Experimental Models to Study Immune Dysfunction in the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is a chronic, age-related, progressive multisystem disease associated with neuroinflammation and immune dysfunction. This review discusses the methodological approaches used to study the changes in central and peripheral immunity in PD, the advantages and limitations of the techniques, and their applicability to humans. Although a single animal model cannot replicate all pathological features of the human disease, neuroinflammation is present in most animal models of PD and plays a critical role in understanding the involvement of the immune system (IS) in the pathogenesis of PD. The IS and its interactions with different cell types in the central nervous system (CNS) play an important role in the pathogenesis of PD. Even though culture models do not fully reflect the complexity of disease progression, they are limited in their ability to mimic long-term effects and need validation through in vivo studies. They are an indispensable tool for understanding the interplay between the IS and the pathogenesis of this disease. Understanding the immune-mediated mechanisms may lead to potential therapeutic targets for the treatment of PD. We believe that the development of methodological guidelines for experiments with animal models and PD patients is crucial to ensure the validity and consistency of the results.

Different Alterations of Hippocampal and Reticulo-Thalamic GABAergic Parvalbumin-Expressing Interneurons Underlie Different States of Unconsciousness.

We traced the changes in GABAergic parvalbumin (PV)-expressing interneurons of the hippocampus and reticulo-thalamic nucleus (RT) as possible underlying mechanisms of the different local cortical and hippocampal electroencephalographic (EEG) microstructures during the non-rapid-eye movement (NREM) sleep compared with anesthesia-induced unconsciousness by two anesthetics with different main mechanisms of action (ketamine/diazepam versus propofol). After 3 h of recording their sleep, the rats were divided into two experimental groups: one half received ketamine/diazepam anesthesia and the other half received propofol anesthesia. We simultaneously recorded the EEG of the motor cortex and hippocampus during sleep and during 1 h of surgical anesthesia. We performed immunohistochemistry and analyzed the PV and postsynaptic density protein 95 (PSD-95) expression. PV suppression in the hippocampus and at RT underlies the global theta amplitude attenuation and hippocampal gamma augmentation that is a unique feature of ketamine-induced versus propofol-induced unconsciousness and NREM sleep. While PV suppression resulted in an increase in hippocampal PSD-95 expression, there was no imbalance between inhibition and excitation during ketamine/diazepam anesthesia compared with propofol anesthesia in RT. This increased excitation could be a consequence of a lower GABA interneuronal activity and an additional mechanism underlying the unique local EEG microstructure in the hippocampus during ketamine/diazepam anesthesia.

Hippocampal and Reticulo-Thalamic Parvalbumin Interneurons and Synaptic Re-Organization during Sleep Disorders in the Rat Models of Parkinson's Disease Neuropathology.

We investigated the alterations of hippocampal and reticulo-thalamic (RT) GABAergic parvalbumin (PV) interneurons and their synaptic re-organizations underlying the prodromal local sleep disorders in the distinct rat models of Parkinson's disease (PD). We demonstrated for the first time that REM sleep is a predisposing state for the high-voltage sleep spindles (HVS) induction in all experimental models of PD, particularly during hippocampal REM sleep in the hemiparkinsonian models. There were the opposite underlying alterations of the hippocampal and RT GABAergic PV+ interneurons along with the distinct MAP2 and PSD-95 expressions. Whereas the PD cholinopathy enhanced the number of PV+ interneurons and suppressed the MAP2/PSD-95 expression, the hemiparkinsonism with PD cholinopathy reduced the number of PV+ interneurons and enhanced the MAP2/PSD-95 expression in the hippocampus. Whereas the PD cholinopathy did not alter PV+ interneurons but partially enhanced MAP2 and suppressed PSD-95 expression remotely in the RT, the hemiparkinsonism with PD cholinopathy reduced the PV+ interneurons, enhanced MAP2, and did not change PSD-95 expression remotely in the RT. Our study demonstrates for the first time an important regulatory role of the hippocampal and RT GABAergic PV+ interneurons and the synaptic protein dynamic alterations in the distinct rat models of PD neuropathology.

