Η παρεγκεφαλίδα των πτηνών, μια μορφολογικά μοναδική δομή του εγκεφάλου, αποτελείται από 10 λοβούς (μερικοί από τους οποίους φέρουν δευτερογενείς υποδιαιρέσεις), οι οποίοι σχηματίζονται κατά τα ενδιάμεσα εμβρυϊκά αναπτυξιακά στάδια. Ο πολλαπλασιασμός και η μετανάστευση των κυττάρων της εξωτερικής κοκκώδους στιβάδας είναι το κύριο γεγονός κατά την διαδικασία ανάπτυξης των παρεγκεφαλιδικών λοβών. Σκοπός της παρούσας μελέτης είναι η διερεύνηση του μηχανισμού σχηματισμού των λοβών στην αναπτυσσόμενη παρεγκεφαλίδα. Για τον προσδιορισμό του ρυθμού του πολλαπλασιασμού των πρόγονων κοκκωδών κύτταρων, εφαρμόσθηκε η ανοσοϊστοχημική μέθοδος της 5-βρωμο-2-δεοξυουριδίνης (BrdU). Η BrdU, που ενσωματώνεται στο DNA κατά την S-φάσης του κυτταρικού κύκλου, ενέθηκε in ovo (100μg/gr βάρους αυγού σε φυσιολογικό ορό) στις εμβρυικές μέρες E10, E13, E15 και E17, και μετά από διαφορετικούς χρόνους επιβίωσης, τα έμβρυα θυσιάστηκαν (1, 2 και 3 ώρες μετά την ένεση), μονιμοποιήθηκαν με ενδοκάρδια έγχυση, και αφαιρέθηκε ο εγκέφαλος, έγινε κρυο-προστασία, μετά –μονιμοποίηση και φύλαξη του εγκεφάλου στους -80°C. Την E10 ημέρα, παρατηρήθηκε πλήρως ανεπτυγμένη πρωτογενής σχισμή, ενώ από την E13 ημέρα, όλοι οι λοβοί της παρεγκεφαλίδας έχουν σχηματιστεί και μέχρι την E17 μέρα όλες οι υποδιαιρέσεις τους είναι εμφανείς. Με πειράματα απλής και διπλής ανοσοϊστοχημείας, σε διατομές 20μm, προσδιορίσθηκε ο αριθμός των μιτωτικά ενεργών κυττάρων στην S-φάση (BrdU σήμανση), ο αριθμός των αποπτωτικών κυττάρων (σήμανση κασπάσης 3). Έγινε χαρτογράφηση των προτύπων πολλαπλασιασμού και απόπτωσης (με τη χρήση camera lucida) και υπολογίστηκε σε συγκεκριμένους λοβούς ο αριθμός των προγονικών κοκκωδών κυττάρων σε διαφορετικούς χρόνους επιβίωσης. Μόνο μικρός αριθμός αποπτωτικών κυττάρων παρατηρήθηκε στη μελλοντική εσωτερική κοκκώδη στιβάδα, τον τελικό προορισμό των κυττάρων της εξωτερικής κοκκώδους στιβάδας, και συνεπώς η απόπτωση δεν παίζει καθοριστικό ρόλο στον σχηματισμό των λοβών. / The cerebellum (Cb), a highly conserved structure of the vertebrate brain, is a morphologically unique laminated structure made up of a set of folia separated by fissures. The avian cerebellum consists of ten folia, (some of them are subdivided in secondary folia) which are mainly developed during mid and late embryonic stages. External granule cell proliferation is the major event that coincides with the fissure formation. The present study aimed to address the question of the mechanisms that underlie the cerebellar fissure development. For this, the proliferation pattern in the external granule cell layer of the developing embryonic chick cerebellum was mapped, by means of 5-bromo-2’-deoxyurinine (BrdU) immunohistochemistry. BrdU, an S-phase marker was injected in ovo, (100μg/gr egg weight in physiological saline) at the embryonic days E10, E13, E15 and E17 and embryos were allowed to survive for different survival times. The embryos were fixed, cryo-protected and adjacent cerebellar sections were processed for BrdU labelling to determine proliferating cells, for caspase 3 labelling to determine the pattern of apoptotic cells and for double immunofluorescence to characterize the phenotype of BrdU+ cells. Cell proliferation and apoptotic patterns were determined throughout the individual lobules, by image analysis and camera lucida mapping. At day E10, mid-sagittal sections of cerebellum showed that primitive fissure is formed, with well infolded the fissure prima. From day E13, all primary folia were present and by E17 day all subdivisions were developed. While mitotic activity characterized all external granule cell layer folia, low numbers of cells undergoing apoptosis were found mainly in the future granule cell layer, the site of the final destination of external granule cells. Estimation of the density of BrdU+ granule cell precursors revealed that differences determined in the proliferation rate between the fissure’s wall and floor, but not in the apoptotic cell death, may account for the formation of fissures and folia.

