Elsevier

Experimental Neurology

Volume 247, September 2013, Pages 562-571
Experimental Neurology

Lesion of the pedunculopontine tegmental nucleus in rat augments cortical activation and disturbs sleep/wake state transitions structure

https://doi.org/10.1016/j.expneurol.2013.02.007Get rights and content

Abstract

The pedunculopontine tegmental nucleus (PPT) represents a major aggregation of cholinergic neurons in the mammalian brainstem, which is important in the generation and maintenance of REM sleep.

We investigated the effects of unilateral and bilateral PPT lesions on sleep and all the conventional sleep-state related EEG frequency bands amplitudes, in an attempt to find the EEG markers for the onset and progression of PPT cholinergic neuronal degeneration.

The experiments were performed on 35 adult male Wistar rats, chronically implanted for sleep recording. During the surgical procedure for EEG and EMG electrodes implantation, the unilateral or bilateral PPT lesion was produced under ketamine/diazepam anesthesia, by the stereotaxically guided microinfusion of 100 nl 0.1 M ibotenic acid (IBO) into PPT. We applied Fourier analysis to signals acquired throughout 6 h of recordings, and each 10 s epoch was differentiated as a Wake, NREM or REM state. We also calculated the group probability density estimates (PDE) of all Wake, NREM and REM conventional EEG frequency amplitudes, and the number of all the transition states using MATLAB 6.5.

Our results show that the unilateral or bilateral PPT lesions did not change the sleep/wake architecture, but did change the sleep/wake state transitions structure and the sleep/state related “EEG microstructure”.

Unilateral or bilateral PPT lesions sustainably increased Wake/REM and REM/Wake transitions from 14 to 35 days after lesions. This was followed by decreased NREM/REM and REM/NREM transitions from 28 days only in the case of the bilateral PPT lesion.

The unilateral PPT lesion augmented both Wake theta and REM beta while it also attenuated the relative amplitude of the Wake delta frequency, with a delay of one week. Following a bilateral PPT lesion there was augmentation of the relative amplitude of the Wake, NREM, and REM beta and REM gamma frequency which occurred simultaneously to NREM and Wake delta attenuation.

We have shown that the PPT cholinergic neuronal loss sustainably increased the number of the Wake/REM and REM/Wake transitions and augmented sleep-states related cortical activation that was simultaneously expressed by the high frequency amplitude augmentation, as well as Wake and NREM delta frequency attenuation.

Highlights

► We studied the pedunculopontine tegmental nucleus (PPT) lesion impact on sleep. ► Unilateral or bilateral PPT lesion was done in rat using ibotenic acid microinfusion. ► PPT cholinergic neuronal loss did not change sleep/wake architecture. ► Impaired PPT cholinergic control disturbed the sleep/state transitions structure. ► PPT cholinergic cells neurodegeneration augmented cortical activation during sleep.

Introduction

The pedunculopontine tegmental nucleus (PPT) is postulated to have important functions relevant to the regulation of rapid eye movement sleep (REM) (Lu et al., 2006, McCarley and Hobson, 1975), arousal (Bringmann, 1995, Bringmann, 1997, Datta, 2002, Datta and MacLean, 2007, Datta and Siwek, 1997, Datta et al., 2001, Kleiner and Bringmann, 1996, Rye, 1997, Vertes, 1984), and various motor control systems (Inglis et al., 1994, Saper and Loewy, 1982, Takakusaki et al., 2004), including breathing control (Lydic and Baghdoyan, 1993, Radulovacki et al., 2004, Saponjic et al., 2003, Saponjic et al., 2005a, Saponjic et al., 2005b, Saponjic et al., 2006). It is also postulated that PPT is the high relay nucleus for overall REM sleep phenomenon control, and that each REM sleep event, executed by distinct cell groups within the brainstem, may be triggered and modulated by the activation of the PPT (Datta, 1995, Datta, 1997, Garcia-Rill, 1991). Anatomical studies support the central role of the PPT in controlling the REM sleep phenomenon: each individual REM-sleep-sign generating nucleus receives afferent inputs from PPT (Rye, 1997, Semba, 1993).