Diversity of simultaneous sleep in the motor cortex and hippocampus in rats.

We investigated the homogeneity/heterogeneity of spontaneous sleep, simultaneously recorded in the motor cortex and the hippocampus of control rats, and particularly analysed simultaneous and non-simultaneous motor cortical and hippocampal non-rapid eye movement (NREM)/rapid eye movement (REM) sleep. We demonstrate that the sleep architectures of the motor cortex and hippocampus are different in control rats. There was an increase of NREM duration and a decrease of REM duration in the hippocampus versus the motor cortex. In terms of duration, NREM state is the most heterogeneous in the hippocampus, whereas the REM state is the most heterogeneous in the motor cortex. Whereas the hippocampal NREM duration was increased due to the prolongation of NREM episodes, the hippocampal REM duration decreased due to the decreased number of REM episodes. The heterogeneity of sleep in the motor cortex and hippocampus in control rats was particularly expressed through the inverse alteration of sigma amplitude during NREM sleep and beta/gamma amplitudes during REM sleep in the hippocampus, along with the delta, sigma, beta and gamma amplitudes only during non-simultaneous NREM/REM sleep in the hippocampus. We demonstrated the brain structure-related and NREM/REM state-related heterogeneity of the motor cortical and hippocampal local sleep in control rats. The distinctly altered local NREM/REM states, alongside their episode dynamics and electroencephalographic (EEG) microstructures, suggest the importance of both the local neuronal network substrate and the NREM/REM neurochemical substrate in the control mechanisms of sleep.

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

We investigated the prodromal alterations of local sleep, particularly the motor cortical and hippocampal sleep, along with spontaneous locomotor activity in the rat models of Parkinson's disease (PD). We performed our experiments in adult, male Wistar rats, chronically implanted for sleep recording and divided into four experimental groups: the control (implanted controls), the bilateral pedunculopontine tegmental nucleus (PPT) lesions (PD cholinopathy), the unilateral substantia nigra pars compacta (SNpc) lesions (hemiparkinsonism) and the unilateral SNpc/bilateral PPT lesions (hemiparkinsonism with PD cholinopathy). We followed their sleep, basal locomotor activity and spatial habituation for 14 days following the surgical procedures. Severe prodromal local sleep disturbances in the hemiparkinsonian rats were expressed as sleep fragmentation and distinct local NREM/REM EEG microstructure alterations in both the motor cortex and the hippocampus. Alongside the state-unrelated role of the dopaminergic control of theta oscillations and NREM/REM related sigma and beta oscillations, we demonstrated that the REM neurochemical regulatory substrate is particularly important in the dopaminergic control of beta oscillations. In addition, hippocampal prodromal sleep disorders in the hemiparkinsonian rats were expressed as NREM/REM fragmentation and the opposite impact of dopaminergic versus cholinergic control of the NREM delta and beta oscillation amplitudes in the hippocampus, likewise in the motor cortex versus the hippocampus. All these distinct prodromal local sleep disorders and the dopaminergic vs. cholinergic impact on NREM/REM EEG microstructure alterations are of fundamental importance for the further development and follow-up of PD-modifying therapies, and for the identification of patients who are at risk of developing PD.

Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells.

We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.

Novel application of the gray-level co-occurrence matrix analysis in the parvalbumin stained hippocampal gyrus dentatus in distinct rat models of Parkinson's disease.

To reveal the best choice of algorithm for parvalbumin-immunostained images of the hippocampal gyrus dentatus in two distinct rat models of Parkinson's disease (PD), particularly in terms of extracting the crucial information from the image, we tested whether the impact of experimentally induced dopaminergic (hemiparkinsonism) vs. cholinergic (PD cholinopathy) innervation impairment on the parvalbumin stained GABA interneurons could be detected using two separate algorithms, the fractal box-count and the gray-level co-occurrence matrix analysis (GLCM) algorithms. For the texture and fractal analysis of the hippocampal gyrus dentatus images, we used.tif images from three experimental groups of adult male Wistar rats: control rats, rats with Parkinson disease (PD) cholinergic neuropathology (with a PPT lesion), and hemiparkinsonian rats (with a SNpc lesion). For the suprapyramidal layer of the gyrus dentatus ASM and Entropy differentiated the images of the SNpc lesion versus the images of the control and the PPT lesion subjects, with significantly higher ASM and lower Entropy, indicating the homogenization of the images and their lower gray-level complexity. The infrapyramidal images of the SNpc group were differentiated versus the images from the control and PPT groups in terms of all the GLCM parameters: they showed lower mean Entropy and Contrast and higher ASM, Correlation and IDM. These results strongly suggest a rise in the uniformity, homogeneity and orderliness in the gray-levels of images from the SNpc group. Our results indicate that GLCM analysis is a more sensitive tool than fractal analysis for the detection of increased dendritic arborization in histological images.

