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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.)

The reticular activating system (RAS) is not an amorphous region but distinct nuclei with specific membrane properties that dictate their firing during waking and sleep. The locus coeruleus and raphe nucleus fire during waking and slow wave sleep, with the pedunculopontine nucleus (PPN) firing during both waking and REM sleep, the states manifesting arousal-related EEG activity. Two important discoveries in the PPN in the last 10 years are, 1) that some PPN cells are electrically coupled, and 2) every PPN cell manifests high threshold calcium channels that allow them to oscillate at beta/gamma band frequencies. The role of arousal in drug abuse is considered here in terms of the effects of drugs of abuse on these two mechanisms. Drug abuse and the perception of withdrawal/relapse are mediated by neurobiological processes that occur only when we are awake, not when we are asleep. These relationships focus on the potential role of arousal, more specifically of RAS electrical coupling and gamma band activity, in the addictive process as well as the relapse to drug use., (Copyright © 2017. Published by Elsevier B.V.)

The relationship between arousal, perception, and visual neglect was examined in this case study. Cold pressor stimulation (CPS: immersing the foot in iced water) was used to manipulate arousal and to determine its effects on contralesional neglect, perception of stimulus intensity (magnitude estimation), reaction time, and an electrophysiological correlate of ascending reticular activating system activity (i.e., the P50 potential). Measures that normalized from baseline following CPS included contralesional neglect on a clock drawing test, perception of stimulus magnitude, and P50 amplitude. The P50 amplitude returned to its abnormally low baseline level 20 min after CPS ended, indicating that CPS increased arousal.

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.

Objective: Quantitative measures of pre-attentional, attentional and frontal lobe processes were compared to evaluate quantitative measures of these deficits in Ex-Preterm vs. Ex-Term adolescents., Methods: We compared 43 Ex-Preterm with 26 Ex-Term adolescents using the P50 auditory potential, the Psychomotor Vigilance Task (PVT), a reaction time (RT) test, and Near Infrared Spectroscopy (NIRS)., Results: The mean amplitude (+/-SE) of the P50 amplitude was similar in the Ex-Preterm (1.8+/-1.4 microV) vs. Ex-Term adolescents (1.8+/-0.6 microV, df = 68, F = 0.05, p = 0.8), but the Ex-Preterm group showed a trimodal distribution in amplitude (High, 3.3+/-0.4 microV, df=42.25, F=19.2, p < 0.01; Medium, 1.7+/-0.1 microV, df = 39, F = 0.41, p = 0.53; Low, 0.7+/-0.1 microV, df = 40, F = 49.5, p < 0.01) suggested by statistically significant variance between populations (Kolmogorov-Kuiper test, df = 42.25, F = 5.4, p < 0.01). Mean RT was longer in Ex-Preterm (250+/-8 ms) vs. Ex-Term subjects (200+/-5 ms, df = 68, F = 18.8, p < 0.001). PVT lapses were increased in Ex-Preterm subjects, and varied inversely with P50 amplitude (Overall Mean 17+/-5 lapses, df = 67, F = 5.34, p < 0.05; Low P50 amplitude, 25+/-10, df = 40, F = 8.8, p < 0.01; Medium, 21+/-11, df = 38, F = 5.37, p < 0.05; High, 6+/-2, df = 39, F = 6.78, p < 0.01) vs. Ex-Term subjects (2+/-0.4 lapses, p < 0.01). NIRS levels did not differ statistically, but tended to correlate with P50 amplitude in the Ex-Preterm group., Conclusions: These findings suggest differential pre-attentional, attentional and frontal lobe dysfunction in Ex-Preterm adolescents., Significance: These measures could provide a means to objectively assess differential dysregulation of arousal and attention in Ex-Preterm adolescents, allowing optimization of therapeutic designs.

We investigated the effects of tinnitus on measures of arousal and attention at various levels of the neuraxis to derive a profile of the pathophysiology of tinnitus. Individuals with tinnitus of at least 6 months' duration (14 male, 15 female) and healthy controls (14 male, 21 female) were tested for arousal and habituation to repetitive stimulation at the brainstem-thalamus level by measuring the P50 potential, a scalp-recorded, auditory-evoked response, using pairs of click stimuli. We used the psychomotor vigilance task, a reaction-time test, to assess attentional processes mediated by thalamocortical functions. We then correlated deficits in arousal and attention, as measured by these tests, with perceived tinnitus severity. Results showed no difference between tinnitus patients and controls in level of arousal or habituation to repetitive sensory stimulation, as measured by the amplitude of the P50 potential and the ability to suppress a second, closely paired stimulus, respectively. However, reaction-time assessments showed that patients with tinnitus have attentional deficits relative to controls (p = .02). We found no significant correlation between sleep disturbance or tinnitus severity and reaction-time testing.

