Microinjection of neostigmine into the pontine reticular formation of C57BL/6J mouse enhances rapid eye movement sleep and depresses breathing.
OBJECTIVE
The cholinergic model of rapid eye movement (REM) sleep has contributed significantly to understanding sleep neurobiology and sleep-dependent respiratory depression. The model has been used extensively in cat and rat, but no previous studies have demonstrated cholinergic REM sleep enhancement in mouse. The present study used microinjection of neostigmine into pontine reticular formation of mouse to test the hypothesis that enhancing pontine cholinergic neurotransmission would cause increased REM sleep and sleep disordered breathing.


METHODS
Mice (n=8) were anesthetized and implanted with electrodes for measuring cortical electroencephalogram (EEG). Stainless steel cannulae were stereotaxically implanted to permit subsequent microinjections of 50 nl neostigmine (0.133 microg; 8.8 mM) or saline into the pontine reticular formation. Following recovery, an intensive within-subjects design was used to obtain measures of sleep/wake states, breathing, and locomotor activity. Inferential statistics were provided by t-tests. A probability value of < 0.05 indicated statistical significance.


METHODS
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METHODS
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METHODS
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RESULTS
Behavioral observations and manual scoring of polygraphic recordings showed that neostigmine produced a REM sleep-like state. EEG power analysis using Fast Fourier Transformation confirmed that pontine neostigmine caused EEG activation. Plethysmography demonstrated significantly disordered breathing. Compared to waking, pontine microinjection of neostigmine decreased respiratory rate (-64%) and minute ventilation (-75%). Pontine neostigmine significantly increased duration of inspiration (138%) and expiration (140%) above waking levels and decreased inspiratory flow (-69%). Additional studies showed that pontine neostigmine significantly depressed locomotor activity.


CONCLUSIONS
This study is the first to demonstrate cholinergic REM sleep enhancement in unanesthetized, intact mouse. The results encourage future studies to characterize similarities and differences in cholinergic REM sleep enhancement in additional inbred strains and in transgenic mice. Such comparisons will help characterize sleep and breathing as intermediate phenotypes that are determined, in part, by the lower level phenotype of pontine cholinergic neurotransmission.
Lydic
Ralph
R
Department of Anesthesiology, University of Michigan, Ann Arbor, MI 48109-0615, USA. rlydic@umich.edu
Douglas
Christopher L
CL
Baghdoyan
Helen A
HA
D000818:Animals; D002800:Cholinesterase Inhibitors / Q000008:administration & dosage / Q000009:adverse effects*; D004567:Electrodes, Implanted; D004569:Electroencephalography; D005133:Eye Movements / Q000187:drug effects*; D005583:Fourier Analysis; D008124:Locomotion / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections; D009388:Neostigmine / Q000008:administration & dosage / Q000009:adverse effects*; D010991:Plethysmography / Q000379:methods; D011149:Pons / Q000187:drug effects*; D012154:Reticular Formation / Q000187:drug effects*; D012891:Sleep Apnea Syndromes / Q000139:chemically induced / Q000175:diagnosis; D012895:Sleep, REM / Q000187:drug effects*; D012965:Sodium Chloride / Q000008:administration & dosage / Q000494:pharmacology; D014851:Wakefulness / Q000187:drug effects
D002800:Cholinesterase Inhibitors; D009388:Neostigmine; D012965:Sodium Chloride