Alterations of Sleep and Sleep Oscillations in the Hemiparkinsonian Rat.

Our previous studies in the rat model of Parkinson's disease (PD) cholinopathy demonstrated the sleep-related alterations in electroencephalographic (EEG) oscillations at the cortical and hippocampal levels, cortical drives, and sleep spindles (SSs) as the earliest functional biomarkers preceding hypokinesia. Our aim in this study was to follow the impact of a unilateral substantia nigra pars compacta (SNpc) lesion in rat on the cortical and hippocampal sleep architectures and their EEG microstructures, as well as the cortico-hippocampal synchronizations of EEG oscillations, and the SS and high voltage sleep spindle (HVS) dynamics during NREM and REM sleep. We performed unilateral SNpc lesions using two different concentrations/volumes of 6-hydroxydopamine (6-OHDA; 12 µg/1 µl or 12 µg/2 µl). Whereas the unilateral dopaminergic neuronal loss >50% throughout the overall SNpc rostro-caudal dimension prolonged the Wake state, with no change in the NREM or REM duration, there was a long-lasting theta amplitude augmentation across all sleep states in the motor cortex (MCx), but also in the CA1 hippocampus (Hipp) during both Wake and REM sleep. We demonstrate that SS are the hallmarks of NREM sleep, but that they also occur during REM sleep in the MCx and Hipp of the control rats. Whereas SS are always longer in REM vs. NREM sleep in both structures, they are consistently slower in the Hipp. The dopaminergic neuronal loss increased the density of SS in both structures and shortened them in the MCx during NREM sleep, without changing the intrinsic frequency. Conversely, HVS are the hallmarks of REM sleep in the control rats, slower in the Hipp vs. MCx, and the dopaminergic neuronal loss increased their density in the MCx, but shortened them more consistently in the Hipp during REM sleep. In addition, there was an altered synchronization of the EEG oscillations between the MCx and Hipp in different sleep states, particularly the theta and sigma coherences during REM sleep. We provide novel evidence for the importance of the SNpc dopaminergic innervation in sleep regulation, theta rhythm generation, and SS/HVS dynamics control. We suggest the importance of the underlying REM sleep regulatory substrate to HVS generation and duration and to the cortico-hippocampal synchronizations of EEG oscillations in hemiparkinsonian rats.

Sleep spindle dynamics during NREM and REM sleep following distinct general anaesthesia in control rats and in a rat model of Parkinson's disease cholinopathy.

On the basis of our previous studies and the important role of the thalamo-cortical network in states of unconsciousness, such as anaesthesia and sleep, and in sleep spindles generation, we investigated sleep spindles (SS) and high-voltage sleep spindle (HVS) dynamics during non-rapid eye movement (NREM) and rapid eye movement (REM) sleep following different types of general anaesthesia in both physiological controls and in a rat model of Parkinson's disease (PD) cholinopathy, to follow the impact of anaesthesia on post-anaesthesia sleep at the thalamo-cortical level through an altered sleep spindle dynamics. We recorded 6 hr of spontaneous sleep in all rats, both before and 48 hr after ketamine/diazepam or pentobarbital anaesthesia, and we used 1 hr of NREM or REM sleep from each to validate visually the automatically detected SS or HVS for their extraction and analysis. In the controls, SS occurred mainly during NREM, whereas HVS occurred only during REM sleep. Ketamine/diazepam anaesthesia promoted HVS, prolonged SS during NREM, induced HVS of increased frequency during REM, and increased SS/HVS densities during REM versus NREM sleep. Pentobarbital anaesthesia decreased the frequency of SS during NREM and the HVS density during REM sleep. Although the pedunculopontine tegmental nucleus lesion prolonged SS only during NREM sleep, in these rats, ketamine/diazepam anaesthesia suppressed HVS during both sleep states, whereas pentobarbital anaesthesia promoted HVS during REM sleep. The different impacts of two anaesthetic regimens on the thalamo-cortical regulatory network are expressed through their distinct sleep spindle generation and dynamics that are dependent on the NREM and REM state regulatory neuronal substrate.