The different regulatory functions of the PPT are reflected in electroencephalographic (EEG/ECoG) or electromyographic (EMG) events and rhythms. For example, EEG desynchronization or gamma activity reflects cortical activation (Maloney et al., 1997). The activation states of the mammalian neocortex, measured by electroencephalogram (EEG) or electrocorticogram (ECoG), are regulated by a complex interplay of cortical and subcortical networks. While slow EEG/ECoG oscillations (< 1 Hz) are present in neocortical isolated tissue, suggesting that intracortical networks are sufficient to maintain this type of deactivated activity pattern (Dringenberg and Olmstead, 2003, Timofeev et al., 2000), the high-frequency oscillations within the beta and gamma ranges are not present in the isolated cortex, which suggests a dependence on subcortical–cortical impulse flow.

Human development, maturation, healthy aging and numerous neurological diseases are associated with profound changes in sleep/wake states distribution, and with a variety of sleep-related behavioral disorders. Sleep-related behavioral disorders very frequently go unnoticed in patients with neurodegenerative diseases, and as a symptom, precede the onset of motor and cognitive disturbances by years or even decades (Boeve et al., 2007, Simic et al., 2009, Whitwell et al., 2007). REM behavioral disorders (RBD) in Alzheimer's and Parkinson's Disease (AD and PD) involve the selective loss of specific neuronal populations within the brain, and reflects an underlying synucleinopathy with the presence of the α-synuclein protein pathology within the REM sleep-related regulatory structures of the dorsal midbrain and pons at the onset of disease, and with ascending pattern of neurodegeneration progression from brainstem to basal areas of the brain (Raggi and Ferri, 2010, Simic et al., 2009, Whitwell et al., 2007).

The cholinergic afferent fibers system of the basal forebrain plays a critical role in switching cortical activity from deactivated slow to activated high-frequency EEG/ECoG patterns (Dringenberg and Olmstead, 2003). Inputs to the cortex originating in the thalamus constitute the second major system involved in regulation of cortical EEG/ECoG. The thalamic local network, consisting of thalamic interneurons in the reticular nucleus (RT), and thalamocortical projection neurons, generates spindle oscillations in the thalamocortical network, that are associated with reduced behavioral activation in early sleep stages, and block incoming sensory information (Steriade, 2000, Steriade, 2003). However, thalamic and RT lesions in rats and cats abolished spindles, even though high frequency activation was maintained (Buzsaki et al., 1988, Vanderwolf and Stewart, 1993). All the above mentioned experimental evidence suggests that thalamic integrity is not essential to the occurrence of cortical activation, although it can modulate the ability of other (e.g. cholinergic) systems to induce cortical activation. The rat thalamus receives cholinergic projections exclusively from the brainstem, with the exception of the RT which receives cholinergic innervation from both the basal forebrain as well as the mesopontine tegmentum (Williams et al., 1994).

Anatomical and electrophysiological studies have demonstrated that the activating influences of the basal forebrain and thalamus are under the powerful neuromodulatory control of the fibers from the diencephalon and the brainstem. While the dorsal pathway from the brainstem innervates the thalamus, the ventral pathway ascends through the subthalamus and the hypothalamus toward the basal forebrain (Steriade, 2000). It has been shown that the cholinergic (PPT) and monoaminergic (locus coeruleus — LC; dorsal raphe nucleus — DR) inputs from the brainstem to the thalamus suppress spindle oscillations and facilitate thalamocortical transmission (Steriade, 2000). Also, the direct projections from PPT reach both the basal forebrain and the thalamus (Loiser and Semba, 1993), and therefore the PPT represents the control relay nucleus for intergrated contributions of these two cholinergic systems to the regulation of cortical activation (Dringenberg and Olmstead, 2003, Sarter and Bruno, 2000).

This study aims to investigate the effects of unilateral and bilateral PPT lesions on sleep–wake states and all the conventional sleep-state related EEG frequency bands amplitudes, in an attempt to find the EEG markers for the onset and progression of PPT cholinergic neuronal degeneration.

Section snippets

Material and methods

We performed the experiments on 35 adult, male Wistar rats, chronically instrumented for sleep recording, and randomly assigned to one of five experimental groups: physiological controls (n = 8), unilateral sham controls (n = 8), bilateral sham controls (n = 5), unilaterally PPT lesioned rats (n = 7), and bilaterally PPT lesioned rats (n = 7).

