolepsy type 1 (NT1). While the number of histamine neurons is increased in patients with NT1, results on orexin-deficient mouse models of NT1 are inconsistent. On the other hand, the effect of histamine deficiency on orexin neuron number has never been tested on mammals, even though histamine has been reported to be essential for the development of a functional orexin system in zebrafish. The aim of this study was to test whether histamine neurons are increased in number in orexin-deficient mice and whether orexin neurons are decreased in number in histamine-deficient mice. The hypothalamic neurons expressing L-histidine decarboxylase (HDC), the histamine synthesis enzyme, and those expressing orexin A were counted in four orexin knock-out mice, four histamine-deficient HDC knock-out mice, and four wild-type C57BL/6J mice. The number of HDC-positive neurons was significantly higher in orexin knock-out than in wild-type mice (2,502 ± 77 vs. 1,800 ± 213, respectively, one-tailed t-test, P = 0.011). Conversely, the number of orexin neurons was not significantly lower in HDC knock-out than in wild-type mice (2,306 ± 56 vs. 2,320 ± 120, respectively, one-tailed t-test, P = 0.459). These data support the view that orexin peptide deficiency is sufficient to increase histamine neuron number, supporting the involvement of the histamine waking system in the pathophysiology of NT1. Conversely, these data do not support a significant role of histamine in orexin neuron development in mammals.

Keywords: histamine; mouse; narcolepsy; neurons; orexins/hypocretins.

FIGURE 1

L-histidine decarboxylase-positive cells in orexin knockout and wild-type mice. (A,B) Show representative sections of L-histidine decarboxylase (HDC) immunostaining (cells with red cytoplasm) in orexin knockout (KO-ORX), and control wild-type (WT) mice, respectively. In both panels, the bottom left insets show a higher magnification of the delimited area (white square) in the main panel. 3V: third ventricle. Scale bars = 100 μm for main panels and upper insets and 12.5 μm for the lower insets. For the sole purpose of figure publication, the contrast, and brightness were adjusted to resemble the appearance of the labeling seen through the microscope using Adobe Photoshop CS3 Extended 10.0 software (Adobe Systems, San Jose, CA, United States). (C) Shows the total number of HDC- positive neurons in four WT control and four KO-ORX mice. (D) Shows the number of HDC-positive neurons in four KO-ORX and four WT mice as a function of distance from bregma along the rostrocaudal axis, with reference to the mouse brain atlas (Franklin and Paxinos, 2007). Gray and black dots represent individual animals. The horizontal line and whiskers indicate means ± SEM. *P < 0.05 vs. WT, one-sample independent t-test.

FIGURE 2

Orexin-A positive cells in L-histidine decarboxylase knockout and wild-type mice. (A,B) Show representative sections of orexin immunostaining (cells with red cytoplasm) in L-histidine decarboxylase knockout mice (KO-HIST) and WT mice, respectively. In both panels, the bottom left insets show a higher magnification of the delimited area (white square) in the main panel. 3V: third ventricle. Scale bars = 100 μm for main panels and upper insets and 12.5 μm for the lower insets. For the sole purpose of figure publication, the contrast and brightness were adjusted to resemble the appearance of the labeling seen through the microscope using Adobe Photoshop CS3 Extended 10.0 software (Adobe Systems, San Jose, CA, United States). (C) Shows the total number of orexin-positive neurons in four WT control and four KO-HIST mice. Gray and black dots represent individual animals. The horizontal line and whiskers indicate means ± SEM.

FIGURE 3

L-histidine decarboxylase-positive and orexin-A positive cell size. (A) Shows HDC-positive average cell size in four WT mice and four KO-ORX mice. (C) Shows average orexin-A positive cell size in four WT mice and four KO-HIST mice. Gray and black dots represent individual animals. (B) Shows the HDC-positive cell size distribution (50 μm² bins, X axis) in four WT (gray) mice and four KO-ORX (black) mice. (D) Shows the orexin-A positive cell size distribution (50 μm² bins, X axis) in four WT (gray) mice and four KO-HIST (black) mice. (A,C) The horizontal line and whiskers indicate means ± SEM. (B,D) Data are reported as mean + SEM.