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

In order to find out the possible earliest biomarkers of Parkinson's disease (PD) cholinopathy, we followed the impact of bilateral pedunculopontine tegmental nucleus (PPT) lesion in rat on: the cortical and hippocampal sleep/wake states architectures, all sleep states related EEG microstructures, sleep spindles, the basal and stimulated locomotor activity. Sleep and basal locomotor activity in adult Wistar rats were followed during their inactive circadian phase, and throughout the same aging period. The bilateral PPT lesions were done by 0.1M ibotenic acid (IBO) during the surgical procedure for implantation of the electroencephalographic (EEG) and electromyographic (EMG) electrodes for chronic sleep recording. The cholinergic neuronal loss was identified by NADPH - diaphorase histochemistry. After all sleep and behavioral recording sessions, the locomotor activity was stimulated by d-amphetamine (d-AMPH) and the neuronal activity of striatum was followed by c-Fos immunolabeling. Impaired cholinergic innervation from the PPT was expressed earlier as sleep disorder then as movement disorder, and it was the earliest and long-lasting at hippocampal and thalamo-cortical level, and followed by a delayed "hypokinesia". This severe impact of a tonically impaired PPT cholinergic innervation was evidenced as the cholinergic interneuronal loss of the caudate putamen and as a suppressed c-Fos expression after stimulation by d-AMPH. In order how they occurred, the hippocampal non rapid eye movement (NREM) sleep disorder, altered high voltage sleep spindle (HVS) dynamics during rapid eye movement (REM) sleep in the hippocampus and motor cortex, and "hypokinesia" may serve as the biomarkers of PD cholinopathy onset and progression.

Slower EEG alpha generation, synchronization and "flow"-possible biomarkers of cognitive impairment and neuropathology of minor stroke.

BACKGROUND: We investigated EEG rhythms, particularly alpha activity, and their relationship to post-stroke neuropathology and cognitive functions in the subacute and chronic stages of minor strokes. METHODS: We included 10 patients with right middle cerebral artery (MCA) ischemic strokes and 11 healthy controls. All the assessments of stroke patients were done both in the subacute and chronic stages. Neurological impairment was measured using the National Institute of Health Stroke Scale (NIHSS), whereas cognitive functions were assessed using the Montreal Cognitive Assessment (MoCA) and MoCA memory index (MoCA-MIS). The EEG was recorded using a 19 channel EEG system with standard EEG electrode placement. In particular, we analyzed the EEGs derived from the four lateral frontal (F3, F7, F4, F8), and corresponding lateral posterior (P3, P4, T5, T6) electrodes. Quantitative EEG analysis included: the group FFT spectra, the weighted average of alpha frequency (αAVG), the group probability density distributions of all conventional EEG frequency band relative amplitudes (EEG microstructure), the inter- and intra-hemispheric coherences, and the topographic distribution of alpha carrier frequency phase potentials (PPs). Statistical analysis was done using a Kruskal-Wallis ANOVA with a post-hoc Mann-Whitney U two-tailed test, and Spearman's correlation. RESULTS: We demonstrated transient cognitive impairment alongside a slower alpha frequency (αAVG) in the subacute right MCA stroke patients vs. the controls. This slower alpha frequency showed no amplitude change, but was highly synchronized intra-hemispherically, overlying the ipsi-lesional hemisphere, and inter-hemispherically, overlying the frontal cortex. In addition, the disturbances in EEG alpha activity in subacute stroke patients were expressed as a decrease in alpha PPs over the frontal cortex and an altered "alpha flow", indicating the sustained augmentation of inter-hemispheric interactions. Although the stroke induced slower alpha was a transient phenomenon, the increased alpha intra-hemispheric synchronization, overlying the ipsi-lesional hemisphere, the increased alpha F3-F4 inter-hemispheric synchronization, the delayed alpha waves, and the newly established inter-hemispheric "alpha flow" within the frontal cortex, remained as a permanent consequence of the minor stroke. This newly established frontal inter-hemispheric "alpha flow" represented a permanent consequence of the "hidden" stroke neuropathology, despite the fact that cognitive impairment has been returned to the control values. All the detected permanent changes at the EEG level with no cognitive impairment after a minor stroke could be a way for the brain to compensate for the lesion and restore the lost function. DISCUSSION: Our study indicates slower EEG alpha generation, synchronization and "flow" as potential biomarkers of cognitive impairment onset and/or compensatory post-stroke re-organizational processes.

