Synaptic aging disrupts synaptic morphology and function in cerebellar Purkinje cells

Synapses are key structures in neural networks,and are involved in learning and memory in the central nervous system.Investigating synaptogenesis and synaptic aging is important in understanding neural development and neural degeneration in diseases such as Alzheimer disease and Parkinson’s disease.Our previous study found that synaptogenesis and synaptic maturation were harmonized with brain development and maturation.However,synaptic damage and loss in the aging cerebellum are not well understood.This study was designed to investigate the occurrence of synaptic aging in the cerebellum by observing the ultrastructural changes of dendritic spines and synapses in cerebellar Purkinje cells of aging mice.Immunocytochemistry,Di I diolistic assays,and transmission electron microscopy were used to visualize the morphological characteristics of synaptic buttons,dendritic spines and synapses of Purkinje cells in mice at various ages.With synaptic aging in the cerebellum,dendritic spines and synaptic buttons were lost,and the synaptic ultrastructure was altered,including a reduction in the number of synaptic vesicles and mitochondria in presynaptic termini and smaller thin specialized zones in pre-and post-synaptic membranes.These findings confirm that synaptic morphology and function is disrupted in aging synapses,which may be an important pathological cause of neurodegenerative diseases.

Role of 5-hydroxytryptamine expression in cerebellar Purkinje cells in obstructive sleep apnea syndrome

In the present study, electrical stimulation to the rat insular cortex induced apnea or respiratory disturbance, reduced amplitude of genioglossal electromyogram, and decreased electromyogram integrals. In addition, arterial blood gas analysis showed arterial blood acidosis, reduced pH values, increased alkali reserve negative values, decreased peripheral blood 5-hydroxytryptamine content, and increased 5-hydroxytryptamine expression in cerebellar Purkinje cells. Following lidocaine injection to block the habenular nucleus, abnormalities in breath, genioglossal electromyogram, and blood gas values disappeared, and peripheral blood 5-hydroxytryptamine content returned to levels prior to electric stimulation. However, 5-hydroxytryptamine expression in cerebellar Purkinje cells remained high. The results suggested that 5-hydroxytryptamine expression in Purkinje cells did not correlate with ventilation function involving insular cortex and habenular nucleus.

Effects of progesterone on T-type-Ca^(2+)-channel expression in Purkinje cells

Plasticity of cerebellar Purkinje cells(PC)is influenced by progeste rone via the classical progeste rone receptors PR-A and PR-B by stimulating dendritogenesis,spinogenesis,and synaptogenesis in these cells.Dissociated PC cultures were used to analyze progeste rone effects at a molecular level on the voltage-gated T-type-Ca^(2+)-channels Ca_(v)3.1,Ca_(v)3.2,and Ca_(v)3.3 as they helped determine neuronal plasticity by regulating Ca^(2+)-influx in neuronal cells.The results showed direct effects of progesterone on the mRNA expression of T-type-Ca^(2+)-channels,as well as on the protein kinases A and C being involved in downstream signaling pathways that play an important role in neuronal plasticity.For the mRNA expression studies of T-type-Ca^(2+)-channels and protein kinases of the signaling cascade,laser microdissection and purified PC cultures of diffe rent maturation stages were used.Immunohistochemical staining was also performed to characte rize the localization of T-type-Ca^(2+)-channels in PC.Expe rimental progesterone treatment was performed on the purified PC culture for 24 and 48 hours.Our results show that progesterone increases the expression of Ca_(v)3.1 and Ca_(v)3.3 and associated protein kinases A and Cin PC at the mRNA level within 48 hours after treatment at latest.These effects extend the current knowledge of the function of progesterone in the central nervous system and provide an explanatory approach for its influence on neuronal plasticity.

