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Alzheimer's disease (AD) involves selective loss of basal forebrain cholinergic neurons, particularly in the nucleus basalis (NB). Similarly, Parkinson's disease (PD) might involve the selective loss of pedunculopontine tegmental nucleus (PPT) cholinergic neurons. Therefore, lesions of these functionally distinct cholinergic centers in rats might serve as models of AD and PD cholinergic neuropathologies. Our previous articles described dissimilar sleep/wake-state disorders in rat models of AD and PD cholinergic neuropathologies. This study further examines astroglial and microglial responses as underlying pathologies in these distinct sleep disorders. Unilateral lesions of the NB or the PPT were induced with rats under ketamine/diazepam anesthesia (50 mg/kg i.p.) by using stereotaxically guided microinfusion of the excitotoxin ibotenic acid (IBO). Twenty-one days after the lesion, loss of cholinergic neurons was quantified by nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, and the astroglial and microglial responses were quantified by glia fibrillary acidic protein/OX42 immunohistochemistry. This study demonstrates, for the first time, the anatomofunctionally related astroglial response following unilateral excitotoxic PPT cholinergic neuronal lesion. Whereas IBO NB and PPT lesions similarly enhanced local astroglial and microglial responses, astrogliosis in the PPT was followed by a remote astrogliosis within the ipslilateral NB. Conversely, there was no microglial response within the NB after PPT lesions. Our results reveal the rostrorostral PPT-NB astrogliosis after denervation of cholinergic neurons in the PPT. This hierarchically and anatomofunctionally guided PPT-NB astrogliosis emerged following cholinergic neuronal loss greater than 17\% throughout the overall rostrocaudal PPT dimension. (c) 2014 Wiley Periodicals, Inc. Ministry of Education, Science, and Technological Development, Republic of Serbia {[}OI 173022, III 41005]

We examined the effects of unilateral and bilateral nucleus basalis (NB) lesion in rat on sleep/wake states, and sleep/wake state-related electroencephalographic (EEG) frequency relative amplitude distributions. We aimed this study to identify the possible EEG markers for the onset and progression of cortical cholinergic neurodegeneration in rats. NB lesion was performed by ibotenic acid (IBO) microinfusion, and identified by NADPH-diaphorase histochemistry. Sleep/wake states related EEG relative amplitude analysis was done using the Probability Density Estimate (PDE) routine supplied with MATLAB 6.5. Bilateral NB lesion transiently altered gross sleep/wake states topography 14 days following lesion. While control rats exhibited equivalent durations of Wake, NREM and REM, as determined by sensorimotor versus motor cortex EEG, bilateral NB lesion decreased Wake duration in both cortices, with NREM duration increased within sensorimotor cortex, and REM duration increased within motor cortex. Also, Wake, NREM and REM theta relative amplitude was lower in motor versus sensorimotor cortex in all groups of animals. In sensorimotor cortex bilateral NB lesion increased only REM theta relative amplitude from 1421 days following lesion, and returned to control value 28 days following lesion. In motor cortex both Wake and REM theta relative amplitude transiently increased 14 days following unilateral and bilateral NB lesion, and returned to control values 21 days after lesions. We demonstrated at functional level, for the first time, the topographically specific impact of NB cholinergic cortical afferent system dysregulation on sleep/wake states, REM and Wake EEG theta relative amplitude. Serbian Ministry of Education, Science and Technological Development [OI 173022]

This study was aimed to explore the sleep/wake states related cortico-pontine theta carrier frequency phase shift following a systemically induced chemical axotomy of the monoaminergic afferents within a brain of the freely moving rats. Our experiments were performed in 14 adult, male Sprague Dawley rats, chronically implanted for sleep recording. We recorded sleep during baseline condition, following sham injection (saline i.p. 1 ml/kg), and every week for 5 weeks following injection of the systemic neurotoxins (DSP-4 or PCA; 1 ml/kg, i.p.) for chemical axotomy of the locus coeruleus (LC) and dorsal raphe (DR) axon terminals. After sleep/wake states identification, FFT analysis was performed on 5 s epochs. Theta carrier frequency phase shift (Delta Phi) was calculated for each epoch by averaging theta Fourier component phase shifts, and the Delta Phi values were plotted for each rat in control condition and 28 days following the monoaminergic lesions, as a time for permanently established DR or LC chemical axotomy. Calculated group averages have shown that Delta Phi increased between pons and cortex significantly in all sleep/wake states (Wake, NREM and REM) following the monoaminergic lesions, with respect to controls. Monoaminergic lesions established the pontine leading role in the brain theta oscillations during all sleep/wake states. Serbian Ministry of Education and Science [OI173022]; NIH [AG016303]