Earlier human and animal studies have indicated abnormal striatal GABAergic interneurons relating to autism spectrum disorder’s (ASD) core features such as stereotypic repetitive behaviors, impaired language and motor skills, and social interactions. Purkinje cells (PCs) in the cerebellum are of great interest in ASD; earlier research has reported a loss of PCs, irregularities within deep cerebellar nuclei, a lower level of GAD67 (glutamic acid decarboxylase) mRNA expressed on PCs, and reduced parvalbumin (PV)-positive interneurons in cortex and hippocampus. In this study, in-situ hybridization was used to quantify the levels of PV mRNA in PCs in post-mortem human autism and control cerebellum sections. Two-tailed t-test analysis of the data showed a significant decrease (p<0.05) in PV mRNA level on PCs in autism compared to control sections. In addition, when comparing two groups (seizure and no seizure) in autism sections, no statistical significance was observed. Post-mortem interval (PMI) and age was compared between the PV mRNA levels in autism and control. Only weak negative correlation was found among age and PV mRNA levels in both groups. This report of decreased PV mRNA level in autism cases further supported previous research findings related to PCs and also confirmed interference with the inhibitory function of PCs to deep cerebellar nuclei and the cortex resulting in behavioral and motor impairments in ASD.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-25Bitstream added on 2014-06-13T18:50:09Z : No. of bitstreams: 1 oliveira_sa_me_botib.pdf: 362575 bytes, checksum: bae4117a32310994991fb14daf0fa593 (MD5) / Universidade Estadual Paulista (UNESP) / Experiências traumáticas na infância estão associadas ao aumento do risco de abuso de álcool e de outras drogas na adolescência e na vida adulta. Crianças e adolescentes maltratados manifestam distúrbios do sistema biológico de resposta ao estresse. A exposição crônica a fatores estressantes aumenta a atividade do eixo hipotálamo-hipófise-adrenal (HHA). Sendo o cerebelo local de ação do etanol, onde o consumo de forma abusiva altera o equilibro e a coordenação motora e que vários aspectos do alcoolismo podem ser frutos do estresse vivido precocemente, este trabalho buscou investigar e avaliar se a separação materna neonatal, aplicada em filhotes machos de ratos UChA e UChB, potencializa os efeitos tóxicos da ingestão crônica de álcool sobre a morfometria dos estratos cerebelares dos animais estudados. Para isso, utilizou-se modelo de alcoolismo experimental (ratos UChA e UChB) e de indução de hiper-reatividade do eixo HHA (Separação Materna). Foram realizadas análises microscópicas e morfométricas. Para morfometria as variáveis estudadas foram: massa corpórea(g), consumo de álcool(g/Kg/dia), altura do córtex cerebelar (μm), altura do estrato molecular(μm), granuloso (μm), purkinjense (μm) e áreas dos citoplasmas das células de Purkinje (μm2). O estresse neonatal não alterou o padrão de consumo de etanol das linhagens UChA e UChB, houve alterações nos níveis plasmáticos de corticoesterona. O córtex cerebelar dos animais UCh mostrou-se menor que no Wistar, sendo a SM fator protetor para atrofia desta região nos grupos SMUChA e SMCO. Os estratos granular e purkinjense mantiveram o padrão de alteração do córtex cerebelar, com a SM atenuando os efeitos do consumo crônico de etanol. Já a camada molecular encontrou-se maior nos animais UChB, onde após indução de hiper-reatividade do eixo HHA ocorreu a atrofia deste grupo. Portanto... / Traumatic experiences in the childhood are associated to an increase in the risk of alcohol and other drugs abuse in the adolescence and in the adulthood. Abused children and adolescents manifest diseases in their biological system in response to that stress. The chronic exposition to stressing factors increases the activity of the hypothalamus-hypophysis-adrenal axis (HHA). The ethanol acts in the cerebellum where the abusive consumption alters the balance and the motor coordination; that is, many aspects of the alcoholism might be consequences of the stress experienced in early years. This research investigated and evaluated if the neonatal maternal separation, applied to UChA and UChB male rats, potentializes the toxic effects of the alcohol chronic ingestion on the cell morphometry of cerebellar extracts in the studied animals. For that, both, the experimental alcoholism model (UChA and UChB rats) and the hipper-reactivity induction of the HHA axis (Maternal Separation) were used. Microscopic and morphometrics analysis were also accomplished. For morphometry, the variables analyzed were: corporal mass (g), alcohol consumption (g/Kg/day), cerebellar cortex height (um), molecular extract height (um), granular (um), Purkinje fibers (um), and cytoplasm areas of the Purkinje cells (um2). The neonatal stress did not alter the ethanol consumption pattern of the UChA and UChB lineages; however, there were alterations in the corticosterone plasmatic levels. The cerebellar cortex of the UCh animals have shown to be smaller than the Wistar ones, and the SM has shown to be a protecting factor for this region atrophy in the SMUChA and SMCO groups. The granular and Purkinje fiber extracts have kept the cerebellar cortex alteration patterns, having the SM lessen the effects of the ethanol chronic consumption. The molecular layer was bigger in the UChB animals, where after the hipperreactivity induction of the HHA axis there was an atrophy of this group.

