Pedunculopontine Nucleus Neurons Research Articles

Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

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 Ca2+ 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 Ca2+ channel types.

Direct presynaptic and indirect astrocyte-mediated mechanisms both contribute to endocannabinoid signaling in the pedunculopontine nucleus of mice.

The pedunculopontine nucleus (PPN), a cholinergic nucleus of the reticular activating system, is known to be involved in the regulation of sleep and wakefulness. Endogenous and exogenous cannabinoids, by systemic or local administration to the pedunculopontine nucleus, can both influence sleep. We previously demonstrated that activation of astrocytes by cannabinoid type 1 (CB1) receptor agonists was able to modulate the membrane potential of PPN neurons, even in the presence of blockers of fast synaptic neurotransmission. In the present work, we provide evidence that synaptic inputs of PPN neurons are also affected by activation of presynaptic and astrocytic CB1 receptors. Using slice electrophysiology combined with calcium imaging, optogenetics and immunohistochemistry, we revealed a direct presynaptic inhibitory action on inhibitory postsynaptic currents, along with a mild increase of excitatory postsynaptic currents during CB1 receptor stimulation. Besides inhibition of excitatory and inhibitory neurotransmission through stimulation of presynaptic CB1 receptors, astrocyte- and mGluR-dependent tonic inhibition and excitation also developed. The mild stimulatory action of CB1 receptor activation on excitatory neurotransmission is the combination of astrocyte-dependent tonic excitation on excitatory neurons and the canonical presynaptic CB1 receptor activation and consequential inhibition of excitatory synaptic neurotransmission, whereas the astrocyte-dependent stimulatory action was not observed in inhibitory neurotransmission within the PPN. Our findings demonstrate that endocannabinoids act in the PPN via a dual pathway, consisting of a direct presynaptic and an indirect, astrocyte-mediated component, regulating synaptic strength and neuronal activity via independent mechanisms.

Reward functions of the basal ganglia.

Besides their fundamental movement function evidenced by Parkinsonian deficits, the basal ganglia are involved in processing closely linked non-motor, cognitive and reward information. This review describes the reward functions of three brain structures that are major components of the basal ganglia or are closely associated with the basal ganglia, namely midbrain dopamine neurons, pedunculopontine nucleus, and striatum (caudate nucleus, putamen, nucleus accumbens). Rewards are involved in learning (positive reinforcement), approach behavior, economic choices and positive emotions. The response of dopamine neurons to rewards consists of an early detection component and a subsequent reward component that reflects a prediction error in economic utility, but is unrelated to movement. Dopamine activations to non-rewarded or aversive stimuli reflect physical impact, but not punishment. Neurons in pedunculopontine nucleus project their axons to dopamine neurons and process sensory stimuli, movements and rewards and reward-predicting stimuli without coding outright reward prediction errors. Neurons in striatum, besides their pronounced movement relationships, process rewards irrespective of sensory and motor aspects, integrate reward information into movement activity, code the reward value of individual actions, change their reward-related activity during learning, and code own reward in social situations depending on whose action produces the reward. These data demonstrate a variety of well-characterized reward processes in specific basal ganglia nuclei consistent with an important function in non-motor aspects of motivated behavior.

Decoding brain state transitions in the pedunculopontine nucleus: cooperative phasic and tonic mechanisms.

Cholinergic neurons of the pedunculopontine nucleus (PPN) are most active during the waking state. Their activation is deemed to cause a switch in the global brain activity from sleep to wakefulness, while their sustained discharge may contribute to upholding the waking state and enhancing arousal. Similarly, non-cholinergic PPN neurons are responsive to brain state transitions and their activation may influence some of the same targets of cholinergic neurons, suggesting that they operate in coordination. Yet, it is not clear how the discharge of distinct classes of PPN neurons organize during brain states. Here, we monitored the in vivo network activity of PPN neurons in the anesthetized rat across two distinct levels of cortical dynamics and their transitions. We identified a highly structured configuration in PPN network activity during slow-wave activity that was replaced by decorrelated activity during the activated state (AS). During the transition, neurons were predominantly excited (phasically or tonically), but some were inhibited. Identified cholinergic neurons displayed phasic and short latency responses to sensory stimulation, whereas the majority of non-cholinergic showed tonic responses and remained at high discharge rates beyond the state transition. In vitro recordings demonstrate that cholinergic neurons exhibit fast adaptation that prevents them from discharging at high rates over prolonged time periods. Our data shows that PPN neurons have distinct but complementary roles during brain state transitions, where cholinergic neurons provide a fast and transient response to sensory events that drive state transitions, whereas non-cholinergic neurons maintain an elevated firing rate during global activation.