We applied a combination of fractal analysis and Independent Component Analysis (ICA) method to detect the sources of fractal complexity in snail Br neuron activity induced by static magnetic field of 2.7 mT. The fractal complexity of Br neuron activity was analyzed before (Control), during (MF), and after (AMF) exposure to the static magnetic field in six experimental animals. We estimated the fractal dimension (FD) of electrophysiological signals using Higuchi's algorithm, and empirical FD distributions. By using the Principal Component Analysis (PCA) and FastICA algorithm we determined the number of components, and defined the statistically independent components (ICs) in the fractal complexity of signal waveforms. We have isolated two independent components of the empirical FD distributions for each of three groups of data by using FastICA algorithm. ICs represent the sources of fractal waveforms complexity of Br neuron activity in particular experimental conditions. Our main results have shown that there could be two opposite intrinsic mechanisms in single snail Br neuron response to static magnetic field stimulation. We named identified ICs that correspond to those mechanisms - the component of plasticity and the component of elasticity. We have shown that combination of fractal analysis with ICA method could be very useful for the decomposition and identification of the sources of fractal complexity of bursting neuronal activity waveforms. (C) 2011 Elsevier Ireland Ltd. All rights reserved. Ministry of Education and Science, Republic of Serbia [OI 173027, OI 173022]

The complexity, entropy and other non-linear measures of the electroencephalogram ( EEG), such as Higuchi fractal dimension (FD), have been recently proposed as the measures of anesthesia depth and sedation. We hypothesized that during unconciousness in rats induced by the general anesthetics with opposite mechanism of action, behaviorally and poligraphically controlled as appropriately achieved stable anesthesia, we can detect distinct inter-structure brain dynamic using mean FDs. We used the surrogate data test for nonlinearity in order to establish the existence of nonlinear dynamics, and to justify the use of FD as a nonlinear measure in the time series analysis. The surrogate data of predefined probability distribution and autocorrelation properties have been generated using the algorithm of statically transformed autoregressive process (STAP). FD then is applied to quantify EEG signal complexity at the cortical, hippocampal and pontine level during stable general anesthesia (ketamine/xylazine or nembutal anesthesia). Our study showed for the first time that global neuronal inhibition caused by different mechanisms of anesthetic action induced distinct brain inter- structure complexity gradient in Sprague Dawley rats. EEG signal complexities were higher at cortical and hippocampal level in ketamine/ xylazine vs. nembutal anesthesia, with the dominance of hippocampal complexity. In nembutal anesthesia the complexity dominance moved to pontine level, and ponto- hippocampocortical decreasing complexity gradient was established. This study has proved the Higuchi fractal dimension as a valuable tool for measuring the anesthesia induced inter- structure EEG complexity. Serbian Ministry of Science and Technological Development [173022]; NIH [AG16303]

An accumulating body of evidence from animal investigations supports the potential importance of vagal afferent pathways in the pathogenesis and/or therapy of SRBD. The neurochemistry and neuropharmacology of this pathway offers numerous possible targets for pharmacologic modulation. Defining the ultimate clinical relevance of these pathways in SRBD pathogenesis and treatment can only be accomplished with significant ongoing clinical and basic investigation. Značajan napredak u našem razumevanju poremećaja disanja u vezi sa spavanjem (SRBD) je načinjen u pogledu epidemiologije i faktora rizika, patogeneze i kliničkih i bihevioralnih posledica. Ipak, napori da se ovi faktori shvate u smislu ćelijskih, molekularnih i neuromodulatornih procesa još uvek su u povoju, što nas sprečava da razvijemo delotvorne a prihvatljive strategije dijagnostike i lečenja. SRBD sa prevalencom od ~2% kod žena i ~4% kod