RICERCA ITALIANA     

The relationship between sleep and body adaptive responses to modifications in the internal and external environment.

Università degli Studi di Bologna

Abstract

The project will consider the mechanisms determining at a cellular and systemic level the adaptive responses of the body to changes in the internal and external environment during sleep. In particular, the study will consider from a neurochemical, neuroanatomical and neurophysiological perspective the following structures: cerebral cortex, hypothalamus, and brainstem. The systemic regulation of circulation, respiration and water and salt balance will be studied. <<<

Principal Investigator

Carlo FRANZINI Università degli Studi di BOLOGNA

Research Objectives

The sleep cycle in mammals (Non-Rapid-Eye-Movement sleep, NREM sleep, characterized by absence of rapid eye movements, synchronized electroencephalogram, and fully effective homeostatic regulations; Rapid-Eye-Movement sleep, REM sleep, characterized by presence of rapid eye movements, desynchronized electroencephalogram, and impairment of homeostatic regulations) is the expression of a global behavior involving therefore both somatic and autonomic activity. The research project aims at studying the relationship between sleep and body adaptive responses to modifications in the internal and external environment. Both the effect of different stimulations on the sleep cycle and the effect of the sleep state on body adaptive responses will be studied. To this end, the different but complementary expertise of sleep researchers of the Universities of Bologna and Milan has been joined to present an integrated proposal approaching issues of the sleep-wake cycle. The study will consider: a) the relationship between sleep and water-salt balance, assessing the regulation of vasopressin secretion following an hyperosmotic challenge; b) the relationship between sleep and cardiovascular regulation following acoustic and hypoxic challenges; c) the relationship between sleep and the immune system following Interleukin-1 microinjections; d) electroencephalographic changes following transcranial magnetic stimulation during sleep. <<<

First Results

a) Sleep and the regulation of water-salt balance. Phase 1: vasopressin release following hyperosmotic stimulation. A reduced or absent release of vasopressin during REM sleep with respect to NREM sleep or wakefulness will stand for a generalized impairment of hypothalamic structures in REM sleep.
b) Sleep and perturbations in cardiovascular regulation. Phase 1: experiments performed in control conditions and after acoustic and hypoxic stimulation in normotensive rats. The acoustic and hypoxic stimulation in normotensive rats should entail a reduction in sleep quality assessed on the basis of EEG, EMG, and cardiorespiratory changes. Moreover, cardiovascular and respiratory changes, secondary to the experimental challenge, may show a dependence from the sleep-wake state. Finally, this will allow to assess the sleep-state dependent cardiorespiratory risk in pathophysiological conditions.
c) Changes in the sleep-wake cycle following an immunological challenge. Phase 1: technical refinement of the stereotaxic approach, of the patch clamp recordings and of the immunohistochemical characterization. The precise determination of the stereotaxic coordinates for the placement of the cannula needed for IL-1 microinjections in vivo and the direct administration of IL-1 to cholinergic neurons of the pontine tegmentum will be performed. An inhibition of bioelectric activity would favor the hypothesis that the effect of IL-1 on REM sleep may result from an inhibition of cholinergic neurons in the pontine tegmentum.
d) Perturbations of cortical excitability during sleep. Phase 1: EEG changes following transcranial magnetic stimulation during sleep. The experiment will clarify the possibility of TMS to interfere with the genesis and propagation of slow waves in NREM sleep. In particular, a significant difference in the number and topographic distribution of slow waves and in their propagation pathways when TMS is applied would indicate that cortical stimulation modifies slow EEG waves in NREM sleep. An increased number of recorded waves, a change in their sites of origin, a preferential propagation pattern originating in the stimulated area would confirm that TMS is capable of generating and reinforcing the slow EEG waves in NREM sleep in humans.a) Sleep and the regulation of water-salt balance. Phase 2: functional identification of neural circuits involved in vasopressin release during the sleep-wake cycle. Whilst observations concerning osmoregulation during sleep are lacking, in wakefulness a dose-dependent increase in c-fos expression following ICV administration of hypertonic saline has been observed in the supraoptic and paraventricular nuclei, in sub fornical organ, organum vasculosum laminae terminalis and in the median preoptic area. We expect a confirmation of the behavioral and biochemical results obtained in Phase 1. In particular, the relevance of identifying an immunocytochemical correlate of a reduced response to osmotic challenge during REM sleep is evident.
b) Sleep and perturbations in cardiovascular regulation. Phase 2: experiments performed in control conditions and in the presence of acoustic and hypoxic challenges in Spontaneously Hypertensive Rats. It can be foreseen that in Spontaneously Hypertensive Rats, like in normotensive subjects, acoustic and hypoxic challenges may entail a reduction of the sleep quality indexes defined on the basis of EEG, EMG, and cardiorespiratory changes. Moreover, the assessment of both the modifications of cardiorespiratory function and of the cardiorespiratory changes to environmental challenges will shed light on the specific effect of hypertensive on cardiovascular regulation during sleep.
c) Changes in the sleep-wake cycle following an immunological challenge. Phase 2. In vivo studies: sleep changes induced by IL-1 microinjection in the cholinergic nuclei of the pontine tegmentum. In vitro studies: effects of IL-1 administration on bioelectrical activity of cholinergic neurons in the pontine tegmentum. The hypothesis that REM sleep inhibition resulting from IL-1 administration can be explained on the basis of the inhibition of cholinergic neurons of the pontine tegmentum will be verified.
d) Perturbations of cortical excitability during sleep. Phase 2: functional effects of transcranial magnetic stimulation on the genesis and propagation of slow EEG waves during NREM sleep. We will evaluate the possibility that a local modification of the slow EEG waves in NREM sleep resulting from the TMS challenge may be responsible of behavioral and cognitive changes after awakening even if the challenge per se did not determine an arousal from sleep. <<<

