Gamma oscillations in the pedunculopontine nucleus are regulated by F-actin: neuroepigenetic implications.

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.

HDAC superfamily promoters acetylation is differentially regulated by modafinil and methamphetamine in the mouse medial prefrontal cortex.

Dysregulation of histone deacetylases (HDAC) has been proposed as a potential contributor to aberrant transcriptional profiles that can lead to changes in cognitive functions. It is known that METH negatively impacts the prefrontal cortex (PFC) leading to cognitive decline and addiction whereas modafinil enhances cognition and has a low abuse liability. We investigated if modafinil (90 mg/kg) and methamphetmine (METH) (1 mg/kg) may differentially influence the acetylation status of histones 3 and 4 (H3ac and H4ac) at proximal promoters of class I, II, III, and IV HDACs. We found that METH produced broader acetylation effects in comparison with modafinil in the medial PFC. For single dose, METH affected H4ac by increasing its acetylation at class I Hdac1 and class IIb Hdac10, decreasing it at class IIa Hdac4 and Hdac5. Modafinil increased H3ac and decreased H4ac of Hdac7. For mRNA, single-dose METH increased Hdac4 and modafinil increased Hdac7 expression. For repeated treatments (4 d after daily injections over 7 d), we found specific effects only for METH. We found that METH increased H4ac in class IIa Hdac4 and Hdac5 and decreased H3/H4ac at class I Hdac1, Hdac2, and Hdac8. At the mRNA level, repeated METH increased Hdac4 and decreased Hdac2. Class III and IV HDACs were only responsive to repeated treatments, where METH affected the H3/H4ac status of Sirt2, Sirt3, Sirt7, and Hdac11. Our results suggest that HDAC targets linked to the effects of modafinil and METH may be related to the cognitive-enhancing vs cognitive-impairing effects of these psychostimulants.

Neuroepigenetics of arousal: Gamma oscillations in the pedunculopontine nucleus.

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.

Bottom-up gamma and bipolar disorder, clinical and neuroepigenetic implications.

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.

The Critical Role of Intrinsic Membrane Oscillations.

Intrinsic, rhythmic subthreshold oscillations have been described in neurons of regions throughout the brain and have been found to influence the timing of action potentials induced by synaptic inputs. Some oscillations are sodium channel-dependent while others are calcium channel-dependent. These oscillations allow neurons to fire coherently at preferred frequencies and represent the main mechanism for maintaining high frequency network activity, especially in the cortex. Because cortical circuits are incapable of maintaining high frequency activity in the gamma range for prolonged periods, those processes dependent on continuous gamma band activity are subserved by subthreshold oscillations. As such, intrinsic oscillations, coupled with synaptic circuits, are essential to prolonged maintenance of such functions as sensory perception and "binding", problem solving, memory, waking, and rapid eye movement (REM) sleep.

Modulation of GABA release from the thalamic reticular nucleus by cocaine and caffeine: role of serotonin receptors.

Serotonin receptors are targets of drug therapies for a variety of neuropsychiatric and neurodegenerative disorders. Cocaine inhibits the re-uptake of serotonin (5-HT), dopamine, and noradrenaline, whereas caffeine blocks adenosine receptors and opens ryanodine receptors in the endoplasmic reticulum. We studied how 5-HT and adenosine affected spontaneous GABAergic transmission from thalamic reticular nucleus. We combined whole-cell patch clamp recordings of miniature inhibitory post-synaptic currents (mIPSCs) in ventrobasal thalamic neurons during local (puff) application of 5-HT in wild type (WT) or knockout mice lacking 5-HT2A receptors (5-HT2A -/-). Inhibition of mIPSCs frequency by low (10 µM) and high (100 µM) 5-HT concentrations was observed in ventrobasal neurons from 5-HT2A -/- mice. In WT mice, only 100 µM 5-HT significantly reduced mIPSCs frequency. In 5-HT2A -/- mice, NAN-190, a specific 5-HT1A antagonist, prevented the 100 µM 5-HT inhibition while blocking H-currents that prolonged inhibition during post-puff periods. The inhibitory effects of 100 µM 5-HT were enhanced in cocaine binge-treated 5-HT2A -/- mice. Caffeine binge treatment did not affect 5-HT-mediated inhibition. Our findings suggest that both 5-HT1A and 5-HT2A receptors are present in pre-synaptic thalamic reticular nucleus terminals. Serotonergic-mediated inhibition of GABA release could underlie aberrant thalamocortical physiology described after repetitive consumption of cocaine. Our findings suggest that both 5-HT1A , 5-HT2A and A1 receptors are present in pre-synaptic TRN terminals. 5-HT1A and A1 receptors would down-regulate adenylate cyclase, whereas 5-HT1A would also increase the probability of the opening of G-protein-activated inwardly rectifying K(+) channels (GIRK). Sustained opening of GIRK channels would hyperpolarize pre-synaptic terminals activating H-currents, resulting in less GABA release. 5-HT2A -would activate PLC and IP3 , increasing intracellular [Ca(2+) ] and thus facilitating GABA release.