Prior to surgery and consistently throughout the experimental protocol, the animals were maintained on a 12-hour light–dark cycle, and were housed at 25 °C with free

Surgical procedure

The surgical procedures employed for the chronic electrode implantation for sleep recording have previously been described (Carley and Radulovacki, 2003, Saponjic et al., 2007), and are outlined below. We implanted two epidural parietal stainless-steel screw electrodes for EEG cortical activity recording from motor (A/P: + 1.0; R/L: 2), and sensorimotor (A/P: − 3.0; R/L: 2) cortex (Paxinos and Watson, 2005) under ketamine/diazepam anesthesia (Zoletil 50, VIRBAC, France, 50 mg/kg; i.p). Bilateral

Recording procedure

At the end of the surgical procedure, the scalp wounds were sutured and the rats were given a recovery period of 2 weeks before adapting to the recording cable and plexiglass chamber (30 cm × 30 cm × 30 cm) for one day. EEG and EMG activities were carried from the connector plug on the rat head by cable, and passed through the sealed port of the recording box. They were displayed on a computer monitor, and stored on disk for further off-line analysis. After conventional amplification and filtering

Tissue processing and histochemistry

At the end of the recording sessions the PPT lesion was identified by NADPH-diaphorase histochemistry (Datta et al., 2001, Paxinos et al., 2009, Sabbatini et al., 1999, Vincent, 1992, Vincent et al., 1983). The rats were deeply anesthetized and perfused transcardially, starting with a vascular rinse until the liver had been cleared (200 ml of 0.9% saline; perfusion speed of 40 ml/min); followed by a 4% paraformaldehyde solution in 0.1 M PBS (200 ml; 100 ml at 40 ml/min, and then 30 ml/min), and

Data analysis

We included the signals recorded from the control rats and all rats with positively identified unilateral or bilateral PPT lesions in the data analysis. Analysis of the recorded signals was conducted with software we developed using MATLAB 6.5. We applied Fourier analysis to signals acquired throughout each 6-hour recording (2160 10 s Fourier epochs), and each 10 s epoch was differentiated as Wake, NREM or REM state for further analysis of the Wake, NREM and REM related EEG amplitudes of all the

Results

Unilateral and bilateral PPT lesions did not change the sleep/wake architecture (Figs. 2A, B) during the 35 days of the lesion follow-up period (χ2  0.78, p  0.17 for Wake; χ2  0.73, p  0.34 for NREM; χ2  0.72, p  0.08 for REM), but they did change the sleep/wake state transitions structure (Figs. 3A, B; Table 1), and the sleep/state related “EEG microstructure” or EEG frequency relative amplitudes (Fig. 4, Fig. 5, Fig. 6). Since we did not find statistically significant sleep/wake state distribution

Discussion

Our study demonstrates that PPT cholinergic control impairment disturbs the sleep/wake state transition structure, and augments cortical activation during all sleep/wake states. PPT cholinergic control impairment was expressed as: a) a sustainable increase in the Wake/REM and REM/Wake transitions for 5 weeks followed by a decrease in the NREM/REM and REM/NREM transitions within last two weeks only following bilateral PPT lesions; and b) augmented cortical activation recognized by the

Conclusion

Our study demonstrates that PPT cholinergic control impairment sustainably disturbs the sleep/wake state transition structure, and shows that PPT cholinergic neuronal loss augmented sleep-state related cortical activation during Wake, NREM and REM. This cortical activation was expressed by the simultaneous state-related high frequency amplitude augmentation, and Wake and NREM delta frequency attenuation.

Acknowledgment

This work was supported by a Serbian Ministry of Education, Science and Technological Development grant OI 173022.

References (58)

  • C.B. Saper et al.

    Projections of the pedunculopontine tegmental nucleus in the rat: evidence for additional extrapyramidal circuitry

    Brain Res.

    (1982)
  • J. Saponjic et al.

    Respiratory pattern modulation by the pedunculopontine tegmental nucleus

    Respir. Physiol. Neurobiol.

    (2003)
  • J. Saponjic et al.

    Monoaminergic system lesions increase post-sigh respiratory pattern disturbance during sleep in rats

    Physiol. Behav.

    (2007)
  • M. Steriade

    Corticothalamic resonance, states of vigilance and mentation

    Neuroscience

    (2000)
  • K. Takakusaki et al.

    Changes in the excitability of hindlimb motoneurons during muscular atonia induced by stimulating the pedunculopontine tegmental nucleus in cats

    Neuroscience

    (2004)
  • R.P. Vertes

    Brainstem control of the events of REM sleep

    Prog. Neurobiol.