muškaraca [1] povećava rizik za kardiovaskularni, metabolički i bihevioralni morbiditet [2]. Bolesnici sa SRBD mogu imati čitav spektar respiratornih poremećaja tokom spavanja kao što su centralna apneja (prestanak respiratornog napora); opstruktivna apneja (inspiratorni napor protiv okludiranog gornjeg disajnog puta); mešovita apneja (prikazuje se inicijalnom centralnom komponentom kojoj neposredno sledi opstruktivna komponenta); hipopneja (parcijalni kolaps gornjih disajnih puteva); i razbuđivanja povezana sa respiratornim događajem (otežan inspiratorni napor koji dovodi do razbuđivanja). Premda učestalost poremećaja disanja ili 'indeksa respiratornog poremećaja' (RDI) retko premašuje pet događaja na sat kod zdravih, do sada nije ustanovljen jasan prag RDI iznad koga nastaje bihevioralna ili klinička bolest. null

Sleep is a complex, global and reversible behavioral state of all mammals, that is homeostatically regulated. Generally it is also defined as a rapidly reversible state of immobility and reduced sensory responsiveness. Still, there is no definition that has succeeded in satisfying all aspects of sleep. The failure to define sleep as a single behavior lies in several facts: (1) sleep is not a homogenous state, but continuum of number of mixed states; (2) the control mechanisms of sleep are manifested at all levels of biological organization - from genes and intracellular mechanisms to the networks of neuronal populations within the central nervous system that control movement, arousal, autonomic functions, behavior and cognition; (3) the activity and interactions of these neurochemically greatly heterogenous neuronal populations are dependent of two biological rhythms - the circadian rhythm of wake/sleep and periodic cycles of NREM/REM sleep as two main sleep states. There are several levels of sleep control. The brain forebrain areas serve to control neuropsychology of dreaming; thalamo-cortical system controls NREM sleep rhythms, EEG activation and deactivation; hippocampo-cortical system controls memory consolidation; hypothalamic nuclei are the sources of circadian rhythm and sleep onset control; the control of periodic NREM/REM cycling is within the pons. The wake promoting neuronal populations are within the brainstem, midbrain, hypothalamus and basal forebrain. The main pontine wake-promoting centers are the noradrenergic neurons of locus coeruleus, the serotonergic neurons of dorsal raphe nucleus and the cholinerigic neurons of pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus. The reciprocal connections and interactions of these neurons, and their opposite discharge pattern activity from wake to NREM and REM sleep have been the background of reciprocal interaction hypothesis of REM sleep generation. The wake-promoting neurons at the midbrain level are glutamatergic neurons of midbrain reticular formation, dopaminergic neurons of substantia nigra and ventral tegmental area. All pontine and midbrain wake-promoting cells project dorsally to activate thalamocortical system and ventrally to activate hypothalamo-cortical and basalo-cortical systems. There are also hypothalamic wake-promoting histaminegic neurons within the tuberomammilary nucleus of the posterior hypothalamus, and the hypocretinergic neurons of lateral hypothalamus. Cholinergic neurons of the basal forebrain beside control of wakefulness are included in control of many wake-promoting behaviors such as attention, sensory procession and learning. Recent molecular studies suggest the nucleus suprachiasmaticus as a wake-promotin area of the brain. The most important endogenously accumulated metabolite during wakefulness that initiates sleep is adenosine. Beside adenosine, the sleep-initiating factors are also GAB A, glycine, PGD2, and cytokines IL-1beta and TNF-alfa. Also, releasing hormone of growth hormone (GHRH) increases depth and duration of NREM sleep. On the bases of 'flip-flop' hypothesis of REM sleep control two GABA neuronal populations within the pons - REM-off area (ventrolateral periaqueductal grey matter and lateral pontine tegmentum), and REM-on area (sublaterodorsal nucleus and precoeruleus) are reciprocally interconnected and inhibit each other. REM-off area is activated by orexinergic neurons of lateral hypothalamus, and inhibited by GABAergic and galanin neurons of hypothalamic ventrolateral preoptic nucleus. Monoaminergic neurons of dorsal raphe nucleus and locus coeruleus are excitatory, while cholinergic neurons of PPT/LDT are inhibitory modulators of REM-off area of 'flip-flop' switch. Although a lot of knowledge and progress has been accumulated in sleep research, we still do not have a meaningful explanation for the actual function of sleep. Sleep deprivation for 2-3 weeks in rats is fatal. Many experimental evidence suggest a role of REM sleep in brain development and maturation, synaptic homeostasis, memory and learning, but sleep in not universal state with the same underlying vital function in all species. Even within the species that do meet the behavioral definitions of sleep behavior (about 50 of 60 000 vertebrates tested) still is questionable do they sleep for the same reason. null

