Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep

Abstract

Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of REM sleep. In the present study performed on rats, phosphorylated cAMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high REM sleep (HR, ~27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With high REM sleep, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low REM sleep (LR, ~2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr = 0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr = 0.88), PnO (Rsqr = 0.87), and SubCD (Rsqr = 0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr = 0.70) and DRN (Rsqr = 0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are inactive. More importantly, the regression analysis indicated that pCREB activation in approximately 98% of PPT cholinergic neurons, was caused by REM sleep. Moreover the results indicate that during REM sleep, PPT intracellular PKA activation and a transcriptional cascade involving pCREB occurs exclusively in the cholinergic neurons.

REM sleep and dreaming: towards a theory of protoconsciousness

“…confirmed that the cholinergic pedunculopontine tegmental neurons are REM-on cells and serotonergic dorsal raphe nucleus and noradrenergic locus coeruleus neurons are REM-off cells 98 . It has also become clear that GABA (γ-aminobutyric acid) and glutamate participate in this process 66 .…”

Cholinergic and glutamatergic agonists induce gamma frequency activity in dorsal subcoeruleus nucleus neurons

“…Lesion of this area produced REM sleep without muscle atonia or ponto-geniculo-occipital (PGO) waves (21,26,29,33), or diminished REM sleep (24). The SubCD is most active during REM sleep (5,13), and injection of the nonspecific acetylcholine receptor agonist carbachol (CAR) or the glutamatergic receptor agonist kainic acid (KA) into this area induced a REM sleep-like state with muscle atonia (2,28,38,44). The SubCD receives afferents from several nuclei, including the pedunculopontine nucleus (PPN) (11).…”

Mechanisms underlying sleep–wake disturbances in alcoholism: Focus on the cholinergic pedunculopontine tegmentum

“…Interestingly, one of the major brain areas in the S-W regulating network that actively regulates both W and REM sleep is the cholinergic cell compartment of the PPT (CCC-PPT) [12, 13, 136, 159]. For example, neuropharmacological and physiological studies have shown that glutamatergic activation of kainate receptors on PPT cholinergic cells induces REM sleep by raising neuronal activity to a medium level, whereas glutamatergic activation of NMDA receptors on these same PPT cholinergic cells induces W by increasing neuronal activity to its highest level [12, 130–133, 159, 160].…”

“…It is already known that the majority of BF cholinergic cells are involved in the induction of W while the other cells are involved in the induction of SWS, but both of these cell groups suppress REM sleep [12, 171, 172]. On the other hand, activation of PPT cholinergic cells increases REM sleep [136]. Therefore, in alcohol-dependent individuals, phase advance in REM sleep could potentially be caused by the over-activation of cholinergic cells in the PPT (but not in the BF).…”