Chromatin remodeling enzymes are instrumental for neural development as evidenced by their identification as disease genes underlying human disorders characterized by intellectual-disability. In this regard, the murine Snf2h and Snf2l genes show differential expression patterns during embryonic development, with a unique pattern in the brain where Snf2h is predominant in neural progenitors, while Snf2l expression peaks at the onset of differentiation. These observations led me to investigate the role of Snf2h and Snf2l in brain development by using conditionally targeted Snf2h and Snf2l mice. I selectively ablated Snf2h expression in cortical progenitors, cerebellar progenitors, or postmitotic Purkinje neurons of the cerebellum, while Snf2l was deleted in the germline. I found that Snf2h plays diverse roles in neural progenitor expansion and postmitotic gene expression control, while Snf2l is involved in the precise timing of neural differentiation onset. Gene expression studies revealed that Snf2h and Snf2l co-modulate the FoxG1 and En1 transcription factors during cortical and cerebellar neurogenesis, respectively, to precisely control the transition from a progenitor to a differentiated neuron. Moreover, Snf2h is essential for the postmitotic neural activation of the clustered protocadherin genes, and does so by functionally interacting with the matrix-attachment region protein Satb2. My neurobehavioral studies also provided insight into how Snf2h loss in cerebellar progenitors results in cerebellar ataxia, while Snf2h loss in cortical progenitors, or in postmitotic Purkinje neurons of the cerebellum, resulted in learning and memory deficits, and hyperactive-like behavior. Molecularly, Snf2h plays an important role in linker histone H1e dynamics and higher order chromatin packaging, as evidenced by loss of chromatin ultrastructure upon Snf2h deletion in progenitor and postmitotic neurons. I further demonstrated that Snf2h loss in a neuronal cell culture model results in reduced H1e deposition, and that overexpression of human SNF2H or SNF2L upon Snf2h knockdown rescues this biochemical dysfunction. My experiments suggest that Snf2h and Snf2l are regulatory nucleosome remodeling engines that co-modulate the gene expression programs necessary for proper brain development, maturation and function.