REM sleep disorder following general anesthesia in rats.

Postoperative sleep disorders, particularly the REM sleep disorder, may have a significant deleterious impact on postoperative outcomes and may contribute to the genesis of certain delayed postoperative complications. We have followed the effect of distinct anesthesia regimens (ketamine/diazepam vs. pentobarbital) over 6days following the induction of a stable anesthetized state in adult male Wistar rats, chronically instrumented for sleep recording. In order to compare the effect of both anesthetics in the physiological controls vs. the rats with impaired pedunculopontine tegmental nucleus (PPT) cholinergic innervation, during the operative procedure for the implantation of EEG and EMG electrodes, the bilateral PPT lesion was conducted using ibotenic acid (IBO). We have followed in particular post-anesthesia REM sleep. Our results show the distinct EEG microstructure of the motor cortex during the different stable anesthetized states, and their distinct impact on post-anesthesia REM sleep. In contrast to pentobarbital anesthesia, the ketamine/diazepam anesthesia potentiated the long-lasting post-anesthesia REM statewith higher muscle tone (REM1) vs. REM state with atonia (REM2). Whereas both anesthesias prolonged the post-anesthesia REM sleep duration, the long-term prolongation of the REM1 state was demonstrated only after the ketamine/diazepam anesthesia, first due to the increased number of REM1 episodes, and then due to the prolonged REM1 episodes duration. On the other hand, whereas both anesthetic regimens abolished the prolonged post-anesthesia REM/REM1 sleep and the EEG microstructure disorder during REM sleep, only the pentobarbital abolished the increased NREM/REM/NREM transitions, caused by the PPT lesion. In addition, in the PPT lesioned rats, the ketamine/diazepam anesthesia decreased the Wake/NREM/Wake transitions while the pentobarbital anesthesia decreased the Wake/REM/Wake transitions. Our present study suggests pentobarbital anesthesia as being highly beneficial for post-anesthesia REM sleep in the physiological condition as well as during PPT cholinergic neuropathology.

Age-related disorders of sleep and motor control in the rat models of functionally distinct cholinergic neuropathology.

We studied the impact of aging during sleep in the rat models of Alzheimer's (AD) and Parkinson's (PD) disease cholinergic neuropathology to determine the possible different and earlier onset of age-related sleep disorder during the neurodegenerative diseases vs. healthy aging. We used the bilateral nucleus basalis (NB) and pedunculopontine tegmental nucleus (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. 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 rapid eye movement sleep (REM). EEG delta amplitude attenuation within the sensorimotor cortex (SMCx) during REM was the earliest sign of aging in the NB lesion. EEG sigma amplitude augmentation within the motor cortex (MCx) during REM was the earliest sign of aging in the PPT lesion. 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. Our study provided evidence of distinct REM sleep disorders and sleep state related cortical drives as the signs of aging onset during functionally distinct cholinergic neuropathologies (NB lesion vs. PPT lesion).

Aging induced cortical drive alterations during sleep in rats.

