Christen Simon

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.

Gamma band unit activity and population responses in the pedunculopontine nucleus.

The pedunculopontine nucleus (PPN) is involved in the activated states of waking and paradoxical sleep, forming part of the reticular activating system (RAS). The studies described tested the hypothesis that single unit and/or population responses of PPN neurons are capable of generating gamma band frequency activity. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. Regardless of cell type (I, II, or III) or type of response to the nonselective cholinergic receptor agonist carbachol (excitation, inhibition, biphasic), almost all PPN neurons fired at gamma band frequency, but no higher, when subjected to depolarizing steps (50 +/- 2 Hz, mean +/- SE). Nonaccommodating neurons fired at 18-100 Hz throughout depolarizing steps, while most accommodating neurons exhibited gamma band frequency of action potentials followed by gamma band membrane oscillations. These oscillations were blocked by the sodium channel blocker tetrodotoxin (TTX), suggesting that at least some are mediated by sodium currents. Population responses in the PPN showed that carbachol induced peaks of activation in the theta and gamma range, while glutamatergic receptor agonists induced overall increases in activity at theta and gamma frequencies, although in differing patterns. Gamma band activity appears to be a part of the intrinsic membrane properties of PPN neurons, and the population as a whole generates different patterns of gamma band activity under the influence of specific transmitters. Given sufficient excitation, the PPN may impart gamma band activation on its targets.

Coherence and frequency in the reticular activating system (RAS)

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.

Gamma Band Activity in the Reticular Activating System

This review considers recent evidence showing that cells in three regions of the reticular activating system (RAS) exhibit gamma band activity, and describes the mechanisms behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine subcoeruleus nucleus dorsalis (SubCD) all fire in the beta/gamma band range when maximally activated, but no higher. The mechanisms behind this ceiling effect have been recently elucidated. We describe recent findings showing that every cell in the PPN have high-threshold, voltage-dependent P/Q-type calcium channels that are essential, while N-type calcium channels are permissive, to gamma band activity. Every cell in the Pf also showed that P/Q-type and N-type calcium channels are responsible for this activity. On the other hand, every SubCD cell exhibited sodium-dependent subthreshold oscillations. A novel mechanism for sleep–wake control based on well-known transmitter interactions, electrical coupling, and gamma band activity is described. The data presented here on inherent gamma band activity demonstrates the global nature of sleep–wake oscillation that is orchestrated by brainstem–thalamic mechanism, and questions the undue importance given to the hypothalamus for regulation of sleep–wakefulness. The discovery of gamma band activity in the RAS follows recent reports of such activity in other subcortical regions like the hippocampus and cerebellum. We hypothesize that, rather than participating in the temporal binding of sensory events as seen in the cortex, gamma band activity manifested in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking and paradoxical sleep. Most of our thoughts and actions are driven by pre-conscious processes. We speculate that continuous sensory input will induce gamma band activity in the RAS that could participate in the processes of pre-conscious awareness, and provide the essential stream of information for the formulation of many of our actions.

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

The dorsal subcoeruleus nucleus (SubCD) is involved in generating two signs of rapid eye movement (REM) sleep: muscle atonia and ponto-geniculo-occipital (PGO) waves. We tested the hypothesis that single cell and/or population responses of SubCD neurons are capable of generating gamma frequency activity in response to intracellular stimulation or receptor agonist activation. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. All SubCD neurons (n = 103) fired at gamma frequency when subjected to depolarizing steps. Two statistically distinct populations of neurons were observed, which were distinguished by their high (>80 Hz, n = 24) versus low (35-80 Hz, n = 16) initial firing frequencies. Both cell types exhibited subthreshold oscillations in the gamma range (n = 43), which may underlie the gamma band firing properties of these neurons. The subthreshold oscillations were blocked by the sodium channel blockers tetrodotoxin (TTX, n = 21) extracellularly and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314) intracellularly (n = 5), indicating they were sodium channel dependent. Gamma frequency subthreshold oscillations were observed in response to the nonspecific cholinergic receptor agonist carbachol (CAR, n = 11, d = 1.08) and the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA, n = 12, d = 1.09) and kainic acid (KA, n = 13, d = 0.96), indicating that cholinergic and glutamatergic inputs may be involved in the activation of these subthreshold currents. Gamma band activity also was observed in population responses following application of CAR (n = 4, P < 0.05), NMDA (n = 4, P < 0.05) and KA (n = 4, P < 0.05). Voltage-sensitive, sodium channel-dependent gamma band activity appears to be a part of the intrinsic membrane properties of SubCD neurons.