Coexistence of dopamine-beta-hydroxylase and activated protein-2 alpha in rat cerebellar Purkinje cells

BACKGROUND： Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum. Numerous reports have also been published addressing whether dopamine-beta-hydroxylase （DBH） expression exists in cerebellar Purkinje cells. OBJECTIVE： To investigate the coexistence of DBH and activator protein-2α expression in rat cerebellar Purkinje cells. DESIGN, TIME AND SETTING： A cell morphological study was performed at the Institute of Neuroscience, Chongqing Medical University, China in May 2007. MATERIALS： Ten healthy Wistar rats, of either gender, aged 14 weeks, served as experimental animals. Rabbit anti-mouse DBH, goat anti-mouse activator protein-2α and rabbit anti-mouse β-actin （Santa Cruz Biotechnology, Inc., USA）, horseradish peroxidase-labeled goat anti-rabbit IgG, FITC-labeled mouse anti-rabbit IgG, and Cy3-labeled mouse anti-goat IgG （Boster, Wuhan, China）, were used in this study. METHODS： Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α, with double-label immunofluorescence being employed to determine coexpression of both, in the cerebellum of 5 randomly selected rats. Western blot assay was utilized to determine the expression of DBH and activator protein-2α in the cerebellum of the remaining 5 rats. MAIN OUTCOME MEASURES： Expression, localization and coexistence of DBH and activator protein-2α in the cerebellum were measured separately. RESULTS： Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α. Western blot assay also demonstrated DBH and activator protein-2α expression in the cerebellum. Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2α in cerebellar Purkinje cells. CONCLUSION： Norepinephrine and activator protein-2α coexist in rat cerebellar Purkinje cells.

Curcumin alters motor coordination but not total number of Purkinje cells in the cerebellum of adolescent male Wistar rats

OBJECTIVE： The present study aimed at coordination and the estimate of the total investigating the effects of curcumin on the motor number of cerebellar Purkinje cells of adolescent Wistar rats exposed to ethanol. METHODS： The total of 21 male Wistar rats aged 37 d old were divided into three groups, namely ethanol, ethanol-curcumin, and control groups. The ethanol group received 1.5 g/kg ethanol injected intraperitoneally and water given per oral; the ethanol-curcumin group received 1.5 g/kg ethanol injected intraperitoneally and curcumin extract given per oral; the control group received saline injection and oral water. The treatment was carried out daily for one month, after which the motor coordination performance of the rats was examined using revolving drum apparatus at test days 1, 8, and 15. The rats were finally sacrificed and the cerebellum of the rats was further processed for stereological analysis. The estimate of the total number of Purkinje cells was calculated using physical fractionator method. RESULTS： The ethanol-curcumin group performed better than both ethanol and control groups in the motor coordination ability at day 8 of testing （P〈 0.01）. No Purkinje cell loss was observed as a result of one month intraperitoneal injection of ethanol. CONCLUSION： Curcumin may exert beneficial effects on the motor coordination of adolescent rats exposed to ethanol via undetermined hormetic mechanisms.

Mouse cerebellar Purkinje cell damage induced by diphenylhydantoin acute intoxication

Twenty one days old Swiss albino mice that received diphenylhydantoin(25 mg/kg,i.p.,daily for 15 days)progressively developed gait alterations,changes of behavior and cerebellar ataxia.Cerebellar slices were processed by conventional transmission electron microscopy.The body of Purkinje cells exhibited fragmented limiting plasma membranes,dilated nuclear envelopes,swelling and disassembly of nuclear pores,enlargement of rough and smooth endoplasmic reticulum and a notable detachment of membrane associated ribosomes,to-gether with distorted vacuoles of smooth endoplasmic reticulum,bizarre shaped and swollen mitochondria with dilated cristae,as well as disrupted limiting lysosomal membranes.Degenerated axosomatic synapses apparently corresponding to basket cell axonal endings were recognized.Degenerated Purkinje cell axon initial segments exhibited vacuolar degeneration of myelin sheath,dilated axoplasmic tubular bundles,fragmented axonal mem-branes,swollen mitochondria,and disassembly of cytoskeletal structures.Some edematous and clear secondary and tertiary dendrites exhibited areas of dilated cisterns of smooth endoplasmic reticulum,clear and dark mul-tivesicular bodies,and coated vesicles.Other dendritic ramifications exhibited an electron dense dendroplasm.Degenerated and large climbing fiber endings were observed making axodendritic synapses with edematous Purkinje dendrites.These presynaptic endings appeared depleted or containing few synaptic vesicles.These syn-apses did not exhibit pre-and postsynaptic densities.At the molecular layer,the edematous synaptic varicosities of parallel fibers containing pleomorphic synaptic vesicles and dense extravesicular substance were observed making asymmetric synaptic contacts with swollen Purkinje dendritic spines.These findings are postulated as pathogenic mechanisms of mouse cerebellar ataxia.