Cannabinoid receptors (CBR) are the most abundant G-protein coupled receptors in the mammalian brain with the highest densities within the cerebellum (Herkenham et al., 1990). Cannabinoid manipulations have been reported to cause deficits in cerebellar-dependent learning (Kishimoto and Kano, 2006; Skosnik et al., 2007; Steinmetz and Freeman, 2010; 2013). Cannabinoid receptors-1 (CB1R) have been hypothesized to be important in the establishment of long-term depression within the cerebellar cortex (Levenes et al., 1998; Safo and Regehr, 2005). However, all investigations in vivo have used global manipulations and have not attempted to localize or characterize these receptors during cerebellar-dependent learning. Chapter 2 systematically examined localization within the cerebellar cortex of cannabinoid effects on eyeblink conditioning, a type of cerebellum-dependent learning. Local infusions into a specific portion of the cerebellar cortex, the eyeblink conditioning microzone, resulted in deficits in learning similar to systemic injections. Additionally, infusions of cannabinoids into the eyeblink conditioning microzone, and no other parts of the cerebellar cortex or deep nuclei, were responsible for the deficits. Finally, tetrode recordings were made in Purkinje cells while receiving either CBR agonist or vehicle injections prior to training. Fewer Purkinje cells exhibited learning-related decreases in activity when the rat was administered a CBR agonist as compared to when it was injected with the vehicle. The CBR administered Purkinje cells also showed earlier onsets and smaller amplitudes in their learning-related activity. Purkinje cells that show a learning-related increase in activity were not affected by cannabinoid administration. The impairment in Purkinje cell plasticity was not observed after the rats reached asymptotic levels of learning. These results indicate that CBR agonist administration disrupts the induction of plasticity within the cerebellar cortex and this may account for the behavioral deficit in eyeblink conditioning. Chapter 3 examined whether infusions of the CBR agonist into the cerebellar cortex impaired forebrain-dependent learning as well as forebrain-independent associative learning. Similar to subcutaneous injections, forebrain-dependent trace eyeblink conditioning was unimpaired, whereas forebrain independent delay eyeblink conditioning was impaired. These findings provide evidence that plasticity mechanisms that are modulated by cannabinoids do not play a significant role in trace eyeblink conditioning. Finally, in Chapter 4 the role of CBRs and endocannabinoids during memory consolidation were examined. CBR and endocannabinoid manipulations prior to training resulted in impaired eyeblink conditioning. However, a CBR agonist or a drug increasing endocannabinoid levels resulted in enhanced consolidation when administered 1 hour post-training. In contrast, a CBR antagonist or an endocannabinoid decreasing drug resulted in impairments 1 hour post-training. Thus, CBRs and endocannabinoids appear to be important in learning and consolidation of cerebellar-dependent learning.

KOSTRZEWA, R. M., J. W. KLARA, J. ROBERTSON AND L. C. WALKER. Studies on the mechanism of sprouting of noradrenergic terminals in rat and mouse cerebellum after neonatal 6-hydroxydopa. BRAIN RES. BULL. 3(5) 525-531, 1978.-The effect of various pharmacologic agents on the noradrenergic innervation of rat cerebellum was observed. It was found that the neurotoxin 6-hydroxydopa (6-OHDOPA), when given to rats at birth, caused a 46% reduction at 5 weeks of age in tyrosine hydroxylase activity in the locus coeruleus, the nucleus of origin for noradrenergic fibers innervating the cerebellum. At the same time, however, both tyrosine hydroxylase activity and NE content were elevated by 50% in the cerebellum. By treating gravid mice with the 6-OHDOPA, which crosses the placental barrier to affect the brains of developing pups, a dissociation has been shown between the elevated cerebellar NE levels and reduced telencephalic NE content. None of the other assorted pharmacological agents-namely amphetamine, metaraminol, apomorphine, α-methyl-ρ-tyrosine, L-dihydroxyphenylalanine and tyramine-when given at birth, caused a permanent elevation in cerebellar NE content. This series of studies suggests that a reduced number of noradrenergic perikarya are providing a greater innervation of the cerebellum than in control rats. Also, alteration of the telencephalic noradrenergic fibers, which are also derived from the locus coeruleus, does not appear to be a necessary event for the initiation of sprouting of noradrenergic fibers in the cerebellum. Because none of the acute-acting pharmacological agents caused a permanent elevation of NE in the cerebellum, it appears that damage, and not mere stimulation or blockade, is a necessary event for initiation of sprouting.

Although human congenital cerebellar malformations are common, their molecular and developmental basis is still poorly understood. Recently, cilia-related gene deficiencies have been implicated in several congenital disorders that exhibit cerebellar abnormalities such as Joubert syndrome, Meckel-Gruber syndrome, Bardet-Biedl syndrome, and Orofaciodigital syndrome. The association of cilia gene mutations with these syndromes suggests that cilia may be important for cerebellar development, but the nature of cilia involvement has not been elucidated. To assess the importance of cilia-related proteins during cerebellar development, we studied the effects of CNS-specific inactivation of two mouse genes whose protein products are critical for cilia formation and maintenance, IFT88, (also known as polaris or Tg737), which encodes intraflagellar transport 88 homolog, and Kif3a, which encodes kinesin family member 3a. We showed that loss of either of these genes caused severe cerebellar hypoplasia and foliation abnormalities, primarily attributable to a failure of expansion of the neonatal granule cell progenitor population. In addition, granule cell progenitor proliferation was sensitive to partial loss of IFT function in a hypomorphic mutant of IFT88 (IFT88orpk), an effect that was modified by genetic background. IFT88 and Kif3a were not required for the specification and differentiation of most other cerebellar cell types, including Purkinje cells. Together, our observations constitute the first demonstration that cilia proteins are essential for normal cerebellar development and suggest that granule cell proliferation defects may be central to the cerebellar pathology in human cilia-related disorders.