We followed the impact of healthy aging on cortical drive during sleep in rats by using the corticomuscular coherence (CMC). We employed the chronic electrodes implantation for sleep recording in adult, male Wistar rats, and followed the aging impact during sleep from 3 to 5.5 months age. We have analyzed the sleep/wake states architecture, and the sleep/wake state related EEG microstructure and CMCs. We evidenced the topographically distinct impact of aging on sleep/wake states architecture within the sensorimotor (SMCx) vs. motor cortex (MCx) from 4.5 to 5.5 months age. Healthy aging consistently altered only the SMCx sleep/wake states architecture, and increased the delta and beta CMCs through both cortical drives during Wake, but only through the MCx drive during REM. According to the delta and beta CMCs values, aging impact through the SMCx drive was opposite, but it was convergent through the MCx drive during Wake vs. REM, and there was a dual and inverse mode for the motor control during REM.

Glial response in the rat models of functionally distinct cholinergic neuronal denervations.

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.

REM sleep diversity following the pedunculopontine tegmental nucleus lesion in rat.

The aim of this study was to demonstrate that two REM clusters, which emerge following bilateral pedunculopontine tegmental nucleus (PPT) lesions in rats, are two functionally distinct REM states. We performed the experiments in Wistar rats, chronically instrumented for sleep recording. Bilateral PPT lesions were produced by the microinfusion of 100 nl of 0.1M ibotenic acid (IBO). Following a recovery period of 2 weeks, we recorded their sleep for 6h. Bilateral PPT lesions were identified by NADPH - diaphorase histochemistry. We applied Fourier analysis to the signals acquired throughout the 6h recordings, and each 10s epoch was differentiated as a Wake, NREM or REM state. We analyzed the topography of the sleep/wake states architecture and their transition structure, their all state-related EEG microstructures, and the sensorimotor (SMCx) and motor (MCx) cortex REM related cortico-muscular coherences (CMCs). Bilateral PPT lesion in rats increased the likelihood of the emergence of two distinct REM sleep states, specifically expressed within the MCx: REM1 and REM2. Bilateral PPT lesion did not change the sleep/wake states architecture of the SMCx, but pathologically increased the duration of REM1 within the MCx, alongside increasing Wake/REM1/Wake and NREM/REM2/NREM transitions within both cortices. In addition, the augmented total REM SMCx EEG beta amplitude and REM1 MCx EEG theta amplitude was the underlying EEG microstructure pathology. PPT lesion induced REM1 and REM2 are differential states with regard to total EMG power, topographically distinct EEG microstructures, and locomotor drives to nuchal musculature.

Topography of the sleep/wake states related EEG microstructure and transitions structure differentiates the functionally distinct cholinergic innervation disorders in rat.

In order to identify the differences for the onset and progression of functionally distinct cholinergic innervation disorders, we investigated the effect of bilateral nucleus basalis (NB) and pedunculopontine tegmental nucleus (PPT) lesions on sleep/wake states and electroencephalographic (EEG) microstructure in rats, chronically implanted for sleep recording. Bilateral NB lesion transiently altered Wake/NREM duration within the sensorimotor cortex, and Wake/REM duration within the motor cortex, while there was no change in the sleep/wake states distributions following the bilateral PPT lesion. Bilateral PPT lesion sustainably increased the Wake/REM and REM/Wake transitions followed by inconsistent dysregulation of the NREM/REM and REM/NREM transitions in sensorimotor cortex, but oppositely by their increment throughout four weeks in motor cortex. Bilateral NB lesion sustainably decreased the NREM/REM and REM/NREM transitions during four weeks in the sensorimotor cortex, but oppositely increased them in the motor cortex. We have shown that the sustained beta and gamma augmentation within the sensorimotor and motor cortex, and across all sleep/wake states, simultaneously with Wake delta amplitude attenuation only within the sensorimotor cortex, were the underlying EEG microstructure for the sleep/wake states transitions structure disturbance following bilateral PPT lesion. In contrast, the bilateral NB lesion only augmented REM theta in sensorimotor cortex during three weeks. We have shown that the NB and PPT lesions induced differing, structure-related EEG microstructure and transition structure disturbances particularly expressed in motor cortex during NREM and REM sleep. We evidenced for the first time the different topographical expression of the functionally distinct cholinergic neuronal innervation impairment in rat.