This study demonstrates that unilateral excitotoxic lesionin of thepontine ITR increases the frequency of spontaneous central sleep apneas in freely behaving rats without alteration in average respiratory rate, minute ventilation, or sleep architecture. Thus, our findings suggest that the ITR may function specifically to dampen respiratory perturbations and that bilateral integrity of this structure is important. We may also speculate that the ITR functions to maintain stable breathing during sleep in man. null

U ovoj doktorskoj disertaciji ispitivan je uticaj ketamin/diazepam i pentobarbital opšte anestezije na EEG mikrostrukturu i obrazac disanja tokom anestezije, kao i na arhitekturu spavanja i strukturu prelaznih stanja, EEG mikrostrukturu i dinamiku epizoda svih faza spavanja nakon anestezije, kako u fiziološkim kontrolama, tako i u eksperimentalnom modelu holinergičke neuropatologije Parkinsonove bolesti (bilateralna lezija PPT jedra).Rezultati ove doktorske disertacije pokazali su da je ketamin/diazepam anestezija operativnog nivoa izazvala ozbiljan poremećaj respiratornog obrasca i EEG mikrostrukture tokom anestezije. Međutim, jednako vreme potrebno za uspostavljanje stabilne anestezije u pacova sa bilateralnom lezijom PPT jedra kao i dugotrajni suprimirajući efekti na povećanu NREM beta i teta amplitudu (elektrofiziološki markeri deficita holinergičkih neurona PPT jedra) nakon anestezije, ukazuju da je ova anestezija potencijalno povoljnija, kako za ulazak u anesteziju, tako i za NREM fazu spavanja nakon operativnih zahvata kod gerijatrijskih pacijenata, kao i pacijenata obolelih od Parkinsonove i Alchajmerove bolesti.Nasuprot ketamin/diazepam anesteziji, pentobarbital anestezija ima povoljnije dejstvo na REM fazu spavanja nakon anestezije, kako kod fizioloških kontrola, tako i u eksperimentalnom modelu holinergičke neuropatologije Parkinsonove bolesti na osnovu: dugotrajnih poremećaja REM spavanja i strukture prelaznih stanja kod fizioloških kontrola koje izaziva ketamin/diazepam anestezija; suprimirajućih efekata obe anestezije operativnog nivoa na produženu REM/REM1 fazu spavanja i poremećaj njihove EEG mikrostrukture kod bilateralne lezije PPT jedra; suprimirajućeg efekta pentobarbital anestezije na povećani broj NREM/REM/NREM prelaza uzrokovanih lezijom PPT jedra; kao i na osnovu EEG mikrostrukture tokom stabilne pentobarbital anestezije, koja se ne razlikuje u poređenju PPT lezije i fiziološke kontrole. The aim of this doctoral dissertation was to follow the impact of ketamine/diazepam and pentobarbital anesthesia on the EEG microstructure and respiratory pattern during anesthesia, and on the post-anesthesia sleep/wake states architecture and transition structure, EEG microstructure across sleep, and all the sleep state episodes dynamics in the physiological condition, as well as during the PPT cholinergic neuropathology (the experimental model of Parkinson’s disease cholinergic neuropathology). Ketamine/diazepam anesthesia induces more alterations in the EEG microstructure and respiratory pattern than does the pentobarbital anesthesia in the PPT lesioned rats. In addition, the equal time required to establish an anesthetized state in the PPT lesioned vs. control rats, and the long-term suppressive effect on augmented NREM beta and theta amplitudes (the hallmarks of PPT cholinergic neuronal loss in rat) suggest ketamine/diazepam anesthesia as potentially more beneficial both for anesthesia induction and for post-anesthesia NREM sleep in the surgical procedures of the elderly, and Parkinson’s, and Alzheimer’s patients. In contrast to the ketamine/diazepam anesthesia the pentobarbital anesthesia is much more beneficial for the post-anesthesia REM sleep in physiological conditions as well as during the PPT cholinergic neuropathology on the basis of: the long-lasting REM sleep and transition structure disorders in physiological controls, induced by the ketamine/diazepam; the abolishing effects of both anesthetic regimens on the post-anesthesia prolonged REM/REM1 sleep and on their EEG microstructure disorders in the PPT lesioned rats; the abolishing effect of pentobarbital on the increased NREM/REM/NREM transitions, caused by the PPT lesion; and the equal EEG microstructure during stable pentobarbital anesthesia in the PPT lesioned rats versus controls.

