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The pedunculopontine nucleus (PPN) has long been known to be part of the reticular activating system (RAS) in charge of arousal and REM sleep. We previously showed that in vitro exposure to a HDAC Class I and II mixed inhibitor (TSA), or a specific HDAC class IIa inhibitor (MC 1568), decreased PPN gamma oscillations. Given the lack of information on systemic in vivo treatments on neuronal synaptic properties, the present study was designed to investigate the systemic effect of HDAC inhibitors (HDACi) on PPN rhythmicity. Rat pups were injected (acute, single dose) with TSA (4 or 20 mg/kg), MC1568 (4 or 20 mg/kg), or MS275 (20 or 100 mg/kg). Our results show that MC1568 (20 mg/kg) reduced mean frequency of PPN oscillations at gamma band, while increasing mean input resistance (Rm) of PPN neurons. For TSA (4 and 20 mg/kg), we observed reduced mean frequency of oscillations at gamma band and increased mean Rm of PPN neurons. Systemic administration of 20 mg/kg MC1568 and TSA effects on Rm were washed out after 60 min of in vitro incubation of PPN slices, suggesting an underlying functional recovery of PPN calcium-mediated gamma band oscillations over time. In addition, at a lower dose, 4 mg/kg, MC1568 and TSA induced higher mean amplitude gamma oscillations. Blocking HDAC class I might not have deleterious effects on gamma activity, but, more importantly, the inhibition of HDAC class I (at 100 mg/kg) increased gamma band oscillations. In conclusion, the present results in vivo validate our previous findings in vitro and further expand information on the effects of HDAC inhibition on PPN rhythmicity. PPN neurons require normal activity of HDAC class IIa in order to oscillate at gamma band., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The pedunculopontine nucleus (PPN) is part of the reticular activating system (RAS) in charge of arousal and rapid eye movement sleep. The presence of high-frequency membrane oscillations in the gamma-band range in the PPN has been extensively demonstrated both in vivo and in vitro. Our group previously described histone deacetylation (HDAC) inhibition in vitro induced protein changes in F-actin cytoskeleton and intracellular Ca 2+ concentration regulation proteins in the PPN. Here, we present evidence that supports the presence of a fine balance between HDAC function and calcium calmodulin kinase II-F-actin interactions in the PPN. We modified F-actin polymerization in vitro by using jasplakinolide (1 μM, a promoter of F-actin stabilization), or latrunculin-B (1 μM, an inhibitor of actin polymerization). Our results showed that shifting the balance in either direction significantly reduced PPN gamma oscillation as well as voltage-dependent calcium currents.

Four major discoveries on the function of the pedunculopontine nucleus (PPN) have significantly advanced our understanding of the role of arousal in neurodegenerative disorders. The first was the finding that stimulation of the PPN-induced controlled locomotion on a treadmill in decerebrate animals, the second was the revelation of electrical coupling in the PPN and other arousal and sleep-wake control regions, the third was the determination of intrinsic gamma band oscillations in PPN neurons, and the last was the discovery of gene transcription resulting from the manifestation of gamma activity in the PPN. These discoveries have led to novel therapies such as PPN deep brain stimulation (DBS) for Parkinson's disease (PD), identified the mechanism of action of the stimulant modafinil, determined the presence of separate mechanisms underlying gamma activity during waking versus REM sleep, and revealed the presence of gene transcription during the manifestation of gamma band oscillations. These discoveries set the stage for additional major advances in the treatment of a number of disorders., (© 2019 Wiley Periodicals, Inc.)

Objectives: This limited review examines the role of the reticular activating system (RAS), especially the pedunculopontine nucleus (PPN), one site of origin of bottom-up gamma, in the symptoms of bipolar disorder (BD)., Methods: The expression of neuronal calcium sensor protein 1 (NCS-1) in the brains of BD patients is increased. It has recently been found that all PPN neurons manifest intrinsic membrane beta/gamma frequency oscillations mediated by high threshold calcium channels, suggesting that it is one source of bottom-up gamma. This review specifically addresses the involvement of these channels in the manifestation of BD., Results: Excess NCS-1 was found to dampen gamma band oscillations in PPN neurons. Lithium, a first line treatment for BD, was found to decrease the effects of NCS-1 on gamma band oscillations in PPN neurons. Moreover, gamma band oscillations appear to epigenetically modulate gene transcription in PPN neurons, providing a new direction for research in BD., Conclusions: This is an area needing much additional research, especially since the dysregulation of calcium channels may help explain many of the disorders of arousal in, elicit unwanted neuroepigenetic modulation in, and point to novel therapeutic avenues for, BD., (© 2018 The Authors. Bipolar Disorders Published by John Wiley & Sons Ltd.)