Much of the controversy surrounding the induction of locomotion following stimulation of mesopontine sites, including the pedunculopontine nucleus (PPN), appears based on procedural differences, including stimulus onset, delay preceding stepping, and frequency of stimuli. The results reviewed in this chapter address these issues and provide novel information suggesting that descending projections from the PPN may exert a frequency-dependent effect. Stimulation at approximately 60 Hz (which induces prolonged tonic firing) may exercise a "push" towards locomotion (activation of pontine interneurons) as well as a "pull" away from decreased muscle tone (inhibiting giant pontine reticulospinal cells). Higher frequencies of stimulation (> 100 Hz, which induces phasic burst-like activity) may "push" towards decreases in muscle tone, including the atonia of rapid eye movement sleep (activating giant pontine reticulospinal cells).

An intrinsic function of the reticular activating system (RAS) is its participation in fight vs. flight responses such that alerting stimuli simultaneously activate thalamocortical systems, as well as postural and locomotor systems, in order to enable an appropriate response. The P50 midlatency auditory-evoked potential appears to be an ascending manifestation of the cholinergic arm of the RAS in eliciting changes in arousal state. Abnormalities in the manifestation of the P50 potential are present in disorders which include: (1) dysregulation of sleep-wake cycles; (2) abnormalities in reflex/postural, especially, startle, responses; and (3) malfunctions in flight vs. flight responses. In general, the P50 potential appears to be upregulated (increased amplitude and/or decreased sensory gating) in disorders which are marked by upregulation of RAS outputs (hypervigilance), and downregulated in disorders characterized by decreased RAS outputs (hypovigilance). Many of the disorders discussed have a developmental etiology and a postpubertal age of onset.

The P13 potential is the rodent equivalent of the P50 potential, which is an evoked response recorded at the vertex (Vx) 50 ms following an auditory stimulus in humans. Both the P13 and P50 potentials are only present during waking and rapid eye movement (REM) sleep, and are considered to be measures of level of arousal. The source of the P13 and P50 potentials appears to be the pedunculopontine nucleus (PPN), a brainstem nucleus with indirect ascending projections to the cortex through the intralaminar thalamus, mediating arousal, and descending inhibitory projections to the caudal pontine reticular formation (CPRF), which mediates the auditory startle response (SR). We tested the hypothesis that intracranial microinjection (ICM) of glutamate (GLU) or GLU receptor agonists will increase the activity of PPN neurons, resulting in an increased P13 potential response, and decreased SR due to inhibitory projections from the PPN to the CPRF, in freely moving animals. Cannulae were inserted into the PPN to inject neuroactive agents, screws were inserted into the Vx in order to record the P13 potential, and electrodes inserted into the dorsal nuchal muscle to record electromyograms and SR amplitude. Our results showed that ICM of GLU into the PPN dose-dependently increased the amplitude of the P13 potential and decreased the amplitude of the SR. Similarly, ICM of N-methyl-d-aspartic acid or kainate into the PPN increased the amplitude of the P13 potential. These findings indicate that glutamatergic input to the PPN plays a role in arousal control in vivo, and changes in glutamatergic input, or excitability of PPN neurons, could be implicated in a number of neuropsychiatric disorders with the common symptoms of hyperarousal and REM sleep dysregulation.

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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The pedunculopontine nucleus ( PPN) is part of the Reticular Activating System, and active during waking and REM sleep. Previous results showed that all PPN cells plateau at gamma frequencies and intrinsic membrane oscillations in PPN neurons are mediated by high-threshold N- and P/Q-type Ca2+ channels. The present study was designed to determine whether some PPN cells have only N-, only P/Q-, or both N- and P/Q-type Ca2+ channels. We used patch-clamp recordings in PPN cells in slices from anesthetized rat pups in the presence of synaptic receptor blockers ( SB) and Tetrodotoxin ( TTX), and applied ramps to induce intrinsic membrane oscillations. We found that all PPN cell types showed gamma oscillations in the presence of SB+ TTX when using current ramps. In 50% of cells, the N-type Ca2+ channel blocker ω-Conotoxin- GVIA ( ω-CgTx) reduced gamma oscillation amplitude, while subsequent addition of the P/Q-type blocker ω-Agatoxin- IVA ( ω-Aga) blocked the remaining oscillations. Another 20% manifested gamma oscillations that were not significantly affected by the addition of ω-CgTx, however, ω-Aga blocked the remaining oscillations. In 30% of cells, ω-Aga had no effect on gamma oscillations, while ω-CgTx blocked them. These novel results confirm the segregation of populations of PPN cells as a function of the calcium channels expressed, that is, the presence of cells in the PPN that manifest gamma band oscillations through only N-type, only P/Q-type, and both N-type and P/Q-type Ca2+ channels. [ABSTRACT FROM AUTHOR]