Recent data suggest novel functional roles for cerebellar involvement in a number of neurologic diseases. Function of cerebellar neurons is known to be modulated by norepinephrine and adrenergic receptors. The distribution of adrenergic receptor subtypes has been described in experimental animals, but corroboration of such studies in the human cerebellum, necessary for drug treatment, is still lacking. In the present work we studied cell-specific localizations of α1 adrenergic receptor subtype mRNA (α1a, α1b, α1d), and α2 adrenergic receptor subtype mRNA (α2a, α2b, α2c) by in situ hybridization on cryostat sections of human cerebellum (cortical layers and dentate nucleus). We observed unique neuron-specific α1 adrenergic receptor and α2 adrenergic receptor subtype distribution in human cerebellum. The cerebellar cortex expresses mRNA encoding all six α adrenergic receptor subtypes, whereas dentate nucleus neurons express all subtype mRNAs, except α2a adrenergic receptor mRNA. All Purkinje cells label strongly for α2a and α2b adrenergic receptor mRNA. Additionally, Purkinje cells of the anterior lobe vermis (lobules I to V) and uvula/tonsil (lobules IX/HIX) express α1a and α2c subtypes, and Purkinje cells in the ansiform lobule (lobule HVII) and uvula/tonsil express α1b and α2c adrenergic receptor subtypes. Basket cells show a strong signal for α1a, moderate signal for α2a and light label for α2b adrenergic receptor mRNA. In stellate cells, besides a strong label of α2a adrenergic receptor mRNA in all and moderate label of α2b message in select stellate cells, the inner stellate cells are also moderately positive for α1b adrenergic receptor mRNA. Granule and Golgi cells express high levels of α2a and α2b adrenergic receptor mRNAs. These data contribute new information regarding specific location of adrenergic receptor subtypes in human cerebellar neurons. We discuss our observations in terms of possible modulatory roles of adrenergic receptor subtypes in cerebellar neurons responding to sensory and autonomic input signals, and review species differences in cerebellar adrenergic receptor expression.

Performance motor errors trigger animals’ adaptive learning behaviors to improve the accuracy and efficiency of the movement. The cerebellum is one of the key brain centers for encoding motor performance and motor learning. Climbing fibers relay information related to motor errors to the cerebellar cortex, evoking elevation of intracellular Ca2+ signals at Purkinje cell dendrites and inducing plasticity at coactive parallel fiber synapses, ultimately recalibrating sensorimotor associations to alter behavior. Molecular layer interneurons (MLIs) inhibit Purkinje cells to modulate dendritic excitability and action potential output. How MLIs contribute to the regulation and encoding of climbing fiber-evoked adaptive movements remains poorly understood. In this dissertation, I used genetic tools to manipulate the activity of MLIs while monitoring Purkinje cell dendritic activity during a cerebellum-dependent motor learning task with different contexts to evaluate how MLIs are involved in this process. The results show that by suppressing dendritic Ca2+ signals in Purkinje cells, MLI activity coincident with climbing fiber-mediated excitation prevents the occurrence of learning when adaptation is not necessary. On the other hand, with error signals present, disinhibition onto Purkinje cells, mediated by MLI-MLI microcircuit, unlocked the ability of climbing fibers to induce plasticity and motor learning. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

The cerebellum is well-known for its ability to integrate diverse internal and external stimuli, forming associations which serve as a basis for the construction of predictive models. Though traditionally studied in the context of motor learning, mounting evidence indicates that cerebellar learning may play an analogous role in many non-motor functions, including autonomic regulation and reward processing. Using combinatorial approaches of neuroanatomical tracing, electrophysiology, and behavioral studies, we investigated whether cerebellar learning extends to the domains of feeding behavior and gastrointestinal regulation, wherein prediction is critical for optimally timing food seeking and digestion in order to maximize nutrient acquisition while minimizing risk and energetic cost. Our results show direct anatomical connections between the cerebellum and known feeding centers, including the lateral hypothalamus, nucleus of the solitary tract, and parvocellular reticular nucleus. In addition, a localized area of the rostral cerebellum was found to respond to gastric distension and drive gastric emptying. Finally, we show that cerebellar activation drives robust, short-latency ingestive responses in awake, sated mice. Collectively, our results implicate the specific, interconnected cerebellar regions of Lobules II & III and the rostral fastigial nucleus, which we believe may represent one or more microzones devoted to integrating pre-and post-ingestive signals for control of feeding and digestion.