Epigenetic mechanisms (i.e., histone post-translational modification and DNA methylation) play a role in regulation of gene expression. The pedunculopontine nucleus (PPN), part of the reticular activating system, manifests intrinsic gamma oscillations generated by voltage-dependent, high threshold N- and P/Q-type Ca 2+ channels. We studied whether PPN intrinsic gamma oscillations are affected by inhibition of histone deacetylation. We showed that, a) acute in vitro exposure to the histone deacetylation Class I and II inhibitor trichostatin A (TSA, 1 μM) eliminated oscillations in the gamma range, but not lower frequencies, b) pre-incubation with TSA (1 μM, 90-120 min) also decreased gamma oscillations, c) Ca 2+ currents (I Ca ) were reduced by TSA, especially on cells with P/Q-type channels, d) a HDAC Class I inhibitor MS275 (500 nM), and a Class IIb inhibitor Tubastatin A (150-500 nM), failed to affect gamma oscillations, e) MC1568, a HDAC Class IIa inhibitor (1 μM), blocked gamma oscillations, and f) the effects of both TSA and MC1568 were blunted by blockade of CaMKII with KN-93 (1 μM). These results suggest a cell type specific effect on gamma oscillations when histone deacetylation is blocked, suggesting that gamma oscillations through P/Q-type channels modulated by CaMKII may be linked to processes related to gene transcription.

Bipolar disorder is characterized by sleep dysregulation, suggesting a role for the reticular activating system (RAS). Postmortem studies showed increased expression of neuronal calcium sensor protein 1 (NCS-1) in the brains of some bipolar disorder patients, and reduced or aberrant gamma band activity is present in the same disorder. Lithium (Li + ) has been shown to effectively treat the mood disturbances in bipolar disorder patients. We previously showed that NCS-1 at low levels increased, and at high levels decreased, gamma oscillations in RAS pedunculopontine neurons (PPN), and that Li + decreased these oscillations. We previously described the effects of each agent on oscillations, G-protein mechanisms, and Ca 2+ currents. However, we designed the present experiments to determine the nature of the interaction of NCS-1 and Li + at physiological concentrations that would have an effect within minutes of application. As expected, Li + decreased gamma oscillation amplitude, while NCS-1 increased the amplitude of gamma oscillations. We identified NCS-1 at 2 μmol/L as a concentration that increased gamma oscillations within 5-10 min, and Li + at 10 μmol/L as a concentration that decreased gamma oscillations within 5 min. The combined application of NCS-1 and Li + at these concentrations showed that Li + reduced the effects of NCS-1 on oscillation amplitude within 5-10 min. These results demonstrate that at physiological levels, Li + acts to reduce the effects of NCS-1 so that, given over expression of NCS-1, Li + would have salutary effects., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV. Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Human postmortem studies reported increased expression of neuronal calcium sensor protein 1 (NCS-1) in the brains of some bipolar disorder patients, and reduced or aberrant gamma band activity is present in the same disorder. Bipolar disorder is characterized by sleep dysregulation, suggesting a role for the reticular activating system (RAS). Lithium (Li(+)) has been shown to effectively treat the mood disturbances in bipolar disorder patients and was proposed to act by inhibiting the interaction betweenNCS-1 and inositol 1,4,5-triphosphate receptor protein (InsP3R).NCS-1 is known to enhance the activity of InsP3R, and of Ca(2+)-mediated gamma oscillatory activity in the pedunculopontine nucleus (PPN), part of theRAS This study aimed to determine the nature of some of the intracellular mechanisms of Li(+)on ratPPNcells and to identify the interaction between Li(+)andNCS-1. Since Li(+)has been shown to act by inhibiting the enhancing effects ofNCS-1, we tested the hypothesis that Li(+)would reduced the effects of overexpression ofNCS-1 and prevent the downregulation of gamma band activity. Li(+)decreased gamma oscillation frequency and amplitude by downregulating Ca(2+)channel activity, whereasNCS-1 reduced the effect of Li(+)on Ca(2+)currents. These effects were mediated by a G-protein overinhibition of Ca(2+)currents. These results suggest that Li(+)affected intracellular pathways involving the activation of voltage-gated Ca(2+)channels mediated by an intracellular mechanism involving voltage-dependent activation of G proteins, thereby normalizing gamma band oscillations mediated by P/Q-type calcium channels modulated byNCS-1., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The pedunculopontine nucleus (PPN) region has received considerable attention in clinical studies as a target for deep brain stimulation (DBS) in Parkinson disease. These studies have yielded variable results with an overall impression of improvement in falls and freezing in many but not all patients treated. We evaluated the available data on the surgical anatomy and terminology of the PPN region in a companion paper. Here we focus on issues concerning surgical technique, imaging, and early side effects of surgery. The aim of this paper was to gain more insight into the reasoning for choosing specific techniques and to discuss shortcomings of available studies. Our data demonstrate the wide range in almost all fields which were investigated. There are a number of important challenges to be resolved, such as identification of the optimal target, the choice of the surgical approach to optimize electrode placement, the impact on the outcome of specific surgical techniques, the reliability of intraoperative confirmation of the target, and methodological differences in postoperative validation of the electrode position. There is considerable variability both within and across groups, the overall experience with PPN DBS is still limited, and there is a lack of controlled trials. Despite these challenges, the procedure seems to provide benefit to selected patients and appears to be relatively safe. One important limitation in comparing studies from different centers and analyzing outcomes is the great variability in targeting and surgical techniques, as shown in our paper. The challenges we identified will be of relevance when designing future studies to better address several controversial issues. We hope that the data we accumulated may facilitate the development of surgical protocols for PPN DBS., (© 2016 S. Karger AG, Basel.)