High-threshold Ca2+ channels behind gamma band activity in the pedunculopontine nucleus (PPN).

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.

Cholinergic and endocannabinoid neuromodulatory effects overlap on neurons of the pedunculopontine nucleus of mice.

The pedunculopontine nucleus (PPN) is a part of the reticular activating system and one of the main sources of the cholinergic fibers in the midbrain, while it is also subject to cholinergic modulation. This nucleus is known to be a structure that controls sleep-wake cycles, arousal, and locomotion. Neurons of the PPN are targets of several neuromodulatory mechanisms, which elicit heterogeneous pharmacological responses including hyperpolarization and depolarization, whereas lack of response can also be observed. In agreement with previous findings, we found that PPN neurons respond to the muscarinic agonist carbachol in a heterogeneous manner: they were depolarized and showed increased firing rate, decreased firing frequency, and were hyperpolarized, or showed no response. The heterogeneity of the muscarinic activation was similar to our previous observations with type 1 cannabinoid (CB1) receptor agonists; therefore, we investigated whether muscarinic and endocannabinoid modulatory mechanisms elicit the same action on a certain neuron. To achieve this, whole-cell patch clamp experiments were conducted on midbrain slices containing the PPN. Carbachol was applied first and, after recording the changes in the membrane potential and the firing frequency and achieving washout, the CB1 receptor agonist arachidonyl-2'-chloroethylamide (ACEA) was applied. A marked but not full overlap was observed: all neurons depolarized by carbachol were depolarized by the CB1 receptor agonist ACEA, and all neurons lacking response to carbachol lacked response to ACEA as well. However, neurons hyperpolarized by carbachol were depolarized, hyperpolarized, or not affected by the ACEA. These results indicate that endocannabinoid and muscarinic modulatory effects involve similar mechanisms of action.

The integrative role of the pedunculopontine nucleus in human gait.

The brainstem pedunculopontine nucleus has a likely, although unclear, role in gait control, and is a potential deep brain stimulation target for treating resistant gait disorders. These disorders are a major therapeutic challenge for the ageing population, especially in Parkinson's disease where gait and balance disorders can become resistant to both dopaminergic medication and subthalamic nucleus stimulation. Here, we present electrophysiological evidence that the pedunculopontine and subthalamic nuclei are involved in distinct aspects of gait using a locomotor imagery task in 14 patients with Parkinson's disease undergoing surgery for the implantation of pedunculopontine or subthalamic nuclei deep brain stimulation electrodes. We performed electrophysiological recordings in two phases, once during surgery, and again several days after surgery in a subset of patients. The majority of pedunculopontine nucleus neurons (57%) recorded intrasurgically exhibited changes in activity related to different task components, with 29% modulated during visual stimulation, 41% modulated during voluntary hand movement, and 49% modulated during imaginary gait. Pedunculopontine nucleus local field potentials recorded post-surgically were modulated in the beta and gamma bands during visual and motor events, and we observed alpha and beta band synchronization that was sustained for the duration of imaginary gait and spatially localized within the pedunculopontine nucleus. In contrast, significantly fewer subthalamic nucleus neurons (27%) recorded intrasurgically were modulated during the locomotor imagery, with most increasing or decreasing activity phasically during the hand movement that initiated or terminated imaginary gait. Our data support the hypothesis that the pedunculopontine nucleus influences gait control in manners extending beyond simply driving pattern generation. In contrast, the subthalamic nucleus seems to control movement execution that is not likely to be gait-specific. These data highlight the crucial role of these two nuclei in motor control and shed light on the complex functions of the lateral mesencephalus in humans.