Cilj ove doktorske disertacije bio je da ispita uticaj starenja na spavanje u eksperimentalnim modelima holinergičke neuropatologije Alchajmerove i Parkinsonove bolesti, i da pronađe najranije znake poremećaja spavanja u fiziološkom starenju i u starenju sa neurodegenerativnim bolestima. Uticaj starenja na arhitekturu spavanja, elektroencefalografsku (EEG) mikrostrukturu i motornu kontrolu, u toku svake faze spavanja, je praćen u eksperimentalnim modelima bilateralnih oštećenja jedara nucleus basalis (NB) i nucleus pedunculopontinus tegmentalis (PPT) u pacova, kao eksperimentalnim in vivo modelima funkcionalno različitih holinergičkih neuropatologija. Fiziološko starenje dovodi do topografski različitih promena arhitekture spavanja i motorne kontrole iz senzomotorne kore (SMCx) u odnosu na motornu koru (MCx). Pored promena arhitekture spavanja koje su se javile samo u SMCx, fiziološko starenje dovodi i do povećanja propagacije delta i beta oscilacija iz obe kore za vreme budnosti, ali samo iz MCx za vreme REM faze spavanja. Najraniji znaci starenja u eksperimentalnim modelima funkcionalno različitih holinergičkih neuropatologija, dokazani po prvi put, predstavljaju topografski specifične razlike u EEG mikrostrukturi za vreme REM faze spavanja. Smanjenje delta EEG relativne amplitude u SMCx predstavlja najraniji znak starenja kod NB lediranih pacova, dok povećanje sigma EEG relativne amplitude u MCx predstavlja najraniji znak starenja kod PPT lediranih pacova, za vreme REM faze spavanja. Pored toga, starenjem izazvane promene su različito izražene kroz mišićnu kontrolu iz SMCx, a istovremeno i istovetno izražene iz MCx u toku svih faza spavanja... The aim of the present doctoral dissertation was to evaluate the impact of aging during sleep in the rat models of Alzheimer’s and Parkinson’s disease cholinergic neuropathology, and to determine the possible different and earlier onset of age-related sleep disorder during healthy aging and aging with the neurodegenerative diseases. We used the bilateral nucleus basalis (NB) and nucleus pedunculopontinus tegmentalis (PPT) lesioned rats as the in vivo models of functionally distinct cholinergic neuropathology, and we followed the impact of aging on sleep architecture, the electroencephalographic (EEG) microstructure and motor control across sleep/wake states. We evidenced the topographically distinct impact of healthy aging on sleep architecture and motor control within the sensorimotor (SMCx) vs. motor cortex (MCx). Whereas healthy aging consistently altered only the SMCx sleep architecture, it increased the delta and beta cortical drives from both cortices during Wake, but only through the MCx drive during rapid eye movement sleep (REM). Our results have shown for the first time that the earliest signs of aging during distinct cholinergic neuropathology were expressed through a different and topographically specific EEG microstructure during REM. EEG delta amplitude attenuation within the SMCx was the earliest sign of aging in the NB lesion, whereas EEG sigma amplitude augmentation within the MCx was the earliest sign of aging in the PPT lesion during REM. In addition, aging was differently expressed through the SMCx drive alterations, but it was commonly expressed through the MCx drive alterations during all sleep/wake states...

The aim of the present doctoral dissertation was to evaluate the impact of aging during sleep in the rat models of Alzheimer’s and Parkinson’s disease cholinergic neuropathology, and to determine the possible different and earlier onset of age-related sleep disorder during healthy aging and aging with the neurodegenerative diseases. We used the bilateral nucleus basalis (NB) and nucleus pedunculopontinus tegmentalis (PPT) lesioned rats as the in vivo models of functionally distinct cholinergic neuropathology, and we followed the impact of aging on sleep architecture, the electroencephalographic (EEG) microstructure and motor control across sleep/wake states. We evidenced the topographically distinct impact of healthy aging on sleep architecture and motor control within the sensorimotor (SMCx) vs. motor cortex (MCx). Whereas healthy aging consistently altered only the SMCx sleep architecture, it increased the delta and beta cortical drives from both cortices during Wake, but only through the MCx drive during rapid eye movement sleep (REM). Our results have shown for the first time that the earliest signs of aging during distinct cholinergic neuropathology were expressed through a different and topographically specific