Microinjection of neostigmine into the pontine reticular formation of the mouse: further evaluation of a proposed REM sleep enhancement technique

Microinjections of cholinergic agonists into the pontine reticular formation (PRF) powerfully induce rapid eye movement sleep (REMS) in cats but have comparatively weaker effects in rats. Recently, the cholinomimetic neostigmine has been reported to strongly enhance REMS following microinjection into the PRF of the mouse. That study used behavioral assessments of locomotion in lieu of electrophysiological measures of muscle tone to identify REMS. We sought to confirm that the behavioral state induced in mice by PRF injections of neostigmine meets standard electroencephalogram (EEG) and electromyogram (EMG) criteria for defining REMS. Cortical EEG, nuchal muscle EMG, and PGO waves were recorded from male C57BL/6N mice with chronic indwelling cannulae for the delivery of neostigmine to the PRF. Recordings were made during midday following injections of neostigmine (8.8 mM, 50 nl), 2 h after lights on (LD 12:12). Neostigmine induced a behavioral state characterized by low amplitude, highly desynchronized cortical EEG with little theta, no PGO waves, and a sustained high muscle tone. Behavioral states meeting standard criteria for slow-wave sleep (SWS) and REMS were significantly suppressed compared to baseline recordings, and REMS onset was delayed by 3 h. Consistent with earlier reports, neostigmine did strongly suppress locomotor activity in open field tests and in the home cage. Due to the failure to meet criteria for defining REMS, we conclude that neostigmine microinjection into the PRF of the mouse induces an abnormal waking state rather than REMS.