Arousal from slices to humans

Most psychiatric and neurological disorders exhibit sleep disorders, and in some cases presage the disease. Study of the control of sleep and waking has the potential for making a major impact on a number of disorders, making translational neuroscience research on this area critical. One element of the reticular activating system (RAS) is the pedunculopontine nucleus (PPN), which is the cholinergic arm of the RAS, and projects to the thalamus to trigger thalamocortical rhythms and to the brainstem to modulate muscle tone and locomotion. We developed a research program using brainstem slices containing the PPN to tell us about the cellular and molecular organization of this region. In addition, we developed the P13 midlatency auditory evoked potential, which is generated by PPN outputs, preparation in freely moving rats. This allows the study of PPN cellular and molecular mechanisms at the level of 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 processes detected in vitro, confirmed in the whole animal, and tested in humans. This translational research program led to the discovery of a novel mechanism of sleep-wake control, pointing the way to a number of new clinical applications in the development of novel stimulants and anesthetics.

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.

Developmental Changes in Glutamatergic Fast Synaptic Neurotransmission in the Dorsal Subcoeruleus Nucleus

STUDY OBJECTIVES The dorsal subcoeruleus nucleus (SubCD) is involved in the generation of rapid eye movement sleep (REM), a state distinguished by high-frequency EEG activity, muscle atonia, and ponto-geniculo-occipital (PGO) waves. Activation of the SubCD by injection of the glutamate (GLU) receptor agonist kainic acid (KA) produced a REM sleep-like state with muscle atonia. We tested the hypothesis that developmental changes in the GLU excitability of SubCD neurons could underlie the developmental decrease in REM sleep that occurs in the rat from postnatal days 10-30. DESIGN Sagittal sections containing the SubCD were cut using 9-15 day old rat pups. Whole-cell patch clamp recordings were performed on SubCD neurons and responses were measured following electrical stimulation or bath application of the GLU receptor agonists N-methyl-D-aspartic acid (NMDA) or KA. MEASUREMENTS AND RESULTS Pharmacological or electrical stimulation increased non-cholinergic excitatory postsynaptic currents (EPSCs) in SubCD neurons, which were blocked by GLU receptor antagonists. Although no developmental changes were observed in the relative contribution of AMPA/KA and NMDA receptors to the responses, there was a developmental decrease in the half-width duration of both evoked and miniature EPSCs. Bath application of NMDA or KA revealed a developmental decrease in the direct response of SubCD neurons to these agonists. CONCLUSIONS The SubCD receives glutamatergic input, which may be involved in activation of SubCD neurons during REM sleep. A developmental decrease in the glutamatergic excitability of these neurons could underlie the developmental decrease in REM sleep observed in humans and rodents.

Effects of glutamate receptor agonists on the P13 auditory evoked potential and startle response in the rat

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 EEG and the Discovery of the RAS

Abstract The electroencephalogram was invented by Hans Berger and was used in combination with brain stem transections to ascertain that the reticular activating system (RAS) was the main determinant of the fast activity seen during waking and rapid eye movement (REM) sleep. Lesions of the RAS or disconnection of the RAS from the forebrain generally led to low-frequency activity as seen during slow-wave sleep, but when the RAS was stimulated or remained connected to the forebrain, fast activity such as that observed during waking and REM sleep was manifested. The main nucleus within the RAS in charge of both waking and REM sleep, two states of low-amplitude, high-frequency activity, is the pedunculopontine nucleus.