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.

Pedunculopontine Nucleus Gamma Band Activity-Preconscious Awareness, Waking, and REM Sleep.

The pedunculopontine nucleus (PPN) is a major component of the reticular activating system (RAS) that regulates waking and REM sleep, states of high-frequency EEG activity. Recently, we described the presence of high threshold, voltage-dependent N- and P/Q-type calcium channels in RAS nuclei that subserve gamma band oscillations in the mesopontine PPN, intralaminar parafascicular nucleus (Pf), and pontine subcoeruleus nucleus dorsalis (SubCD). Cortical gamma band activity participates in sensory perception, problem solving, and memory. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. That is, the RAS may play an early permissive role in volition. Our latest results suggest that (1) the manifestation of gamma band activity during waking may employ a separate intracellular pathway compared to that during REM sleep, (2) neuronal calcium sensor (NCS-1) protein, which is over expressed in schizophrenia and bipolar disorder, modulates gamma band oscillations in the PPN in a concentration-dependent manner, (3) leptin, which undergoes resistance in obesity resulting in sleep dysregulation, decreases sodium currents in PPN neurons, accounting for its normal attenuation of waking, and (4) following our discovery of electrical coupling in the RAS, we hypothesize that there are cell clusters within the PPN that may act in concert. These results provide novel information on the mechanisms controlling high-frequency activity related to waking and REM sleep by elements of the RAS.

The use of three-dimensional printing to produce in vitro slice chambers.

In recent years, 3D printing technology has become inexpensive and simple enough that any lab can own and use one of these printers. We explored the potential use of 3D printers for quickly and easily producing in vitro slice chambers for patch clamp electrophysiology. Slice chambers were produced using five available plastics: ABS, PLA, Nylon 618, Nylon 680, and T-glase. These "lab-made" chambers were also made using stereolithography through a professional printing service (Shapeways). This study measured intrinsic membrane properties of neurons in the brain stem pedunculopontine nucleus (PPN) and layer V pyramidal neurons in retrosplenial cortex. Nylon 680 and T-glase significantly hyperpolarized PPN neurons. ABS increased input resistance, decreased action potential amplitude, and increased firing frequency in pyramidal cortical neurons. To test long term exposure to each plastic, human neuroblastoma SHSY5Y cell cultures were exposed to each plastic for 1 week. ABS decreased cell counts while Nylon 618 and Shapeways plastics eliminated cells. Primary mouse pituitary cultures were also tested for 24-h exposure. ABS decreased cell counts while Nylon 618 and Shapeways plastics dramatically decreased cell counts. Chambers can be quickly and inexpensively printed in the lab. ABS, PLA, Nylon 680, and T-glase plastics would suffice for many experiments instead of commercially produced slice chambers. While these technologies are still in their infancy, they represent a powerful addition to the lab environment. With careful selection of print material, slice chambers can be quickly and inexpensively manufactured in the lab.

The physiology of the pedunculopontine nucleus: implications for deep brain stimulation.

This brief review resolves a number of persistent conflicts regarding the location and characteristics of the mesencephalic locomotor region, which has in the past been described as not locomotion-specific and is more likely the pedunculopontine nucleus (PPN). The parameters of stimulation used to elicit changes in posture and locomotion we now know are ideally suited to match the intrinsic membrane properties of PPN neurons. The physiology of these cells is important not only because it is a major element of the reticular activating system, but also because it is a novel target for the treatment of gait and postural deficits in Parkinson's disease (PD). The discussion explains many of the effects reported following deep brain stimulation (DBS) of the PPN by different groups and provides guidelines for the determination of long-term assessment and effects of PPN DBS. A greater understanding of the physiology of the target nuclei within the brainstem and basal ganglia, amassed over the past decades, has enabled increasingly better patient outcomes from DBS for movement disorders. Despite these improvements, there remains a great opportunity for further understanding of the mechanisms through which DBS has its effects and for further development of appropriate technology to effect these treatments. We review the scientific basis for one of the newest targets, the PPN, in the treatment of PD and other movement disorders, and address the needs for further investigation.