EEG microstructure during REM. EEG delta amplitude attenuation within the SMCx was the earliest sign of aging in the NB lesion, whereas EEG sigma amplitude augmentation within the MCx was the earliest sign of aging in the PPT lesion during REM. In addition, aging was differently expressed through the SMCx drive alterations, but it was commonly expressed through the MCx drive alterations during all sleep/wake states. This doctoral dissertation provided for the first time an evidence of distinct REM sleep disorders and sleep state related cortical drives as the signs of aging onset during functionally distinct cholinergic neuropathologies. Cilj ove doktorske disertacije bio je da ispita uticaj starenja na spavanje u eksperimentalnim modelima holinergičke neuropatologije Alchajmerove i Parkinsonove bolesti, i da pronađe najranije znake poremećaja spavanja u fiziološkom starenju i u starenju sa neurodegenerativnim bolestima. Uticaj starenja na arhitekturu spavanja, elektroencefalografsku (EEG) mikrostrukturu i motornu kontrolu, u toku svake faze spavanja, je praćen u eksperimentalnim modelima bilateralnih oštećenja jedara nucleus basalis (NB) i nucleus pedunculopontinus tegmentalis (PPT) u pacova, kao eksperimentalnim in vivo modelima funkcionalno različitih holinergičkih neuropatologija. Fiziološko starenje dovodi do topografski različitih promena arhitekture spavanja i motorne kontrole iz senzomotorne kore (SMCx) u odnosu na motornu koru (MCx). Pored promena arhitekture spavanja koje su se javile samo u SMCx, fiziološko starenje dovodi i do povećanja propagacije delta i beta oscilacija iz obe kore za vreme budnosti, ali samo iz MCx za vreme REM faze spavanja. Najraniji znaci starenja u eksperimentalnim modelima funkcionalno različitih holinergičkih neuropatologija, dokazani po prvi put, predstavljaju topografski specifične razlike u EEG mikrostrukturi za vreme REM faze spavanja. Smanjenje delta EEG relativne amplitude u SMCx predstavlja najraniji znak starenja kod NB lediranih pacova, dok povećanje sigma EEG relativne amplitude u MCx predstavlja najraniji znak starenja kod PPT lediranih pacova, za vreme REM faze spavanja. Pored toga, starenjem izazvane promene su različito izražene kroz mišićnu kontrolu iz SMCx, a istovremeno i istovetno izražene iz MCx u toku svih faza spavanja. Ova doktorska disertacija je po prvi put dokazala različite poremećaje REM faze spavanja i motorne kontrole u toku spavanja, kao najranije znake početka starenja u funkcionalno različitim holinergičkim neuropatologijama. University of Belgrade, Faculty of Biology (2017)

Alchajmerova bolest (AB) i Parkinsonova bolest (PB) su najčešće neurodegenerativne bolesti starenja... Alzheimer's disease (AD)and Parkinson's disease are the most common neurodegenerative diseases in elderly...

Alchajmerova bolest (AB) i Parkinsonova bolest (PB) su najčešće neurodegenerativne bolesti starenja. Neuropatologija AB i PB podrazumeva selektivan gubitak specifičnih populacija neurona, uključujući i holinergičke neurone bazalnog prednjeg mozga i moždanog stabla, kao i ushodni obrazac progresije neurodegeneracije od struktura u moždanom stablu uključenih u regulaciju REM („rapid eye movement”) spavanja ka prednjemoždanim regionima. Poremećaji ponašanja u toku REM spavanja („REM sleep behavior disorders” – RBD) veoma često ostaju neprimećeni kod AB i PB pacijenata i mogu se javiti godinama pa čak i decenijama pre motornih i kognitivnih poremećaja. Ovo ukazuje na značaj istraživanja poremećaja ponašanja u toku REM spavanja kao mogućeg ranog znaka pojave najčešćih neurodegenerativnih bolesti starenja. Predmet istraživanja ove doktorske disertacije bio je da u eksperimentalnim modelima AB i PB holinergičke neuropatologije po prvi put ispita uticaj unilateralnih i bilateralnih lezija glavnog izvora kortikalne holinergičke inervacije (nucleus basalis, NB jedro) i glavnog izvora talamo-kortikalne holinergičke inervacije (nucleus pedunculopontinus tegmentalis, PPT jedro) velikog mozga pacova na spavanje i elektroencefalografske (EEG) ritmove. U ovim eksperimentalnim modelima holinergičke neuropatologije humanih bolesti (NB i PPT lezije) unilateralne lezije su predstavljale lakše oblike ili početne stadijume neurodegeneracija, dok su bilateralne lezije predstavljale teže oblike ili kasnije stadijume neurodegeneracija, a sama progresija bolesti je praćena ukupno 5 nedelja zavisno od eksperimentalne grupe. U skladu sa osnovnom hipotezom ove doktorske disertacije, da funkcionalno različiti centralni holinergički sistemi imaju različite regulatorne