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 mani- fested 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (INa) and h-current (IH) in PPN neurons. TA (100 nM) reduced the blockade of INa (~ 50% reduction) and IH (~ 93% reduction) caused by leptin. Intracellular guanosine 5′-[β-thio]diphosphate trilithium salt (a G-protein inhibitor) significantly reduced the effect of leptin on INa(~ 60% reduction) but not on IH (~ 25% reduction). Intracellular GTPγS (a G-protein activator) reduced the effect of leptin on both INa (~ 80% reduction) and IH (~ 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. [ABSTRACT FROM AUTHOR]

Summary: This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit 1) electrical coupling mainly in GABAergic cells, and 2) gamma band activity in virtually all of the cells. Specifically, cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine dorsal subcoeruleus nucleus dorsalis (SubCD) 1) show electrical coupling, and 2) all fire in the beta/gamma band range when maximally activated, but no higher. The mechanism behind electrical coupling is important because the stimulant modafinil was shown to increase electrical coupling. We also provide recent findings demonstrating that all cells in the PPN and Pf have high threshold, voltage-dependent P/Q-type calcium channels that are essential to gamma band activity. On the other hand, all SubCD, and some PPN, cells manifested sodium-dependent subthreshold oscillations. A novel mechanism for sleep-wake control based on transmitter interactions, electrical coupling, and gamma band activity is described. We speculate that continuous sensory input will modulate coupling and induce gamma band activity in the RAS that could participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. [ABSTRACT FROM AUTHOR]

Abstract: Bray PA, Mamiya N, Fann AV, Gellman H, Skinner RD, Garcia-Rill EE. Modulation of the sleep state–dependent P50 midlatency auditory-evoked potential by electric stimulation of acupuncture points. Objective: To determine if the P50 midlatency auditory evoked potential, a sleep state–dependent waveform thought to be generated by the reticular activating system, is modulated after surface stimulation of acupuncture points (ie, electroacupuncture). Design: P50 potential recordings were carried out before, during, and after electroacupuncture. Setting: A clinical research center. Participants: Eighty healthy subjects ages 25 to 55 were recorded in 7 investigations. Interventions: Stimulation of 3 specific acupuncture points (Pericardium 6, Heart 3, Liver 3) was compared with no stimulation or with stimulation of control points (Gall Bladder 34, Large Intestine 11, Small Intestine 3). We compared different frequencies of stimulation (5, 60, 100Hz), unilateral versus bilateral stimulation, and the effects of repeated episodes of stimulation. Main Outcome Measures: P50 auditory evoked potential latency, amplitude (measure of level of arousal), and habituation (measure of sensory gait) at interstimulus interval of 250ms. Results: Electroacupuncture at specific points decreased P50 potential amplitude versus electroacupuncture at control points (P=.006) or versus no stimulation (P<.001). The optimal effective frequency was 5Hz (P<.05 at 5Hz, P>.05 at 60 and 100Hz), and unilateral electroacupuncture was not as effective as bilateral electroacupuncture (P=.007). Repeated episodes of bilateral electroacupuncture showed additive effects (P<.05). There were no differences in responsiveness across sexes (P=.79), and electroacupuncture did not affect P50 potential habituation (P>.05). Conclusions: Electroacupuncture may be effectively used to decrease arousal levels, perhaps as adjunct therapy for disorders of hypervigilance. [Copyright &y& Elsevier]

Sopite syndrome, characterized by loss of initiative, sensitivity to normally innocuous sensory stimuli, and impaired concentration amounting to a sensory gating deficit, is commonly associated with Space Motion Sickness (SMS). The amplitude of the P50 potential is a measure of level of arousal, and a paired-stimulus paradigm can be used to measure sensory gating. We used the rotary chair to elicit the sensory mismatch that occurs with SMS by overstimulating the vestibular apparatus. The effects of rotation on the manifestation of the P50 midlatency auditory evoked response were then assessed as a measure of arousal and distractibility. Results showed that rotation-induced motion sickness produced no change in the level of arousal but did produce a significant deficit in sensory gating, indicating that some of the attentional and cognitive deficits observed with SMS may be due to distractibility induced by decreased habituation to repetitive stimuli. [ABSTRACT FROM AUTHOR]

