Spavanje i elektroencefalografski ritmovi kao indikatori poremećaja funkcionalno različitih holinergičkih inervacija velikog mozga pacova
Sleep and electroencephalographic rhythms as an indicators of the functionally distinct cholinergic innervation disorders in rat brain
Doctoral thesis (Published version)
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Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases of elderly. AD and PD neuropathology involves selective loss of specific neuronal population within the brain, including the basal forebrain and brainstem cholinergic neurons, with the ascending pattern of neurodegeneration progression from rapid eye movement (REM) sleep-related brainstem regulatory structures to the brain basal areas. REM sleep behavior disorders (RBD) very frequently go unnoticed in the AD and PD patients, and as a symptom, could precede the onset of motor and cognitive disturbances by years or even decades. This indicates the importance of RBD as a potential early marker of the neurodegenerative diseases of elderly. Using the experimental models of AD and PD cholinergic neuropathology, this PhD thesis aimed to investigate for the first time the impact of unilateral and bilateral nucleus basalis (NB – the main source of cortical cholinergic innervation) and nucleus pedunculopontinus tegmentalis (PPT – the main source of thalamo-cortical cholinergic innervation) lesions on sleep and electroencephalographic (EEG) rhythms during sleep in rat. In these experimental models of cholinergic neuropathology (NB and PPT lesions) the unilateral lesions were used as the models of mild or early stage neurodegenerations, whereas the bilateral lesions were used as the models of severe or progressed neurodegenerations, and the follow up period was up to 5 weeks depending on the experimental group. Based on the hypothesis that functionally distinct cholinergic systems have different regulatory functions during sleep, that could be defined by EEG activity, this study aimed to investigate the sleep/wake architecture, sleep/wake state-related transitions structure and EEG microstructure of three main brain states (Wake, NREM – non rapid eye movement, REM) in order to find the possible EEG markers for the onset, severity, and progression of the functionally distinct cholinergic innervations disorders. These EEG markers could be used as a sensitive and reliable methodology of early detection in the patients who are at risk to develop AD or PD, and for a potential prevention and modification of the therapeutic approach. Male Wistar rats were chronically implanted for sleep recording. During operative procedure for the electroencephalographic (EEG) and electromyographic (EMG) electrodes implantation, the selective unilateral or bilateral NB or PPT lesions were performed by stereotaxically guided microinfusion of ibotenic acid (the general glutamate agonist). Sleep recording sessions started after two weeks of recovery period. Sleep was recorded for 6 h, weekly, during 4 weeks in NB lesioned rats, and during 5 weeks in PPT lesioned rats, using DataWave SciWorks Experimenter 8.0 software (Datawave Technologies, Longmont, SAD). At the end of recording sessions the NB and PPT lesions were identified by NADPH – diaphorase histochemistry, and quantified throughout the overall NB or PPT antero-posterior dimensions, using ImageJ 1.46 software (NIH, SAD). Analysis of the signals recorded from the control rats and all rats with positively identified NB or PPT lesion was done in MATLAB 6.5 using originally developed software. Fourier analysis was applied on 6 h recordings, and each 10 s epoch was differentiated as Wake, NREM or REM state. Additionally, for detailed analysis of two distinct REM clusters, that emerged following bilateral PPT lesion, each REM epoch was further differentiated, based on the EMG power, as either REM without atonia (REM1, pathological REM), and REM with atonia (REM2, physiological REM). Further analysis included the topography of sleep/wake states architecture, sleep/wake state-related transitions structure, and EEG microstructure, as well as all REM related cortico-muscular coherences. To calculate the group probability density distribution of all conventional EEG frequency bands relative amplitudes/6 h for the Wake/NREM/REM/REM1/REM2 of each experimental group we used the Probability Density Estimate (PDE) routine within MATLAB 6.5. For the functional REM cluster differentiation the REM/REM1/REM2 cortico-muscular coherences (CMCs) were calculated separately, for each conventional EEG frequency band and each experimental group, between the EEG of sensorimotor or motor cortex and the EMG of the dorsal nuchal muscles using the MATLAB 6.5 cohere routine. All statistical analyses were done using Kruskal-Wallis ANOVA with post-hoc Mann-Whitney U two-tailed tests. Bilateral NB lesion transiently altered sleep/wake states topography 14 days following lesion. While the control rats exhibited equivalent durations of Wake, NREM and REM, as determined by sensorimotor versus motor cortex EEG, the bilateral NB lesion decreased Wake duration in both cortices, with NREM duration increased within the sensorimotor cortex, and REM duration increased within the motor cortex. At