The Oscillating Lateral Hypothalamus and the Orexinergic System

Neurons which release the orexins (OX)/hypocretins peptides and are located in the lateral hypothalamus (LH) are key regulators of energy metabolism, arousal and sleep-wake stability, motivated behaviors. This thesis presents three data sets focused on the synaptic wiring of OX-A cell bodies in relation to state-dependent behavior in mice. The first study (Chapter 2; Laperchia et al. 2017) tested the hypothesis of synaptic plasticity phenomena of OX soma innervation in basal conditions. Adult mice were sacrificed during day or night periods in which sleep or wake predominance, respectively, were assessed by electroencephalography in matched mice. Excitatory and inhibitory terminals on OX somata were evaluated with multiple immunofluorescence. The total number of these terminals did not vary between day and night, but glutamatergic terminals prevailed at night and GABAergic ones at daytime. The findings thus revealed a striking daily fluctuation in the axosomatic wiring of OX neurons, with a switch from prevalent excitatory innervation during wake to prevalent inhibitory innervation during sleep. An addendum to Chapter 2 presents methodological approaches to the analysis of astrocytes surrounding OX neurons, at day and night time points in antiphase as above, and preliminary observations. The second study (Chapter 3) tested the hypotheses that the above diurnal fluctuation could be altered during aging and in the pathology that characterizes Alzheimer's disease (AD). The same paradigm and approaches of the first study were applied to 3 month-old and 20 month-old TASTPM mice, which provide a model of AD and in which main pathological features were investigated, and to matched wild-type (WT) mice. The day/night fluctuation in the inhibitory/excitatory wiring of OX somata was replicated in young WT and TASTPM mice, but was lost in aged WT mice and TASTPM mice. In addition, an overall decrease of presynaptic terminals on OX cell bodies was found in aged WT and TASTPM mice vs young ones, and in TASTPM mice vs WT ones (all sampled during daytime). In 15 month-old TASTPM mice stereological OX cell counts revealed significant loss (34% decrease), and densitometric evaluation showed a significant enhancement OX immunosignal intensity, suggesting a potential compensatory increase of peptide synthesis. The third study (Chapter 4) tested the hypothesis that extracellular matrix (ECM) components, reported to be among the players regulating neural plasticity, could be involved in the daily reorganization of OX cell body wiring. The study was conducted in healthy mice and in a murine model of the parasitic encephalitis African trypanosomiasis or sleeping sickness. The ECM was labelled by Wisteria floribunda agglutinin (WFA) immunofluorescence. Marked day/night variations were observed in confocal microscopy, with a diffuse ECM distribution at daytime, and a more compact organization and condensation around OX cell bodies at night. Furthermore, WFA expression in the LH, evaluated with Western blotting, was significantly enhanced at night compared to day. This diurnal variation of ECM organization was not found in other brain areas (suprachiasmatic nucleus, neocortex, hippocampus), and was lost in the LH after African trypanosome infection. These findings indicate regional day/night fluctuation of the ECM in the LH, and its disruption in a chronic neuroinflammatory pathology which leads to sleep-wake dysregulation.