Methamphetamine blunts Ca(2+) currents and excitatory synaptic transmission through D1/5 receptor-mediated mechanisms in the mouse medial prefrontal cortex.

Psychostimulant addiction is associated with dysfunctions in frontal cortex. Previous data demonstrated that repeated exposure to methamphetamine (METH) can alter prefrontal cortex (PFC)-dependent functions. Here, we show that withdrawal from repetitive non-contingent METH administration (7 days, 1 mg/kg) depressed voltage-dependent calcium currents (ICa ) and increased hyperpolarization-activated cation current (IH ) amplitude and the paired-pulse ratio of evoked excitatory postsynaptic currents (EPSCs) in deep-layer pyramidal mPFC neurons. Most of these effects were blocked by systemic co-administration of the D1/D5 receptor antagonist SCH23390 (0.5 and 0.05 mg/kg). In vitro METH (i.e. bath-applied to slices from naïve-treated animals) was able to emulate its systemic effects on ICa and evoked EPSCs paired-pulse ratio. We also provide evidence of altered mRNA expression of (1) voltage-gated calcium channels P/Q-type Cacna1a (Cav 2.1), N-type Cacna1b (Cav 2.2), T-type Cav 3.1 Cacna1g, Cav 3.2 Cacna1h, Cav 3.3 Cacna1i and the auxiliary subunit Cacna2d1 (α2δ1); (2) hyperpolarization-activated cyclic nucleotide-gated channels Hcn1 and Hcn2; and (3) glutamate receptors subunits AMPA-type Gria1, NMDA-type Grin1 and metabotropic Grm1 in the mouse mPFC after repeated METH treatment. Moreover, we show that some of these changes in mRNA expression were sensitive D1/5 receptor blockade. Altogether, these altered mechanisms affecting synaptic physiology and transcriptional regulation may underlie PFC functional alterations that could lead to PFC impairments observed in METH-addicted individuals.

Pedunculopontine Nucleus Region Deep Brain Stimulation in Parkinson Disease: Surgical Techniques, Side Effects, and Postoperative Imaging.

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.

Pedunculopontine Nucleus Region Deep Brain Stimulation in Parkinson Disease: Surgical Anatomy and Terminology.

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.

Modulation of gamma oscillations in the pedunculopontine nucleus by neuronal calcium sensor protein-1: relevance to schizophrenia and bipolar disorder.

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.

Visualization of fast calcium oscillations in the parafascicular nucleus.

The parafascicular nucleus (Pf) is an ascending target of the pedunculopontine nucleus (PPN) and is part of the "non-specific" intralaminar thalamus. The PPN, part of the reticular activating system, is mainly involved in waking and rapid eye movement sleep. Gamma oscillations are evident in all Pf neurons and 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 synchrony with patch clamp recorded oscillations during depolarizing current ramps. Patch-clamped 9 to 19-day-old rat Pf neurons (n = 148, dye filled n = 61, control n = 87) were filled with Fura 2, Bis Fura, or Oregon Green BAPTA-1. Calcium transients were generated during depolarizing current ramps and visualized with a high-speed, wide-field fluorescence imaging system. Cells manifested calcium transients with oscillations in both somatic and proximal dendrite fluorescence recordings. Fluorescent calcium transients were blocked with the nonspecific calcium channel blocker, cadmium, or the combination of ω-Agatoxin-IVA (AgA), a specific P/Q-type calcium channel blocker and ω-conotoxin-GVIA (CgTx), a specific N-type calcium channel blocker. We developed a viable methodology for studying high-speed oscillations without the use of multi-photon imaging systems.