    (1984)
  • S.R. Vincent et al.

    NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation

    Neurosci. Lett.

    (1983)
  • P. Winn

    How best to consider the structure and function of the pedunculopontine tegmental nucleus: evidence from animal studies

    J. Neurol. Sci.

    (2006)
  • B.F. Boeve et al.

    Pathophysiology of REM sleep behaviour disorder and relevance to neurodegenerative disease

    Brain Rev.

    (2007)
  • N.I. Bohnen et al.

    History of falls in Parkinson disease is associated with reduced cholinergic activity

    Neurology

    (2009)
  • N.I. Bohnnen et al.

    The cholinergic system and Parkinson disease

    Behav. Brain Res.

    (2011)
  • S. Boucetta et al.

    Activity profiles of cholinergic and intermingled GABAergic and putative glutamatergic neurons in the pontomesencephalic tegmentum of urethane-anesthetized rats

    J. Neurosci.

    (2009)
  • A. Bringmann

    Different functions of rat's pedunculopontine tegmental nucleus are reflected in cortical EEG

    Neuroreport

    (1995)
  • A. Bringmann

    Nicotine affects the occipital theta rhythm after lesion of the pedunculopontine tegmental nucleus in rats

    Neuropsychobiology

    (1997)
  • G. Buzsaki et al.

    Nucleus basalis and thalamic control of neocortical activity in the freely moving rat

    J. Neurosci.

    (1988)
  • W.D. Carley et al.

    Pathophysiology of sleep-related breathing disorders: unanswered questions

  • K.A. Chung et al.

    Cholinergic augmentation in frequently fallings subjects with Parkinson's disease

    Mov. Disord.

    (2009)
  • S. Datta

    Cellular basis of pontine ponto-geniculo-occipital wave generation and modulation

    Cell. Mol. Neurobiol.

    (1997)
  • S. Datta

    Evidence that REM sleep is controlled by activation of brain stem pedunculopontine tegmental kainate receptor

    J. Neurophysiol.

    (2002)
  • Cited by (29)

    • Prodromal local sleep disorders in a rat model of Parkinson's disease cholinopathy, hemiparkinsonism and hemiparkinsonism with cholinopathy

      2021, Behavioural Brain Research
      Citation Excerpt :

      The analysis included conventional EEG frequency bans (δ = 0.3−4 Hz; θ = 4.1−8 Hz; σ = 10.1−15 Hz; β = 15.1−30 Hz; γ = 30.1−50 Hz). In addition, for each sleep/wake state and each frequency band, PDE analysis was performed on the assembles of relative amplitudes [14,15,34,35] by pooling the measured values from all the animals belonging to the specific experimental group. For statistical analysis of the relative amplitudes/6 h, we calculated the means during each 30 min for Wake and REM, and during each 60 min for NREM.

    • Sleep disorder and altered locomotor activity as biomarkers of the Parkinson's disease cholinopathy in rat

      2018, Behavioural Brain Research
      Citation Excerpt :

      All experimental procedures were in compliance with EEC Directive (2010/63/EU) on the protection of animals used for experimental and other scientific purposes, and were approved by Ethical Committee for the Use of Laboratory Animals of the Institute for Biological Research “Sinisa Stankovic”, University of Belgrade (Approval No 2-21/10) The surgical procedures employed for the EEG and EMG electrodes implantation for chronic sleep recording and the bilateral PPT lesion have been described previously [6–9,25–29] and are outlined below. We implanted under ketamine/diazepam anesthesia (Zoletil 50, VIRBAC, France, 50 mg/kg; i.p.), in 2 and a one half month old rats, according to Paxinos and Watson [30], two epidural stainless-steel screw electrodes for electroencephalographic (EEG) cortical activity from the motor cortex (MCx; A/P: +1.0 mm from bregma; R/L: 2.0 mm from sagittal suture; D/V: 1 mm from the skull), two pairs of the stainless-steel teflon-coated wires (Medwire, NY, USA) into the CA1 hippocampal regions (A/P: −3.60 mm from bregma; R/L: 2.5 mm from sagittal suture; D/V: 2.5 mm from the brain surface) as well as into the dorsal nuchal musculature to assess skeletal muscle activity (EMG), and a stainless-steel screw electrode in the nasal bone as a ground.

    • Wake–sleep circuitry: an overview

      2017, Current Opinion in Neurobiology
    View all citing articles on Scopus
    View full text