Ralph Lydic, Ph.D. - US grants

The sleep apnea syndromes have been estimated to affect 1 to 5% of the older adult male population, while the sudden infant death syndrome (SIDS) is the leading cause of death in infants between 1 month and 1 year of age. Common to both disorders are respiratory pauses occurring during sleep (apneas) resulting in abnormalities in oxygenation, cardiovascular dysfunction, and sleep disruption. In affected individuals, breathing is particularly disrupted during rapid eye movement (REM) sleep but the exact neuronal mechanisms through which REM sleep induces respiratory abnormalities are unknown. The difficulty of studying breathing during REM sleep is accentuated by the transient occurrence of REM sleep and by the fact that REM sleep is disrupted by many experimental manipulations. The studies proposed here will overcome these difficulties by applying an established pharmacological model of REM sleep to the study of breathing. Microinjecting cholinergic agonists (e.g., carbachol) into the pontine reticular formation of awake, unanesthetized animals produces a state which is polygraphically similar to REM sleep. With this method one gains experimental control over the occurrence of the REM sleep-like state. This pharmacological model has made important contributions to our understanding of the cholinergic control of REM sleep but has not yet been systematically applied to the study of breathing during sleep. Accordingly, the long-term objectives of the proposed research are to determine whether cholinoceptive mechanisms in the pontine reticular formation causally mediate REM sleep- related respiratory alterations. The specific aims of this 5 year research plan are: 1) To test the hypothesis that pontine carbachol administration alters breathing during sleep and upper airway muscle function; 2) To centrally administer cholinergic antagonists (atropine and pirenzepine) prior to carbachol in order to test the hypothesis that reticular M1 and M2 muscarinic cholinergic receptor mechanisms can cause state-dependent respiratory changes; 3) Chemosensitivity is depressed during natural REM sleep but the mechanisms of decreased responsivity to hypoxia are not clear. In order to test the hypothesis that cholinergic mechanisms contribute to this diminished chemosensitivity, Year 3 studies will characterize and compare the hypoxic ventilatory response during natural REM sleep and during the REM sleep-like state caused by carbachol; 4) Since the discharge level of pontine respiratory group (PRG) neurons may be of special relevance for apneic breathing, Year 4 studies will test the hypothesis that the REM sleep depression of PRG neuron discharge will also occur during the carbachol induced REM sleep-like state; 5) To date, there have been no recordings of any respiratory neurons during the carbachol induced REM sleep-like state. Year 5 studies will test the hypothesis that cholinoceptive reticular mechanisms contribute a diminished respiratory drive to PRG neurons during sleep.

DESCRIPTION (provided by applicant): Opioids are widely used for pain m management but one unwanted side effect of acute opioid administration is rapid eye movement (REM) sleep inhibition. Pain itself disrupts sleep, and sleep loss is the most common complaint of acute pain patients. Pontine choilnergic neurotransmission is a major contributor to REM sleep generation, and new data suggest that pontine GABAergic transmission and basal forebrain cholinergic transmission participate in REM sleep regulation. The long-term objectives of this renewal application are to advance scientific knowledge by specifying the cellular and molecular mechanisms through which opioids inhibit REM sleep. The four aims are unified conceptually and related to the long-term goals by focusing on opioid modulation of transmitter release and guanine nucleotide binding protein (G protein) activation. These studies focus on brain stem and forebrain regions known to regulate sleep and breathing. Novel aspects include an emphasis on the interactions of opioids with multiple transmitter systems in multiple sleep-related brain regions. Aims 1, 2, and 3 will use in vivo microdialysis and high performance liquid chromatography. Opioids will be administered systemically and, in separate experiments, directly into brain regions of interest by reverse dialysis. Concentration-response and antagonist blocking studies will evaluate receptor mediation of opioid effects. Aim 4 will use in vitro [35S]GTPgammaS autoradiography to examine the interaction of opioids with other sleep-modulatory neurotransmitters at the level of G protein activation. Aim 1 will test the hypothesis that opioids decrease acetylcholine (ACh) release in medullary hypoglossal nucleus. Aim 2 will test the hypothesis that basal forebrain opioid administration decreases basal forebrain ACh release. The Aim 3 hypothesis is that opioids decrease gamma aminobutyric acid (GABA) release in REM sleep-regulating regions of the pontine reticular formation. Aim 4 will test the hypothesis that opioids alter G protein activation by muscarinic cholinergic agonists and by the novel hypothalamic peptide hypocretin-1/orexin-A. Aim 4 will specify whether combined agonist activation of G proteins in sleep-related nuclei is fully additive, partially additive, or non-additive. Aim 4 will permit inferences concerning activation of independent or common G protein pools by mu opioid, muscarinic cholinergic, and hypocretin/orexin receptor agonists. The potential health relatedness of these basic studies derives from the unwanted side effects of opioids on sleep and breathing.

DESCRIPTION (provided by applicant): This research program was initiated in 1999 in response to RFA HL99001. The goal of the RFA was to stimulate improved molecular, cellular, and systems approaches to investigate sleep in mice. Every human gene has a mouse homologue. This remarkable homology means that the mouse genome may provide unique insights into human disease. Advances in sequencing the mouse genome now require complimentary data regarding normal and abnormal phenotypes. In accord with consensus statements published by The Jackson Laboratory, this application focuses on the C57BL/6J (B6) mouse strain. The long-term objectives are to advance scientific knowledge by providing data not presently available concerning molecules that regulate ACh release and alter electroencephalographic (EEG) excitability, sleep, and breathing. Aim 1 will test the hypothesis that microinjecting neostigmine into the pontine reticular formation of B6 mouse causes a REM sleep-like state and changes in breathing that are concentration-dependent, site-specific within the pons, and blocked by atropine. Aim 2 will use in vivo microdialysis and high performance liquid chromatography (HPLC) to test the hypothesis that dialysis delivery of an adenosine A1 receptor agonist into the prefrontal cortex of B6 mouse will decrease cortical ACh release and EEG power, and delay wake-up time from anesthesia. Aim 3 will use combined microdialysis and microinjection to test the hypothesis that ACh release in the pontine reticular formation of B6 mouse is altered by nitric oxide donors and by inhibitors of nitric oxide synthase. Aim 4 will use a quantitative Western assay to test the hypothesis that brain expression of M2 muscarinic receptor protein varies significantly as a function of mouse strain and brain region. These aims will take this research program in new directions by developing a pharmacological model of rapid eye movement sleep in mouse (Aim 1), quantifying the effects of endogenous neuromodulators on ACh release (Aims 2 and 3), and initiating strain comparisons of muscarinic receptor protein expression (Aim 4). The unifying conceptual scheme of this proposal is that higher level phenotypes such as sleep and breathing (Aim 1) emerge from the expression of lower level phenotypes such as ACh (Aims 2 and 3) and muscarinic cholinergic receptors (Aim 4).