funkcije u toku spavanja, koje se mogu definisati na osnovu EEG aktivnosti, postavljen je i osnovni cilj istraživanja: da se kroz analizu arhitekture spavanja, strukture prelaznih stanja i EEG mikrostrukture osnovnih faza u toku spavanja (budnost, NREM – „non rapid eye movement”, REM) u eksperimentalnim modelima funkcionalno različite holinergičke neuropatologije pronađu potencijalni EEG markeri početka, težine i progresije neurodegeneracije, sa mogućnošću dalje primene ovako senzitivnih metoda (definisanih EEG markera) u eventualnoj prevenciji i promeni terapijskog pristupa u pacijenata sa rizikom razvoja AB i PB. Mužjaci pacova Wistar soja su hronično implantirani za registrovanje spavanja. U toku operativne procedure za implantaciju elektroda za elektroencefalografiju (EEG) i elektromiografiju (EMG), korišćenjem tehnike stereotaksički navođene mikroinfuzije ibotenične kiseline (ekscitotoksina koji je generalni glutamatergički agonista), izvšene su selektivne unilateralne ili bilateralne NB ili PPT lezije. Eksperimentalni protokol registrovanja spavanja je počinjao dve nedelje nakon oporavka od operativne procedure implantacije hroničnih elektroda, sa ili bez unilateralnih ili bilateralnih NB ili PPT lezija. Registrovano je 6 sati spavanja, jednom nedeljno tokom četiri nedelje u eksperimentima NB lezija, odnosno jednom nedeljno tokom pet nedelja u eksperimentima PPT lezija, korišćenjem programa DataWave SciWorks Experimenter 8.0 (Datawave Technologies, Longmont, SAD). Nakon završetka eksperimentalnog protokola registrovanja spavanja, NB i PPT lezije su identifikovane NADPH – diaforaza histohemijskim bojenjem, a zatim i kvantifikovane u celokupnoj antero-posteriornoj dimenziji strukture korišćenjem ImageJ 1.46 programa (NIH, SAD). Analiza spavanja i EEG signala urađena je, kako u kontrolnih pacova tako i u pacova sa identifikovanim NB ili PPT lezijama, u MATLAB 6.5 programu, korišćenjem originalno razvijenog programskog paketa. Na usnimljene šestočasovne signale je najpre primenjena Furijeova transformacija, a zatim je svaka od 2160 Furijeovih epoha dužine 10 s diferencirana kao budnost, NREM ili REM faza spavanja. Pored toga, za potrebe detaljnije analize poremećaja REM faze spavanja izazvanih bilateralnom PPT lezijom, grupisanje REM epoha je na osnovu snage EMG-a dodatno razdvojeno na dva podgrupisanja REM1 (REM epohe sa većom snagom EMG-a, patološki REM) i REM2 (REM epohe sa manjom snagom EMG-a, fiziološki REM). Potom je korišćenjem EEG signala senzomotorne i motorne kore urađena topografska analiza arhitekture spavanja, strukture prelaznih stanja u toku spavanja, EEG mikrostrukture svih osnovnih faza u toku spavanja, kao i analiza kortiko-muskularih koherencija između EEG-a senzomotorne ili motorne kore velikog mozga i EMG-a mišića dorzalne vratne muskulature. Za izračunavanje grupne raspodele gustine verovatnoće relativnih amplituda svih budnost/NREM/REM/REM1/REM2 konvencionalnih EEG frekventnih opsega u toku 6 h spavanja korišćena je PDE („Probability Density Estimate”) funkcija u MATLAB 6.5 programu. U daljoj funkcionalnoj analizi poremećaja REM faze spavanja, za svaki od konvencionalnih EEG frekventnih opsega su korišćenjem „cohere” funkcije u MATLAB 6.5 izračunate REM/REM1/REM2 kortiko-muskularne koherencije između EEG-a senzomotorne ili motorne kore i EMG-a mišića dorzalne vratne muskulature. Za statističku obradu svih podataka korišćena je neparametarska jednofaktorska Kruskal-Wallis ANOVA sa post-hoc Mann-Whitney U kontrastnim testom. Bilateralna NB lezija je privremeno promenila arhitekturu spavanja 14 dana nakon lezije. Iako u slučaju fiziološke kontrole nije bilo razlika u trajanju budnosti, NREM i REM faze spavanja između senzomotorne i motorne kore, bilateralna NB lezija je u senzomotornoj kori smanjila trajanje budnosti i povećala trajanje NREM faze spavanja, dok je u motornoj kori smanjila trajanje budnosti i povećala trajanje REM faze spavanja. Istovremeno, u svim eksperimentalnim grupama, kako u fiziološkoj kontroli, tako i u unilateralnoj i bilateralnoj NB leziji, je EEG teta amplituda budnosti, NREM i REM faze spavanja bila niža u motornoj u odnosu na senzomotornu koru. Pored toga, bilateralna NB lezija je u senzomotornoj kori izazvala povećanje REM teta amplitude u trajanju od tri nedelje, dok su u motornoj kori i unilateralna i bilateralna NB lezija izazvale povećanje teta amplitude i u budnosti i u REM fazi spavanja. PPT lezija nije promenila arhitekturu spavanja, ali je značajno izmenila strukturu prelaznih stanja, kao i EEG mikrostrukturu svih osnovnih faza