Timescale

24 months

National and international background

In mammals, the mode of operation of regulatory mechanisms depends on the sleep-wake states (Parmeggiani, 1980); in particular, during REM sleep, autonomic regulations are significantly impaired. As a consequence: a) changes in the internal or external environments may affect the sleep-wake cycle organization; and b) the adaptive responses following environmental changes may depend on the sleep state. The project aims at studying four representative aspects of the interaction between hypnic and homeostatic regulation.
The first Research Unit will test the relationship between behavioral states and the regulation of salt and water content in body fluids, in particular that of water balance (osmoregulation). Osmoregulation is sustained by the neurohypophyseal reflex release of vasopressin, synthesized in the magnocellular hypothalamic neurons located in the supraoptic and paraventricular nuclei, induced by changes in the osmolality of body fluids. Thus, osmoregulation represents a homeostatic function under a clear hypothalamic control, but which is phylogenetically older than thermoregulation, whose impairment during REM sleep was first shown (Parmeggiani, 1980) as evidence of an homeostatic failure in this state.
The second Research Unit will consider cardiovascular changes during sleep. Peripheral (baroreflex) and central (non-baroreflex) influences converge at the level of HP regulation. The analysis of Heart Period (HP) vs. Arterial Pressure (AP) statistical dependence allows evaluation of the balance of central vs. peripheral influences on HP during sleep (Zoccoli et al. 2001). Environmental stimuli affect both autonomic control and the wake-sleep cycle. In particular, acoustic stimulation affects both sleep quality (Nakagawa 1987) and cardiovascular regulation (Di Nisi 1990). Hypoxia also induces changes in the wake-sleep cycle (Ryan and Megirian 1982) and in cardiovascular control (Kara et al. 2003). Therefore, following acoustic and hypoxic challenges both sleep cycle changes and physiological regulatory responses will be assessed. Cardiovascular effects of environmental stimuli represent risk factors when cardiovascular diseases are associated. Responses may largely depend on behavioral state. For instance, in REM sleep, when autonomic regulations are deeply altered (Parmeggiani 2000) the risk of cardiovascular accidents increases in the presence of little or negligible regulatory reserve.
The third Research Unit will consider changes in the sleep-wake cycle following an immunological challenge. Immune challenge triggers acute phase response and relevant changes in wake-sleep activity (Krueger & Fang, 2000), characterized by an increase in NREM sleep and a decrease in REM sleep. Cytokines such as Interleukin-1(IL-1) play a key role in mediating these changes in sleep. Whereas different studies investigated mechanisms mediating IL-1 stimulation of NREM sleep, mechanisms mediating IL-1 inhibition of REM sleep have not been investigated yet. REM sleep is under the control of cholinergic neurons in the pontine tegmentum. Data in the literature show that IL-1 inhibits acetylcholine release. The aim of the study will be to test the hypothesis that IL-1 inhibits REM sleep because of IL-1 inhibitory effects on cholinergic neurons in the pontine tegmentum.
The fourth Research Unit will stimulate the cerebral cortex of sleeping humans to study the changes induced in the pattern of slow EEG waves. High-density EEG recordings (256 electrodes) recently allowed the possibility to confirm in human sleep the wealth of information derived from earlier animal experiments. In humans, the slow oscillation is a traveling wave that starts locally and than sweeps over the rest of the cerebral cortex at a speed of 2-3 m/s (Massimini et al 2003). Each slow oscillation has a definite site of origin and direction of propagation, both of which vary from one cycle to the next; interestingly, waves originate more frequently at the transition between dorsolateral and orbitofrontal regions and propagate in an antero-posterior direction. On the other hand, Transcranial Magnetic Stimulation (TMS), depending on stimulation timing and parameters, may either trigger or suppress the slow oscillation. The possibility of manipulating the occurrence, the origin and the pathways of propagation of slow waves allows studying their instantaneous homeostatic regulation. Moreover, artificially increasing or suppressing slow waves in sleeping humans may finally reveal their physiological function and may have relevant clinical implications. <<<