Our discovery of low-threshold stimulation-induced locomotion in the pedunculopontine nucleus (PPN) led to the clinical use of deep brain stimulation (DBS) for the treatment of disorders such as Parkinson's disease (PD) that manifest gait and postural disorders. Three additional major discoveries on the properties of PPN neurons have opened new areas of research for the treatment of motor and arousal disorders. The description of (a) electrical coupling, (b) intrinsic gamma oscillations, and (c) gene regulation in the PPN has identified a number of novel therapeutic targets and methods for the treatment of a number of neurological and psychiatric disorders. We first delve into the circuit, cellular, intracellular, and molecular organization of the PPN, and then consider the clinical results to date on PPN DBS. This comprehensive review will provide valuable information to explain the network effects of PPN DBS, point to new directions for treatment, and highlight a number of issues related to PPN DBS. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Propofol (2,6-diisopropylphenol) is a widely used anesthetic agent, but its mechanisms of action are poorly understood. In this report, the effects of three dose levels of propofol (5, 7.5, and 10 mg/kg) on the amplitude of the vertex-recorded, sleep state-dependent P13 midlatency evoked potential were investigated. The P13 potential is generated, at least in part, by the ascending cholinergic reticular activating system (RAS). The RAS is known to be affected by anesthetic agents. Intravenous injections of propofol were found to reduce the amplitude of the P13 potential in a dose- and time-dependent manner. At 2 min post-injection, the mean P13 amplitude was suppressed to 40% of its pre-injection level by the lowest dose, but was suppressed to 10% of pre-injection levels by the two higher doses of propofol. The duration of the suppression of mean P13 potential amplitude was also dose-dependent such that complete recovery occurred by 5 min using 5 mg/kg, by 15 min using 7.5 mg/kg and by 30 min using 10 mg/kg of propofol. Using a paired stimulus paradigm, transient effects on habituation of the P13 potential were observed but only after the highest dose. Thus, one of the mechanisms of propofol may be to affect portions of the RAS which modulate the level of arousal. It may only transiently affect higher systems known to modulate the degree of habituation of responses by the RAS (i.e. processes which modulate habituation and may participate in sensory gating and distractibility). [Copyright &y& Elsevier]

The percent of rapid eye movement (REM) sleep decreases dramatically between 10 and 30 days postnatally in the rat. The pedunculopontine nucleus (PPN) is known to modulate waking and REM sleep. We recorded intracellularly from 127 neurons in the PPN in 12–21-day rat brainstem slices maintained in warmed, oxygenated artificial cerebrospinal fluid. We identified three types of PPN neurons based on intrinsic membrane properties, type I (LTS), type II (A) and type III (A+LTS), as previously described. The percent of type I neurons increased from 6% at 12 days to 17% by 21 days, while the percent of type III neurons decreased from 21% at 12–17 days, to 4% after 17 days. Thus, PPN neurons may differentiate into type I bursting neurons and type II slow-firing neurons across this critical stage in development. The 5-HT1 receptor agonist 5-carboxyamido-tryptamine (5-CT) was found to hyperpolarize 58% of 12–16-day PPN neurons, did not affect 25% and depolarized 17%. However, a higher percentage of 17–21-day PPN neurons were hyperpolarized (85%), and a lower percentage unaffected (10%) or depolarized (5%), suggesting that serotonergic responses switched from both excitatory and inhibitory before, to almost purely inhibitory after, 17 days. These findings indicate a reorganization of PPN intrinsic membrane properties and serotonergic responses occur across this stage, in keeping with the proposed presence of a REM sleep inhibitory process during development. We suggest that disturbances in this developmental process may lead to disorders marked by increased REM sleep drive. [Copyright &y& Elsevier]

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. Red arrows represent statistical significant inhibition. Blue arrows represent statistical significant enhancement. ~ Represents absence of change.Our findings suggest that gamma oscillations in the PPN are finely tuned in vivo by the activity of HDAC class IIa. Sustained blocking of HDAC IIa blunted gamma band oscillations of PPN neurons. Blocking HDAC class I showed higher gamma band amplitudes. In conclusion, systemic administration of HDAC inhibitors might have either negative or positive effects on PPN rhythmicity depending of dose and group of HDAC affected. Image 105643 • HDAC class IIa inhibitors (acutely, i.p.) blunted PPN oscillations at gamma band. • HDAC class I inhibitors (100 mg/kg; i.p.) increased PPN gamma band oscillations. • We suggest the existence of a physiological HDAC class I/IIa balance in the PPN. [ABSTRACT FROM AUTHOR]