spavanja. Obe lezije, i unilateralna i bilateralna PPT lezija, su dugotrajno povećale broj budnost/REM i REM/budnost prelaza tokom celokunog perioda od 5 nedelja. Ova promena u strukturi prelaznih stanja je od 28. dana bila praćena smanjenjem broja NREM/REM i REM/NREM prelaza, ali samo nakon bilateralne PPT lezije, kao težeg oblika holinergičke neurodegeneracije. Unilateralna PPT lezija je izazvala povećanje EEG teta amplitude budnosti i EEG beta amplitude REM faze spavanja, a od treće nedelje nakon lezije i pad EEG delta amplitude budnosti. Bilateralna PPT lezija je izazvala povećanje EEG beta amplitude tokom svih faza spavanja, kao i povećanje EEG gama amplitude REM faze spavanja i smanjenje EEG delta amplitude budnosti i NREM faze spavanja. Poređenje efekata bilateralne NB i bilateralne PPT lezije (modeli težih, odnosno progresivnijih oblika holinergičke neurodegeneracije) na arhitekturu spavanja, strukturu prelaznih stanja i EEG mikrostrukturu svih osnovnih faza spavanja je pokazalo postojanje topografski specifičnih razlika u poremećajima nastalim kao posledica degeneracije funkcionalno različitih holinergičkih inervacija velikog mozga pacova. Za razliku od bilateralne NB lezije koja je privremeno izmenila trajanje budnosti i NREM faze spavanja u senzomotornoj kori, odnosno trajanje budnosti i REM faze spavanja u motornoj kori, bilateralna PPT lezija nije izmenila arhitekturu spavanja. Bilateralna PPT lezija je u obe kore dugotrajno (tokom 4 nedelje) povećala broj budnost/REM i REM/budnost prelaza i ovaj porast je bio praćen nekonzistentnim promenama u broju NREM/REM i REM/NREM prelaza u senzomotornoj kori, a u motornoj kori povećanjem broja ovih prelaza. Bilateralna NB lezija je u senzomotornoj kori smanjila broj NREM/REM i REM/NREM prelaza, dok je u motornoj kori povećala broj ovih prelaza tokom četiri nedelje. Promene u EEG mikrostrukturi nakon bilateralne PPT lezije bile su i u senzomotornoj i u motornoj kori izražene kao dugotrajno (tokom 4 nedelje) povećanje beta i gama amplitude budnosti, NREM i REM faze spavanja, odnosno kao smanjenje delta amplitude budnosti, ali samo u senzomotornoj kori. Nasuprot tome, bilateralna NB lezija je izazvala samo povećanje REM teta amplitude u senzomotornoj kori koje je trajalo tri nedelje. Pored promena u strukturi prelaznih stanja u toku spavanja i EEG mikrostrukturi svih osnovnih faza spavanja, bilateralna PPT lezija je dodatno uslovila izdvajanje dva funkcionalno različita REM stanja u okviru REM faze spavanja, REM1 (PPT lezijom prouzrokovan patološki REM, REM bez atonije, REM sigma koherencije) i REM2 (fiziološki REM, REM sa atonijom, REM teta koherencije), naročito u motornoj kori. Iako nije promenila arhitekturu spavanja senzomotorne kore, bilateralna PPT lezija je u motornoj kori produžila trajanje patološkog REM1 stanja, a povećala broj budnost/REM1/budnost i NREM/REM2/NREM prelaza u obe kore. U osnovi izmenjene REM EEG mikrostrukture su bile povećana beta amplituda senzomotorne kore za vreme celokupnog REM stanja i povećana teta amplituda motorne kore samo za vreme REM1 stanja. Bilateralna PPT lezija je izazvala generalizovan pad REM/REM1/REM2 beta kortiko-muskularne koherencije i dovela do izmene kontrole mišića dorzalne vratne muskulature dominantno iz senzomotorne kore. Ova doktorska disertacija je, koristeći po prvi put nov eksperimentalni model holinergičke PB neuropatologije (PPT lezija), stereotaksički navođenu mikroinfuziju kao nov metodološki pristup za selektivne lezije moždanih jedara, nov histohemijski metod identifikacije deficita holinergičkih neurona, nov pristup u kvantifikaciji neuronskog deficita, kao i nov način analize EEG signala za vreme različitih faza spavanja, po prvi put dokazala topografski specifične razlike u arhitekturi spavanja, strukturi prelaznih stanja i EEG mikrostrukturi osnovnih faza spavanja (budnost, NREM, REM), koje su nastale usled poremećaja funkcionalno različitih holinergičkih inervacija velikog mozga pacova (NB lezija u odnosu na PPT leziju). U ovom novom eksperimentalnom modelu holinergičke PB neuropatologije po prvi put su dokazani dugotrajni i veoma ozbiljni poremećaji u spavanju izraženi kao: povećanje broja budnost/REM i REM/budnost prelaza, povećana kortikalna aktivacija tokom svih osnovnih faza spavanja, izdvajanje dva različita REM stanja (pojava patološkog REM stanja pored fiziološkog REM stanja), kao i dominantna izmena kontrole mišića dorzalne vratne muskulature iz senzomotorne kore, iskazana kao pad REM/REM1/REM2 beta koherencije. 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.