Parkinson's Disease: What role do pedunculopontine nucleus cholinergic neurons play?

5 ISSN 1479-6708 10.2217/FNL.13.61 © 2014 Future Medicine Ltd Future Neurol. (2014) 9(1), 5–8 There is increasing evidence that cholinergic system dysfunction contributes significantly to both nonmotor and motor morbidities in Parkinson’s disease (PD) [1]. Important brain cholinergic projection systems include the well-characterized basal forebrain cortico petal system and cholinergic neurons within the pedunculopontine–lateral dorsal tegmental complex. Located at the mesopontine border, the pedunculopontine nucleus (PPN) is a phylogenetically ancient structure composed of a number of different neuronal populations [2]. Cholinergic projection neurons are prominent PPN components with strong inter connections with the basal ganglia and thalamus, and substantial descending projections to important medullary motor centers. The PPN is a key node in the regulation of both consciousness and motor function. The midbrain locomotor region, a physiologically defined region crucial for normal gait and posture, encompasses the PPN [3]. PET ligands targeting markers of cholinergic terminals enable in vivo assessment of the integrity of cholinergic projections. Combined with post-mortem studies and experimental animal work, a much clearer picture of the nature of cholinergic projection system changes and their clinical correlates is now emerging. Nonmotor symptoms such as rapid eye movement sleep behavior disorder (RBD) and possibly visual hallucinations may ref lect degeneration of PPN cholinergic neurons and their thalamic afferents in PD. Degeneration of basal forebrain cholinergic neurons and their centripetal cortical projections is a determinant of cognitive decline in PD and Alzheimer’s disease (AD) [1]. A major distinction in cholinergic systems degeneration between PD and AD is that PPN neurons and their thalamic projections degenerate in PD but remain relatively spared in AD [4]. RBD is a parasomnia that has been linked almost exclusively to a-synucleinopathies [5]. We showed recently that PD subjects reporting symptoms of RBD had evidence of more prominent cholinergic terminal loss, including within the thalamus, reflecting impaired integrity of PPN–thalamic

Spatiotemporal properties of high-speed calcium oscillations in the pedunculopontine nucleus

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.

Gait Disorders in Parkinsonian Monkeys with Pedunculopontine Nucleus Lesions: A Tale of Two Systems

Gait and balance disorders unresponsive to dopaminergic drugs in Parkinson's disease (PD) are secondary to lesions located outside the dopaminergic system. However, available animal models of PD fail to display l-3,4-dihydroxyphenylalanine (DOPA)-responsive parkinsonism and drug-resistant gait and balance disorders, and this lack of appropriate model could account for the deficit of efficient treatments. Because the pedunculopontine nucleus (PPN) plays an important role in locomotion control, we conducted the present study to investigate the consequences of combined dopaminergic and PPN lesions in a same animal. We used macaques that received first 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication to render them parkinsonian and then local stereotaxic lesion of the PPN. Adding bilateral PPN lesions in MPTP-lesioned macaques induced dopamine-resistant gait and balance disorders but unexpectedly improved hypokinesia. Additional MPTP injections resulted in the association of a severe DOPA-responsive parkinsonism together with DOPA-unresponsive gait disorders. Histological examination assessed a severe dopaminergic degeneration and a significant loss of PPN cholinergic neurons. We observed similar results in aged monkeys intoxicated with MPTP: they developed severe DOPA-responsive hypokinesia and tremor together with unresponsive gait and balance disorders and displayed dopaminergic lesion and a weak but significant cholinergic PPN lesion. Our results highlight the complex role of the cholinergic PPN neurons in the pathophysiology of PD because its lesion induces a dual effect with an improvement of hypokinesia contrasting with a worsening of DOPA-unresponsive gait and balance disorders. Thus, we obtained a primate model of PD that could be useful to test symptomatic treatments for these heavily disabling symptoms.