Role of G-proteins in the effects of leptin on pedunculopontine nucleus neurons.

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'-[ß-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 GDPß.

Differential effects of methylphenidate and cocaine on GABA transmission in sensory thalamic nuclei.

Methylphenidate (MPH) is widely used to treat children and adolescents diagnosed with attention deficit/hyperactivity disorder. Although MPH shares mechanistic similarities to cocaine, its effects on GABAergic transmission in sensory thalamic nuclei are unknown. Our objective was to compare cocaine and MPH effects on GABAergic projections between thalamic reticular and ventrobasal (VB) nuclei. Mice (P18-30) were subjected to binge-like cocaine and MPH acute and sub-chronic administrations. Cocaine and MPH enhanced hyperlocomotion, although sub-chronic cocaine-mediated effects were stronger than MPH effects. Cocaine and MPH sub-chronic administration altered paired-pulse and spontaneous GABAergic input differently. The effects of cocaine on evoked paired-pulse GABA-mediated currents changed from depression to facilitation with the duration of the protocols used, while MPH induced a constant increase throughout the administration protocols. Thalamic reticular nucleus GAD67 and VB Ca(V) 3.1 protein levels were measured using western blot to better understand their link to increased GABA release. Both proteins were increased by sub-chronic administration of cocaine. MPH showed effects on GABAergic transmission that seems less disruptive than cocaine. Unique effects of cocaine on postsynaptic VB calcium currents might explain deleterious cocaine effects on sensory thalamic nuclei. These results suggest that cocaine and MPH produced distinct presynaptic alterations on GABAergic transmission.

Effects of leptin on pedunculopontine nucleus (PPN) neurons.

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.

Gamma band activity in the developing parafascicular nucleus.

The parafascicular nucleus (Pf) receives cholinergic input from the pedunculopontine nucleus, part of the reticular activating system involved in waking and rapid eye movement (REM) sleep, and sends projections to the cortex. We tested the hypothesis that Pf neurons fire maximally at gamma band frequency (30-90 Hz), that this mechanism involves high-threshold voltage-dependent P/Q- and N-type calcium channels, and that this activity is enhanced by the cholinergic agonist carbachol (CAR). Patch-clamped 9- to 25-day-old rat Pf neurons (n = 299) manifested a firing frequency plateau at gamma band when maximally activated (31.5 ± 1.5 Hz) and showed gamma oscillations when voltage-clamped at holding potentials above -20 mV, and the frequency of the oscillations increased significantly with age (24.6 ± 3.8 vs. 51.6 ± 4.4 Hz, P < 0.001) but plateaued at gamma frequencies. Cells exposed to CAR showed significantly higher frequencies early in development compared with those without CAR (24.6 ± 3.8 vs. 41.7 ± 4.3 Hz, P < 0.001) but plateaued with age. The P/Q-type calcium channel blocker ω-agatoxin-IVA (ω-Aga) blocked gamma oscillations, whereas the N-type blocker ω-conotoxin-GVIA (ω-CgTx) only partially decreased the power spectrum amplitude of gamma oscillations. The blocking effect of ω-Aga on P/Q-type currents and ω-CgTx on N-type currents was consistent over age. We conclude that P/Q- and N-type calcium channels appear to mediate Pf gamma oscillations during development. We hypothesize that the cholinergic input to the Pf could activate these cells to oscillate at gamma frequency, and perhaps relay these rhythms to cortical areas, thus providing a stable high-frequency state for "nonspecific" thalamocortical processing.

Improvement in arousal, visual neglect, and perception of stimulus intensity following cold pressor stimulation.

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.

Responses of developing pedunculopontine neurons to glutamate receptor agonists.

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.