The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system (RAS), is known to modulate waking and rapid eye movement (REM) sleep. REM sleep decreases between 10 and 30 days postnatally in the rat, with the majority occurring between 12 and 21 days. We investigated the possibility that changes in the cholinergic, muscarinic and/or nicotinic, input to PPN neurons could explain at least part of the developmental decrease in REM sleep. We recorded intracellularly from PPN neurons in 12-21 day rat brainstem slices maintained in artificial cerebrospinal fluid (aCSF) and found that application of the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) depolarized PPN neurons early in development, then hyperpolarized PPN neurons by day 21. Most of the effects of DMPP persisted following application of the sodium channel blocker tetrodotoxin (TTX), and in the presence of glutamatergic, serotonergic, noradrenergic and GABAergic antagonists, but were blocked by the nicotinic antagonist mecamylamine (MEC). The mixed muscarinic agonist carbachol (CAR) hyperpolarized all type II (A current) PPN cells and depolarized all type I (low threshold spike-LTS current) and type III (A + LTS current) PPN cells, but did not change effects during the period known for the developmental decrease in REM sleep. The effects of CAR persisted in the presence of TTX but were mostly blocked by the muscarinic antagonist atropine (ATR), and the remainder by MEC. We conclude that, while the nicotinic inputs to the PPN may help modulate the developmental decrease in REM sleep, the muscarinic inputs appear to modulate different types of cells differentially.

Rapid eye movement sleep decreases between 10 and 30 days postnatally in the rat. The pedunculopontine nucleus is known to modulate waking and rapid eye movement sleep, and pedunculopontine nucleus neurons are thought to be hyperpolarized by noradrenergic input from the locus coeruleus. The goal of the study was to investigate the possibility that a change in α-2 adrenergic inhibition of pedunculopontine nucleus cells during this period could explain at least part of the developmental decrease in rapid eye movement sleep. We, therefore, recorded intracellularly in 12-21 day rat brainstem slices maintained in oxygenated artificial cerebrospinal fluid. Putative cholinergic vs. non-cholinergic pedunculopontine nucleus neurons were identified using nicotinamide adenine dinucleotide phosphate diaphorase histochemistry and intracellular injection of neurobiotin (Texas Red immunocytochemistry). Pedunculopontine nucleus neurons also were identified by intrinsic membrane properties, type I (low threshold spike), type II (A) and type III (A+low threshold spike), as previously described. Clonidine (20 μM) hyperpolarized most cholinergic and non-cholinergic pedunculopontine nucleus cells. This hyperpolarization decreased significantly in amplitude (mean±S.E.) from -6.8±1.0 mV at 12-13 days, to -3.0±0.7 mV at 20-21 days. However, much of these early effects (12-15 days) were indirect such that direct effects (tested following sodium channel blockade with tetrodotoxin (0.3 μM)) resulted in hyperpolarization averaging -3.4±0.5 mV, similar to that evident at 16-21 days. Non-cholinergic cells were less hyperpolarized than cholinergic cells at 12-13 days (-1.6±0.3 mV), but equally hyperpolarized at 20-21 days (-3.3±1.3 mV). In those cells tested, hyperpolarization was blocked by yohimbine, an α-2 adrenergic receptor antagonist (1.5 μM). These results suggest that the α-2 adrenergic receptor on cholinergic pedunculopontine nucleus neurons activated by clonidine may play only a modest role, if any, in the developmental decrease in rapid eye movement sleep. Clonidine blocked or reduced the hyperpolarization-activated inward cation conductance, so that its effects on the firing rate of a specific population of pedunculopontine nucleus neurons could be significant. In conclusion, the α-2 adrenergic input to pedunculopontine nucleus neurons appears to consistently modulate the firing rate of cholinergic and non-cholinergic pedunculopontine nucleus neurons, with important effects on the regulation of sleep-wake states.