Effects of Maternal Marginal Iodine Deficiency on Interactions between Cerebellar Bergmann Glia Cells and Purkinje Cells in Rat Offspring

Iodine deficiency （ID） during early pregnancy has an adverse effect on children＇s psychomotor and motor function but the mechanism has not been clarified. Therefore, our aim was to study the effect of maternal marginal ID on cerebellar neurodevelopment and the underlying mechanism. After obtaining marginal ID rats, we examined interactions between Bergmann gila cells （BGs） and Purkinje cells （PCs） using immunofluorescence and expression of the glutamate transporter and receptor by western blot. Our results showed that marginal ID reduced the number of contacted points between BGs and PCs,

Loss of Wtap results in cerebellar ataxia and degeneration of Purkinje cells

N^(6)-methyladenosine(m^(6)A)modification,which is achieved by the METTL3/METTL14/WTAP methyltransferase complex,is the most abundant internal mRNA modification.Although recent evidence indicates that m^(6)A can regulate neurodevelopment as well as synaptic function,the roles of m^(6)A modification in the cerebellum and related synaptic connections are not well established.Here,we report that Purkinje cell(PC)-specific WTAP knockout mice display early-onset ataxia concomitant with cerebellar atrophy due to extensive PC degeneration and apoptotic cell death.Loss of Wtap also causes the aberrant degradation of multiple PC synapses.WTAP depletion leads to decreased expression levels of METTL3/14 and reduced m^(6)A methylation in PCs.Moreover,the expression of GFAP and NF-L in the degenerating cerebellum is increased,suggesting severe neuronal injuries.In conclusion,this study demonstrates the critical role of WTAP-mediated m^(6)A modification in cerebellar PCs,thus providing unique insights related to neurodegenerative disorders.

Contribution of CD3 ε Epitope and Oxidative Type of Copper-Zinc Superoxide Dismutase to the Degeneration Processes of Cerebellar Purkinje Cells in Patients with Multiple System Atrophy-Cerebellar Type (MSA-C: Olivopontocerebellar Atrophy, OPCA): An Immunohistochemical Study

Objective: This study aimed to investigate the contribution of CD3 epsilon (ε) epitope and oxidative type of copper-zinc superoxide dismutase to the degeneration processes of cerebellar Purkinje cells in patients with Multiple System Atrophy-Cerebellar type (MSA-C). Methods: This retrospective study was carried out on autopsy specimens of 17 patients with sporadic MSA-C and 10 normal individuals. Paraffin sections of autopsied cerebella and pontes were immunostained with polyclonal antibodies against CD3 ε epitope and oxidative modification to cysteine sulfonic acid of cys111 in human copper-zinc superoxide dismutase (Ox-SOD1). With respect to the areas of CD3-ε-epitope expression, the immunohistochemical study and the quantitative statistical analysis between the areas of CD3-ε-epitope expression in the surviving Purkinje cells of MSA-C patients and their disease duration were performed. Results: The cell bodies and dendritic arborization including primary, secondary, and tertiary dendrites of normal Purkinje cells were intensely immunostained by the antibody against CD3 ε epitope. Both the immunohistochemical study and the quantitative statistical analysis revealed that the areas positive for CD3 ε epitope disappeared in the order from tertiary dendrites, secondary dendrites, primary dendrites toward the cell bodies, along with the disease progression. In addition, Glial Cytoplasmic Inclusions (GCIs) and Neuronal Cytoplasmic Inclusions (NCIs) were strongly positive for CD3 ε epitope. The surviving Purkinje cells in MSA-C showed immunostaining by the anti-Ox-SOD1 antibody, although normal Purkinje cells did not. Conclusion: Based on the oxidative stress that the surviving Purkinje cells in MSA-C express Ox-SOD1, the functions of morphogenesis and morphological maintenance related to CD3-ε-epitope expression of the MSA-C Purkinje cells are impaired from the peripheral dendrites toward the cell bodies as the center of the Purkinje cell system. In addition, GCIs and NCIs that are pathological hallmarks of MSA also intensely express CD3 ε epitope.

