The Hypocretinergic/Orexinergic System: neurotransmission and homeostatic plasticity mechanisms. Modulatory actions in the sleep-wake cycle and other physiological functions, and implications in narcolepsy

• Autores: Carlos Carrera Cañas
• Directores de la Tesis: Miguel Garzón García (dir. tes.), Isabel de Andrés Calle (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2024
• Idioma: inglés
• Número de páginas: 196
• Títulos paralelos:
  • El Sistema Hipocretinérgico/Orexinérgico: mecanismos de neurotransmisión y plasticidad homeostática. Acciones moduladoras en el ciclo vigilia-sueño y otras funciones fisiológicas, e implicaciones en la narcolepsia
• Tribunal Calificador de la Tesis: Angel Núñez Molina (presid.), María García-Amado Sancho (secret.), María Dolores Ledesma Muñoz (voc.), Giancarlo Vanini Souza (voc.), Rafael del Río Villegas (voc.)
• Programa de doctorado: Programa de Doctorado en Neurociencia por la Universidad Autónoma de Madrid
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • THE RATIONALE: The hypocretinergic/orexinergic -Hcrt/Ox- system is a hypothalamic neuromodulatory system comprising two excitatory neuropeptides, Hcrt1/OxA and Hcrt2/OxB, and two G protein-coupled receptors, Hcrt/OxR1 and Hcrt/OxR2. Hcrt/Ox projections extend widely throughout the central nervous system, performing multiple and complex modulatory actions in different physiological functions. Malfunction of Hcrt/Ox transmission results in narcolepsy. In the present doctoral thesis, we chronically administered suvorexant (dual Hcrt/Ox receptor antagonist) in rats to generate an experimental model of narcolepsy, and we aimed: (1) to delve into the transmission type of the Hcrt/Ox system, (2) to study the plasticity of the Hcrt/Ox system (Hcrt1/OxA and Hcrt/Ox receptors levels) after chronic suvorexant treatment, (3) to evaluate the presence/absence of Hcrt/Ox system sexual dimorphism, and to explore the effects of Hcrt/Ox transmission blockade on the regulation of (4) the sleep-wake cycle -SWC-, (5) the circadian rhythmicity, (6) the energy balance of the organism, and (7) motivation.

    METHODS: Daily i.p. injections of suvorexant (10 or 30mg/Kg) or vehicle (DMSO) were carried out in 28 adult male and female rats for 7 days. After treatment, Hcrt1/OxA levels in cerebrospinal fluid -CSF- and in synaptosome preparations from the oral pontine tegmentum -OPT- were measured by ELISA, and Hcrt/OxR1 and Hcrt/OxR2 expression levels in hypothalamic and OPT samples were measured by western blot. In addition, we analysed the central/peripheral location of Hcrt1/OxA+ and Hcrt1/OxA- vesicles in OPT-Hcrt1/OxA+ axons of naïve rats. To perform physiological-behavioural studies, electrodes for SWC polygraphic recordings, and a subcutaneous device for body temperature recording were implanted in 19 male rats. Body weight and peripheral body temperature were monitored throughout suvorexant/DMSO treatment. Twenty-four-hours SWC polygraphic recordings were obtained in the first and the last treatment days and analysed according to the rat SWC patterns. In these rats, a behavioural test to evaluate motivation and the appearance of cataplexy was performed after treatment. All data were statistically treated to obtain the results after each experiment.

    RESULTS AND CONCLUSIONS: Chronic treatment with the high dose of suvorexant produced a significant increase in body weight at the end of the treatment, confirming its effectiveness. In baseline conditions, Hcrt1/OxA levels were higher in CSF than in synaptosomes; Hcrt1/OxA+ vesicles tended to locate peripherally inside Hcrt1/OxA+ axons; and Hcrt/OxR1 was preferentially situated outside synapses, while Hcrt/OxR2 was indistinctly in or out of them. Chronic blockade of Hcrt/Ox transmission (30mg/Kg suvorexant) produced a significant decrease in CSF Hcrt1/OxA levels together with a significant accumulation of the peptide in synaptosomes, and a significant compensatory overexpression of Hcrt/OxR1 at hypothalamus. Baseline levels of Hcrt1/OxA were significantly higher in females, and some of the plastic changes induced by suvorexant were also greater in this sex. Thus, concluding this first part: (1) Hcrt/Ox transmission is mixed but mostly volumetric, and the Hcrt/Ox receptors play differential roles in mediating these responses; (2) at Hcrt/Ox neurons, the Hcrt/OxR1 acts as an autoreceptor controlling peptides release and its own expression; and (3) Hcrt/Ox transmission is greater and shows a stronger homeostatic control in females than in males.

    At the OPT, Hcrt/OxR1 basal levels were significantly higher at its ventral part -VOPT- (involved in REM sleep executive mechanisms) than at its dorsal part -DOPT- (involved in wakefulness -W- and REM preparation), while the opposite occurred for the Hcrt/OxR2 largest isoform. Besides, chronic suvorexant treatment (30mg/Kg) produced a slight overexpression of Hcrt/OxR1 at VOPT, and a significant overexpression of the Hcrt/OxR2 largest isoform at DOPT. Regarding the SWC, acute suvorexant administration induced a significant repression of W (due to decreased duration of episodes) during the dark period, at the expense of a sequential facilitation (increased number of episodes) of light sleep -LS-, slow-wave sleep -SWS-, and finally the intermediate state -IS- and REM. Chronic suvorexant treatment repressed W during the dark period and facilitated LS during the dark and light periods. Unlike acute administration, chronic suvorexant treatment repressed IS (preparatory stage of REM) but enhanced direct entrances into REM avoiding IS, so REM baseline proportions were not modified. No cataplexy episodes were observed at any case. In relation to body temperature, no significant effects of suvorexant were detected for any of the analysed parameters. Finally, regarding motivation, chronic suvorexant treatment entailed alterations in both the direction and the intensity of motivation. Thus, concluding this second part: (4) Hcrt/Ox transmission is essential for W maintenance and for the control of IS-mediated transitions to REM. The Hcrt/OxR2 plays a pivotal role at DOPT, the region promoting W and controlling IS, while the Hcrt/OxR1 is the main actor at VOPT, the region executing REM; (5) Hcrt/Ox transmission is not essential for the regulation of physiological circadian rhythms; (6) Hcrt/Ox transmission controls the organism energy balance through mechanisms other than thermoregulation; (7) Hcrt/Ox transmission acts as a modulator of motivated behaviours, coupling the goal-directed motivational drive and its intensity; and (8) chronic pharmacological blockade of Hcrt/Ox transmission in rats leads to an experimental model that largely mimics the clinical features of human narcolepsy.