the same time, Wake, NREM and REM theta relative amplitude was lower in motor versus sensorimotor cortex in all experimental groups. In sensorimotor cortex the bilateral NB lesion increased only REM theta amplitude for three weeks, whereas in motor cortex both Wake and REM theta amplitude were transiently increased 14 days following both the unilateral and bilateral NB lesion. PPT lesion did not change the sleep/wake architecture but did change the sleep/wake state-related transitions structure and the Wake/NREM/REM EEG microstructures. Both the unilateral and bilateral PPT lesions sustainably increased Wake/REM and REM/Wake transitions during 5 weeks, followed by the decreased NREM/REM and REM/NREM transitions from 28 days only in the case of the bilateral PPT lesion, as a more severe cholinergic neurodegeneration. The unilateral PPT lesion augmented Wake theta and REM beta amplitude, and with a delay of one week it attenuated Wake delta amplitude. The bilateral PPT lesion augmented beta amplitude during all sleep states, and REM gamma amplitude, but simultaneously attenuated Wake and NREM delta amplitude. Comparison of the bilateral NB versus bilateral PPT lesion (the models of severe or progressed cholinergic neurodegenerations) effects on the sleep/wake states architecture, sleep/wake state-related transitions structure and EEG microstructures indicated the topographically specific differentiation of functionally distinct cholinergic innervation disorders. Whereas the bilateral NB lesion transiently altered Wake/NREM duration within the sensorimotor cortex, and Wake/REM duration within the motor cortex, the bilateral PPT lesion did not change the sleep/wake states distributions. Bilateral PPT lesion sustainably (during 4 weeks) increased the Wake/REM and REM/Wake transitions within the both sensorimotor and motor cortex, followed by inconsistent dysregulation of the NREM/REM and REM/NREM transitions within the sensorimotor cortex, but oppositely by their increment within the motor cortex. Bilateral NB lesion sustainably (during 4 weeks) decreased the NREM/REM and REM/NREM transitions within the sensorimotor cortex, but oppositely increased them within the motor cortex. Sleep/wake state-related EEG microstructure following the bilateral PPT lesion was expressed as the sustained (during 4 weeks) Wake/NREM/REM beta and gamma amplitude augmentations within the both sensorimotor and motor cortex, and Wake delta amplitude attenuation, but only within the sensorimotor cortex. In contrast, the bilateral NB lesion augmented only REM theta amplitude within the sensorimotor cortex during three weeks. Alongside the sleep/wake state-related transitions structure and Wake/NREM/REM EEG microstructure alteration, the bilateral PPT lesion in rats additionally potentiated the emergence of two distinct REM sleep states, REM1 (pathological REM, REM without atonia, sigma coherent REM) and REM2 (physiological REM, REM with atonia, theta coherent REM), specifically expressed within the motor cortex. Bilateral PPT lesion did not change the sleep/wake states architecture within the sensorimotor cortex, but pathologically increased the duration of REM1 within the motor cortex, alongside the increased Wake/REM1/Wake and NREM/REM2/NREM transitions within the both cortices. In addition, the augmented total REM beta amplitude within the sensorimotor cortex and REM1 theta amplitude within the motor cortex was the underlying EEG microstructure pathology. Finally, the bilateral PPT lesion dominantly induced sensorimotor cortex-dorsal nuchal muscle drive alteration, expressed throughout the REM/REM1/REM2 beta CMC decrease. Introducing the novel experimental model of PD cholinergic neuropathology (PPT lesion), the stereotaxically guided microinfusion as a novel approach for the selective lesion of the brain nuclei, the novel histochemical method for the cholinergic neuronal loss identification, and the novel methodological approach for neuronal loss quantification, as well as the novel sleep-state related EEG signal analysis methodology, this PhD thesis evidenced for the first time the topographically different expression of the sleep/wake architecture, sleep/wake state-related transitions structure and Wake/NREM/REM EEG microstructure, induced by the functionally distinct cholinergic innervation disorders in rat (NB versus PPT lesion). This study demonstrated for the first time the PPT lesion (novel experimental model of PD cholinergic neuropathology) induced sustained and more severe sleep disturbances expressed as: the Wake/REM and REM/Wake transitions increase, augmented cortical activation across all sleep/wake states, potentiation of two distinct REM clusters, and dominant sensorimotor cortex-dorsal nuchal muscle drive alteration throughout the REM/REM1/REM2 beta CMC decrease.
Keywords:Sleep; Electroencephalography (EEG); Electromyography (EMG); Cholinergic neurons; Nucleus basalis (NB); Nucleus pedunculopontinus tegmentalis (PPT); Ibotenic acid (IBO) lesion; Neurodegeneration; Rat; Cortico-muscular coherence (CMC)
Source:University of Belgrade, Faculty of Biology, 2015, 1-156
- Neurobiology of sleep in aging and disease - electroencephalographic markers and modeling in the estimation of disorder (RS-173022)