CD4 and CD8 T cells mediate distinct lethal meningoencephalitis in mice challenged with Tacaribe arenavirus

Neonates are at increased risk of viral encephalopathies that can result in neurological dysfunction, seizures, permanent disability and even death. The neurological damage results from the combined effect of the virus and the immune response it elicits, thus finding tools to facilitate viral clearance from central nervous system （CNS） while minimizing neuron damage remains a critical challenge. Neonatal mice inoculated intraperitoneally with Tacaribe virus （TCRV） develop seizures, hindlimb paralysis and death within 15 days of inoculation. TCRV localizes to the CNS within days of challenge, primarily infecting astrocytes in the cerebellum and brain stem. We show that infection leads to inflammation, T cell and monocyte infiltration into the cerebellar parenchyma, apoptosis of astrocytes, neuronal degeneration and loss of Purkinje cells. Infiltrating antigen-specific T cells fail to clear the virus but drive the disease, as T-cell-deficient CD3ε KO mice survive TCRV infection with minimal inflammation or clinical manifestations despite no difference in CNS viral loads in comparison with T-cell sufficient mice. CD8＋ T cells drive the pathology, which even in the absence of CD4＋ T-cell help, infiltrate the parenchyma and mediate the apoptotic loss of cerebellar astrocytes, neurodegeneration and loss of Purkinje cells resulting in loss of balance, paralysis and death. CD4＋ T cells are also pathogenic inducing gliosis and inflammation in the cerebellum and cerebrum that are associated with wasting and death several weeks after CD4＋ T-cell transfer. These data demonstrate distinct pathogenic effects of CD4＋ and CD8＋ T cells and identify them as possible therapeutic targets.

Neuronal Activity in the Cerebellum During the Sleep-Wakefulness Transition in Mice

Cerebellar malfunction can lead to sleep disturbance such as excessive daytime sleepiness,suggesting that the cerebellum may be involved in regulating sleep and/or wakefulness.However,understanding the features of cerebellar regulation in sleep and wakefulness states requires a detailed characterization of neuronal activity within this area.By performing multiple-unit recordings in mice,we showed that Purkinje cells(PCs)in the cerebellar cortex exhibited increased firing activity prior to the transition from sleep to wakefulness.Notably,the increased PC activity resulted from the inputs of lowfrequency non-PC units in the cerebellar cortex.Moreover,the increased PC activity was accompanied by decreased activity in neurons of the deep cerebellar nuclei at the nonrapid eye-movement sleep-wakefulness transition.Our results provide in vivo electrophysiological evidence that the cerebellum has the potential to actively regulate the sleep-wakefulness transition.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish

Joubert syndrome is characterized by unique malformation of the cerebellar vermis.More than thirty Joubert syndrome genes have been identified,including ARL13 B.However,its role in cerebellar development remains unexplored.We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae.Granule cells were selectively reduced in the corpus cerebelli,a structure homologous to the mammalian vermis.Purkinje cell progenitors were also selectively disturbed dorsomedially.The expression of atoh1 and ptf1,proneural genes of granule and Purkinje cells,respectively,were selectively down-regulated along the dorsal midline of the cerebellum.Moreover,wnt1,which is transiently expressed early in cerebellar development,was selectively reduced.Intriguingly,activating Wnt signaling partially rescued the granule cell defects in arl13b mutants.These findings suggested that Arl13 b is necessary for the early development of cerebellar granule and Purkinje cells.The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

A bacterial artificial chromosome transgenic mouse model for visualization of neurite growth

Class Ⅲ β-tubulin （Tubb3） is a component of the microtubules in neurons and contributes to microtubule dynamics that are required for axon outgrowth and guidance during neuronal development. We here report a novel bacterial artificial chromosome （BAC） transgenic mouse line that expresses Class Ⅲ β-tubulin fused to mCherry, an improved monomeric red fluorescent protein, for the visualization of microtubules during neuronal development. A BAC containing Tubb3 gene was modified by insertion of mCherry complementary DNA downstream of Tubb3 coding sequence via homologous recombination, mCherry fusion protein was expressed in the nervous system and testis of the transgenic animal, and the fluorescent signal was observed in the neurons that located in the olfactory bulb, cerebral cortex, hippocampal formation, cerebellum, as well as the retina. Besides, Tubb3-mCherry fusion protein mainly distributed in neurites and colocalized with endogenous Class Ⅲ β-tubulin The fusion protein labels Purkinje cell dendrites during cerebellar circuit formation. Therefore, this transgenic line might be a novel tool for scientific community to study neuronal development both in vitro and in vivo.