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

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 0.1M ibotenic acid (IBO) into PPT. We applied Fourier analysis to signals acquired throughout 6h of recordings, and each 10s 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.

[Selective stimulations and lesions of the rat brain nuclei as the models for research of the human sleep pathology mechanisms].

Many complex behavioral phenomena such as sleep can not be explained without multidisciplinary experimental approach, and complementay approaches in the animal models "in vivo" and human studies. Electrophysiological, pharmacological, anatomical and immunohistochemical techniques, and particularly stereotaxically guided local nanovolume microinjection technique, enable us to selectively stimulate and lesion the brain nuclei or their specific neuronal subpopulation, and to reslove the mechanisms of certain brain structure regulatory role, and its afferent-efferent connectivity within the brain. Local stereotaxically guided nanovolume microinjection technique enable us to investigate in animals the brain nulcei functional topography with a resolution of < or = 10 microM, and at a level of 300 microM of effective radius within the brain tissue "in vivo". The advantage of local glutamate or DL- homocysteic acid microinjection stimulation or local excitotoxic (glutamate, ibotenic acid, IgG saporin) microinjection lesion over electrical stimulation/lesion of the same neuronal population are that they reduces the likelihood of activation/lesion of fibers of passage. Much of our knowledge of the sleep neuronal substrates is based on animal studies primarly in cat and rat. Selective pharmacological stimulation of the pedunculopontine tegmentum (PPT) in freely moving rat, using glutamate microinjection, proved that excitation of its cholinergic part is necessary for induction of wakefulness or REM (Datta S, 2001). Local nanovolume glutamate microinjection into PPT of anesthetized rats (Saponjic et al, 2003a) additionally evidenced P-wave and respiratory regulating neuronal subpopulation within the cholinergic compartment of PPT (apneogenic neuronal zone). Local microinjection of serotonin and noradrenaline into cholinergic PPT apneogenic zone evidenced their opposed impact through PPT on breathing, in contrast to their convergent regulatory role in behavioral state control (Saponjic et al., 2005a). Also, selective pharmacological stimulation by microinjection of DL-homocysteic acid defined four neuronal micro-circuitry approximately 500 microm in lenght of breathing-related neurons within the ventral respiratory group of medulla oblongata, which when stimulated produce different effects on respiratory rate, rhythm and amplitude, and on blood pressure. This study was the first high resolution study in order to understand anatomical and functional neuronal system organization (Monnier et al., 2003). Recently, local glutamate microinjection stimulation technique enabled detailed functional topography of respiratory, cardiovascular and pontine-wave responses within the PPT (Topchiy et al., 2010). Discovery of "flip-flop" switch for REM sleep control is based on the experiments in rats using local stereotaxically guided microinjection of excitotoxins (ibotenic acid, IgG saporin), and the anterograde and retrograde tracers for selective lesion, and identifying "REM-off" and "REM- on" regions and their afferent-efferent connections, and for identifying pathways for REM atonia and REM EEG activation (Lu et al., 2006). Recently, selective lesion of SLD part of "REM-on" region in rat established an animal model of RBD, as well as a selective ibotenic acid lesion of PC part of "REM-on" region abolished theta during REM (Lu et al., 200; Anaclet et al., 2010). Selective ablation targeted to pre-Bötzinger complex neurons of ventrolateral respiratory group of medulla in rat induced REM related respiratory disorder up to 10 days, when this respiratory disorder became spreaded to all sleep phases, and even during wakefulness, due to long-lasting intermitent hypoxia, and an increase of the threshold for hypoxia/hypercapnea induced arousal response (McKay et al., 2005). Human development, maturation, healthy aging and many neurological diseases are associated with profound changes in sleep/wake states distribution and with variety of the sleep-related behavioral disorders. Sleep and sleep-related respiratory disorders (insomnia, hypersomnia, parasomnias, excessive nocturnal motor activity, circadian sleep-wake rhythm disturbances, respiratory dysrhythmias, RBD) are very frequently unnoticed in patients with neurodegenerative diseases (Boeve et al., 2007; Whitwell et al., 2007). Alzheimer's and Parkinson's disease (AD, PD) are the most common neurodegenerative diseases, with prevalence of 0.5-1%; increasing to 1-3% for Parkinson, and up to 50% for Alzheimer's disease in ages over 69 (Nussbaum and Christopher, 2003). In spite of a long knowledge of their clinical description and brain pathology (lesions of the NB cholinergic neurons in basal forebrain, dopaminergic neurons in substantia nigra, etc.), they remain incurable with only limited success in temporal amelioration of their symptoms. Clinical symptoms first appear at 65-69 years on average, but there are indications that subclinical features may start many years earlier. Patients with REM-sleep behavior disorder (RBD) face close to a 20% 5-year risk of developing PD or dementia, and that risk rises to more than 40% after 10 years, and exceeds 50% after 12 years. Human studies evidenced that sleep/wake cycle disturbance, as no cognitive symptom of dementia, precedes on average 3 years before the clinical diagnosis of the AD (Simic et al., 2009), and that RBD, precedes as symptom the onset of motor and cognitive disturbances by years or decades. AD and PD involve the selective loss of specific neuronal populations within the brain. RBD in those patients reflects an underlying synucleinopathy, with presence of the alpha-synuclein protein pathology within the REM sleep-related regulatory structures of the dorsal midbrain and pons at the onset of disease, with ascending pattern of neurodegeneration progression from brainstem to basal areas of the brain (Whitwell et al., 2007; Simic et al., 2009: Raggi and Ferri, 2010). On the base of hypothesis that basal forebrain cholinergic system plays an important role in the etiology of the most common neurodegenerative diseases of elderly (AD, PD), the lesion of the nucleus basalis in rat presents the most utilized "in vivo" animal model to study the disorders of cortical cholinergic innervation, and its impact on higher central nervous system functions. Our knowledge of the neural substrates for sleep/wake states and sleep-related behavior disorders regulation in health and the diseases, over more than 50 years of sleep research, is based on animal models, pharmacotherapy, central nervous system lesions, and the neuropathological studies in humans. Today we have many complementary animal models of human sleep pathology, and further work in fundamental multidisciplinary and clinical research between sleep and neurodegenerative disease investigators is promising to enable us understand normal and abnormal sleep, and may provide new insights into preventive or disease-altering approaches for therapy. Obviously counseling and prevention of AD or PD would be highly enriched by the development of a practical, sensitive and reliable methodology of detecting those patients with RBD, or other sleep disorders, who are at risk for developing AD or PD.