Several lines of evidence over the last few years have been important in ascertaining that the pedunculopontine nucleus (PPN) region could be considered as a potential target for deep brain stimulation (DBS) to treat freezing and other problems as part of a spectrum of gait disorders in Parkinson disease and other akinetic movement disorders. Since the introduction of PPN DBS, a variety of clinical studies have been published. Most indicate improvements in freezing and falls in patients who are severely affected by these problems. The results across patients, however, have been variable, perhaps reflecting patient selection, heterogeneity in target selection and differences in surgical methodology and stimulation settings. Here we outline both the accumulated knowledge and the domains of uncertainty in surgical anatomy and terminology. Specific topics were assigned to groups of experts, and this work was accumulated and reviewed by the executive committee of the working group. Areas of disagreement were discussed and modified accordingly until a consensus could be reached. We demonstrate that both the anatomy and the functional role of the PPN region need further study. The borders of the PPN and of adjacent nuclei differ when different brainstem atlases and atlas slices are compared. It is difficult to delineate precisely the PPN pars dissipata from the nucleus cuneiformis, as these structures partially overlap. This lack of clarity contributes to the difficulty in targeting and determining the exact localization of the electrodes implanted in patients with akinetic gait disorders. Future clinical studies need to consider these issues., (© 2016 S. Karger AG, Basel.)

Reduced levels of gamma-band activity are present in schizophrenia and bipolar disorder patients. In the same disorders, increased neuronal calcium sensor protein-1 (NCS-1) expression was reported in a series of postmortem studies. These disorders are also characterized by sleep dysregulation, suggesting a role for the reticular activating system (RAS). The discovery of gamma-band activity in the pedunculopontine nucleus (PPN), the cholinergic arm of the RAS, revealed that such activity was mediated by high-threshold calcium channels that are regulated by NCS-1. We hypothesized that NCS-1 normally regulates gamma-band oscillations through these calcium channels and that excessive levels of NCS-1, such as would be expected with overexpression, decrease gamma-band activity. We found that PPN neurons in rat brain slices manifested gamma-band oscillations that were increased by low levels of NCS-1 but suppressed by high levels of NCS-1. Our results suggest that NCS-1 overexpression may be responsible for the decrease in gamma-band activity present in at least some schizophrenia and bipolar disorder patients., (Copyright © 2015 the American Physiological Society.)