A non-cholinergic neuronal loss in the pedunculopontine nucleus of toxin-evoked Parkinsonian rats

The pedunculopontine nucleus (PPN) controls various physiological functions, whilst being deemed a suitable target for low-frequency stimulation therapy for alleviating aspects of Parkinson's disease (PD). Previous studies showed that the PPN contains mainly cholinergic, γ-aminobutyric acid (GABA)ergic and glutamatergic neurons. Here we report on the total number of PPN neurons in laboratory rats, a species frequently used as an experimental model for simulating aspects of human PD. Moreover, the study reports that the number of PPN neurons decreases under toxic conditions that mimic in animals the core pathology seen in human PD. Immunohistochemical detection methods combined with unbiased stereology served to estimate that the PPN of healthy rats unilaterally contains ~19,028 NeuN-immunopositive neurons. The identified neurons revealed a distinct distribution pattern consisting of high cell density in the most rostral and caudal sections of the PPN nucleus, contrasting with lower densities in the medial segments. Our data also show a significant loss which affected PPN non-cholinergic cells, but not cholinergic ones in rats lesioned unilaterally in the Substantia Nigra pars compacta (SNpc) with a single injection of 6-hydroxydopamine (6-OHDA) compared to control animals. This result differs from previous studies which reported a substantial cholinergic cell loss in the PPN of post-mortem PD brains and in 6-OHDA-lesioned monkeys. Since a noted demise of dopaminergic neurons residing in the SN was confirmed in the 6-OHDA-lesioned rats, the current study suggests that a “dying-back” mechanism may underlie the cell death affecting non-cholinergic PPN neurons.

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'-[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.

Muscarinic modulation of high frequency oscillations in pedunculopontine neurons.

We previously reported that persistent application of the non-specific cholinergic agonist carbachol (CAR) increased the frequency of calcium channel-mediated oscillatory activity in pedunculopontine nucleus (PPN) neurons, which we identified as dependent on voltage-gated, high-threshold P/Q-type channels. Here, we tested the hypothesis that M2 muscarinic receptors and G-proteins associated with M2 receptors mediate the increase in oscillatory frequency in PPN neurons. We found, using depolarizing ramps, that patch clamped 9–12 day old rat PPN neurons (n = 189) reached their peak oscillatory activity around −20 mV membrane potential. Acute (short duration) application of CAR blocked the oscillatory activity through M2 muscarinic receptors, an effect blocked by atropine. However, persistent (long duration) application of CAR significantly increased the frequency of oscillatory activity in PPN neurons through M2 receptors [40 ± 1 Hz (with CAR) vs. 23 ± 1 Hz (without CAR); p < 0.001]. We then tested the effects of the G-protein antagonist guanosine 5′-[β-thio] diphosphate trilithium salt (GDP-β-S), and the G-protein agonist 5′-[γ-thio] triphosphate trilithium salt (GTP-γ-S). We found, using a three-step protocol in voltage-clamp mode, that the increase in the frequency of oscillations induced by M2 cholinergic receptors was linked to a voltage-dependent G-protein mechanism. In summary, these results suggest that persistent cholinergic input creates a permissive activation state in the PPN that allows high frequency P/Q-type calcium channel-mediated gamma oscillations to occur.

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.

Mechanism behind gamma band activity in the pedunculopontine nucleus

The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking and paradoxical sleep, EEG responses are characterized by low-amplitude, high-frequency oscillatory activity in the beta-gamma band range (~20-80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted to identify the voltage-gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8-14 day) PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage-clamped at holding potentials above -30 mV, suggesting that their origin may be spatially located beyond voltage-clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N-type calcium channel blocker ω-conotoxin-GVIA partially reduced gamma oscillations, while the P/Q-type blocker ω-agatoxin-IVA abolished them. Both ω-CgTX and ω-Aga blocked voltage-dependent calcium currents, by 56 and 52% respectively. The delayed rectifier-like potassium channel blocker α-dendrotoxin also abolished gamma oscillations. In carbachol-induced PPN population responses, ω-agatoxin-IVA reduced higher, and ω-CgTx mostly lower, frequencies. These results suggest that voltage-dependent P/Q- and, to a lesser extent, N-type calcium channels mediate gamma oscillations in PPN.

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.