Surrogate data modeling the relationship between high frequency amplitudes and Higuchi fractal dimension of EEG signals in anesthetized rats.

We used spectral analysis and Higuchi fractal dimension (FD) to correlate the EEG spectral characteristics of the sensorimotor cortex, hippocampus, and pons with their corresponding EEG signal complexities in anesthetized rats. We have explored the quantitative relationship between the mean FDs and EEG wide range high frequency (8-50 Hz) activity during ketamine/xylazine versus nembutal anesthesia at surgical plane. Using FD we detected distinct inter-structure complexity pattern and uncovered for the first time that the polygraphically and behaviorally defined anesthetized state at surgical plane as equal during experiment in two anesthetic regimens, is not the same with respect to the degree of neuronal activity (degree of generalized neuronal inhibition achieved) at different brain levels. Using the correlation of certain brain structure EEG spectral characteristics with their corresponding FDs, and the surrogate data modeling, we determined what particular frequency band contributes to EEG complexities in ketamine/xylazine versus nembutal anesthesia. In this study we have shown that the quantitative relationship between higher frequency EEG amplitude and EEG complexity is the best-modeled by surrogate data as a 3rd order polynomial. On the base of our EEG amplitude/EEG complexity relationship model, and the evidenced spectral differences in ketamine versus nembutal anesthesia we have proved that higher amplitudes of sigma, beta, and gamma frequency in ketamine anesthesia yields to higher FDs.