The pedunculopontine nucleus (PPN) is a component of the reticular activating system (RAS), and is involved in the activated states of waking and rapid eye movement (REM) sleep. Gamma oscillations (approximately 30-80 Hz) are evident in all PPN neurons and are mediated by high-threshold voltage-dependent N- and P/Q-type calcium channels. We tested the hypothesis that high-speed calcium imaging would reveal calcium-mediated oscillations in dendritic compartments in synchrony with patch-clamp recorded oscillations during depolarizing current ramps. Patch-clamped 8- to 16-day-old rat PPN neurons (n = 67 out of 121) were filled with Fura 2, Bis Fura, or OGB1/CHR. This study also characterized a novel ratiometric technique using Oregon Green BAPTA-1 (OGB1) with coinjections of a new long-stokes-shift dye, Chromeo 494 (CHR). Fluorescent calcium transients were blocked with the nonspecific calcium channel blocker cadmium, or by the combination of ω-agatoxin-IVA, a specific P/Q-type calcium channel blocker, and ω-conotoxin-GVIA, a specific N-type calcium channel blocker. The calcium transients were evident in different dendrites (suggesting channels are present throughout the dendritic tree) along the sampled length without interruption (suggesting channels are evenly distributed), and appeared to represent a summation of oscillations present in the soma. We confirm that PPN calcium channel-mediated oscillations are due to P/Q- and N-type channels, and reveal that these channels are distributed along the dendrites of PPN cells.

The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system, regulates waking and rapid eye movement sleep. Here, we demonstrate immunohistochemical labeling of the leptin receptor signaling isoform in PPN neurons, and investigated the effects of G-protein modulation and the leptin triple antagonist (TA) on the action of leptin in the PPN. Whole-cell patch clamp recordings were performed in rat brainstem slices from 9 to 17 day old pups. Previous results showed that leptin caused a partial blockade of sodium (I(Na)) and h-current (I(H)) in PPN neurons. TA (100 nM) reduced the blockade of I(Na) (~ 50% reduction) and I(H) (~ 93% reduction) caused by leptin. Intracellular guanosine 5'-[β-thio]diphosphate trilithium salt (a G-protein inhibitor) significantly reduced the effect of leptin on I(Na) (~ 60% reduction) but not on I(H) (~ 25% reduction). Intracellular GTPγS (a G-protein activator) reduced the effect of leptin on both I(Na) (~ 80% reduction) and I(H) (~ 90% reduction). These results suggest that the effects of leptin on the intrinsic properties of PPN neurons are leptin receptor- and G-protein dependent. We also found that leptin enhanced NMDA receptor-mediated responses in single neurons and in the PPN population as a whole, an effect blocked by TA. These experiments further strengthen the association between leptin dysregulation and sleep disturbances. Beck et al. investigated the effects of leptin on the intrinsic properties of neurons from the pedunculopontine nucleus (PPN). Leptin reduced the amplitude of voltage-gated sodium (I(Na)) and hyperpolarization-activated cyclic nucleotide-gated HCN (I(H)) channels. These effects were antagonized by a leptin receptor (OB-R) antagonist and by the G-protein antagonist GDPβ., (© 2013 International Society for Neurochemistry.)

Leptin, a hormone that regulates appetite and energy expenditure, is increased in obese individuals, although these individuals often exhibit leptin resistance. Obesity is characterized by sleep/wake disturbances, such as excessive daytime sleepiness, increased REM sleep, increased nighttime arousals, and decreased percentage of total sleep time. Several studies have shown that short sleep duration is highly correlated with decreased leptin levels in both animal and human models. Arousal and rapid eye movement (REM) sleep are regulated by the cholinergic arm of the reticular activating system, the pedunculopontine nucleus (PPN). The goal of this project was to determine the role of leptin in the PPN, and thus in obesity-related sleep disorders. Whole-cell patch-clamp recordings were conducted on PPN neurons in 9- to 17-day-old rat brainstem slices. Leptin decreased action potential (AP) amplitude, AP frequency, and h-current (I(H)). These findings suggest that leptin causes a blockade of Na⁺ channels. Therefore, we conducted an experiment to test the effects of leptin on Na⁺ conductance. To determine the average voltage dependence of this conductance, results from each cell were equally weighted by expressing conductance as a fraction of the maximum conductance in each cell. I Na amplitude was decreased in a dose-dependent manner, suggesting a direct effect of leptin on these channels. The average decrease in Na⁺ conductance by leptin was ~40 %. We hypothesize that leptin normally decreases activity in the PPN by reducing I(H) and I(Na) currents, and that in states of leptin dysregulation (i.e., leptin resistance) this effect may be blunted, therefore causing increased arousal and REM sleep drive, and ultimately leading to sleep-related disorders.

One element of the reticular activating system (RAS) is the pedunculopontine nucleus (PPN), which projects to the thalamus to trigger thalamocortical rhythms and the brainstem to modulate muscle tone and locomotion. The PPN is a posterior midbrain site known to induce locomotion in decerebrate animals when activated at 40-60 Hz, and has become a target for DBS in disorders involving gait deficits. We developed a research program using brainstem slices containing the PPN to study the cellular and molecular organization of this region. We showed that PPN neurons preferentially fire at gamma band frequency (30-60 Hz) when maximally activated, accounting for the effects of electrical stimulation. In addition, we developed the P13 midlatency auditory evoked potential, which is generated by PPN outputs, in freely moving rats. This allows the study of PPN cellular and molecular mechanisms in the whole animal. We also study the P50 midlatency auditory evoked potential, which is the human equivalent of the rodent P13 potential, allowing us to study PPN-related processes detected in vitro, confirmed in the whole animal, and tested in humans. Previous findings on the P50 potential in PD suggest that PPN output in this disorder is overactive. This translational research program led to the discovery of a novel mechanism of sleep-wake control based on electrical coupling, pointing the way to a number of new clinical applications in the development of novel stimulants (e.g., modafinil) and anesthetics. In addition, it provides methods for monitoring therapeutic efficacy of DBS in humans and animal models.

The pedunculopontine nucleus (PPN) is involved in the generation and maintenance of waking and rapid eye movement (REM) sleep, forming part of the reticular activating system. The PPN receives glutamatergic afferents from other mesopontine nuclei, and glutamatergic input is believed to be involved in the generation of arousal states. We tested the hypothesis that, from postnatal days 9 to 17 in the rat, there are developmental changes in the glutamate receptor subtypes that contribute to the responses of PPN neurons. Whole cell patch-clamp recordings were conducted using brainstem slices from 9- to 17-day-old rats. All cells (types I, II, and III; randomly selected or thalamic-projecting) responded to bath application of the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA) and kainic acid (KA). A developmental decrease in the contribution of the NMDA receptor and developmental increase in the contribution of the KA receptor was observed following electrical stimulation-induced glutamate input. These changes were also observed following bath application in different cell types (randomly selected vs. thalamic-projecting). KA bath application produced an increase in the paired-pulse ratio (PPR) and a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs), suggesting that presynaptic KA autoreceptors may decrease the probability of synaptic glutamate input. In contrast, NMDA application produced no changes in the PPR or mEPSCs. Changes in glutamatergic excitability of PPN cell types could underlie the developmental decrease in REM sleep.

The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and REM sleep. GABAergic modulation of this area appears to regulate sleep-wake cycles. Eszopiclone (ESZ), a nonbenzodiazepine hypnotic agent, appears to modulate GABAergic receptors. However, the action site of ESZ in the brain is still unresolved. We tested the hypothesis that ESZ acts by potentiating GABA(A) receptors on PPN neurons. Wholecell voltage clamp recordings were performed on PPN neurons in 7-15 day rat brainstem slices, and the potentiating effects of ESZ on the responses to the GABA(A) receptor agonist isoguvacine (IGV), and on GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), were determined. In the presence of tetrodotoxin, ESZ (1) increased the amplitude of the outward current induced by IGV, (2) increased its duration, and (3) enhanced the IGV-induced decrease in input resistance (Rin). The GABA(A) receptor antagonist gabazine (GBZ) blocked these effects. ESZ alone did not induce detectable currents or change Rin at a holding potential of -60 mV, but when held at 0 mV, ESZ induced an outward current in 13/21 PPN cells, an effect blocked by GBZ. ESZ also increased the amplitude (n = 18/21), duration (n = 17/21), and frequency (n = 13/15) of IPSCs. ESZ may potentiate GABA(A) inhibition in the PPN via pre- and post-synaptic modulation, which may underlie the hypnotic effects of ESZ. The differential effects of ESZ on both pre- and post-synaptic sites may partially explain why it has less significant side effects compared to other hypnotic agents.

Rapid eye movement sleep decreases dramatically during development. We tested the hypothesis that some of this decrease may be due to GABAergic inhibition of reticular activating system neurons. Recordings of pedunculopontine neurons in vitro showed that the gamma-amino-butyric acid, receptor agonist muscimol depolarized noncholinergic cells early in the developmental decrease in rapid eye movement sleep, and hyperpolarized them later. Most cholinergic cells were hyperpolarized throughout the period tested. The gamma-amino-butyric acid b receptor agonist baclofen hyperpolarized both cholinergic and noncholinergic cells, although the degree of polarization decreased with age. Part of the gradual decrement in rapid eye movement sleep during development may be due in part to the increasing inhibition mediated by gamma-amino-butyric acid, a receptor on pedunculopontine neurons. This influence, however, appears to be mainly on noncholinergic cells.

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.