Effects and mechanism of chronic ethanol consumption on the electrical activity of Purkinje cells in the cerebellar cortex of mice / 中华行为医学与脑科学杂志

Objective:To investigate the effect of long-term chronic ethanol consumption on the spontaneous discharge activity of Purkinje cells in the cerebellar cortex of mice.Methods:Fifty 3-week-old ICR mice, regardless of gender, were divided into control group and ethanol group according to the random number table method, with 25 mice in each group. The mice in ethanol group were administered 20% ethanol (1.6 g/kg, once a day) by gavage, and the control group mice were given the same volume of 0.9% sodium chloride solution by gavage, and the gavage cycle was 28 days.The electrical activity of cerebellar Purkinje cells induced by sensory stimulation was recorded by patch clamp amplifier and data acquisition software. Statistical analysis was conducted by Clampfit 10.3 software and SPSS 22.0 software, t-test and one-way ANOVA were used to compare the data between the two groups and the data before and after intervention of each group. Results:The electrophysiological results showed that the spontaneous simple spike discharge frequency of Purkinje cells in the cerebellar cortex of mice in ethanol group was lower than that of the control group ((26.8±2.5)%, (34.6±4.7)%; t=26.08, P<0.05), and the coefficient of variation was higher than that of the control group ((27.3±3.3)%, (19.2±2.3)%; t=22.95, P<0.05). After cerebral surface perfusion of GABAA receptor antagonist, the frequency of simple peak potentials in the cerebellar cortex of ethanol mice was higher than before administration ( t=10.19, P<0.05), and the coefficient of variation was lower than before administration ( t=28.36, P<0.05). After brain surface perfusion of GABAA receptor antagonist, there was no significant change in the spontaneous simple peak discharge frequency of cerebellar Purkinje cells in the control group( P>0.05), and the coefficient of variation decreased compared to before administration ( t=6.95, P<0.05). After administering AMPA receptor antagonists on the surface of the brain, there were no significant changes in the discharge frequency and coefficient of variation in both the ethanol group and control group compared to before administration (both P>0.05). After simultaneously blocking AMPA and GABAA receptors, it was found that the spontaneous discharge frequency in ethanol group increased after administration compared to before administration((107.3±4.3)%, (99.7±3.7)%, P<0.05), and the increased value of frequency in the ethanol group was also higher than that of control group ( P<0.05). After simultaneously blocking AMPA and GABAA receptors, the coefficient of variation of the alcohol group and the control group mice were both lower than those before administration (both P<0.05), and the decrease in the alcohol group was higher than that in the control group ( P<0.05). Conclusion:Chronic ethanol exposure significantly inhibited the spontaneous discharge of Purkinje cells in the cerebellum, and the enhancement of inhibitory components was achieved by the inhibitory input mediated by GABAA receptors.

Effects of CTNND2 knockout on cerebellar development and motor function in mice / 解剖学报

Objective To investigate the effects of CTNND2 knockout on cerebellar neuronal development and motor function in mice, as well as its possible mechanisms. Methods The mice were divided into two groups (n = 10 in each group), all of them were 7 weeks old : wild-type (WT) C57BL/6J mice were treated as control group, and homozygous of CTNND2 knockout (CTNND2 7) mice were treated as experimental group, the genotype of CTNND2 7 mice were detected with PCR. The motor function of two groups were detected by beam walking test, hanging wire test and gait analysis test. The changes of cerebellar Purkinje cells were detected by immunofluorescence staining and Golgi staining. Western blotting was performed to detect the expression levels of synapse-associated proteins phosphorylated synapsin 1 (p-Synl), synapsin 1 (Synl), ELKS and postsynaptic density protein 95(PSD95), as well as phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), protein kinase B (Akt), phosphorylated mammalian target of rapamycin (p-mTOR) and mammalian target of rapamycin (mTOR). Results Compared with the WT mice, except the increase in time to traverse the beam, there was a decrease in the proportion of pass on the beam, or latency to fall from the hanging wire, or score of hanging wire, or fore-stride length and hind-stride length of CTNND2 7 mice. There was also a decrease in numbers of Purkinje cells and its dendritic arborization in cerebellum of CTNND2 7 mice. The ratio of p-Synl/ Synl, p-Akt/Akt and p-mTOR/mTOR, as well as the expression levels of ELKS, PSD95 and PI3K were lower than those of WT mice. Conclusion CTNND2 knockout can affect the number and dendritic architecture of Purkinje cells, as well as synthesis of synapse-associated proteins in cerebellum by down-regulating PI3K/Akt/mT0R signaling pathway, resulting in cerebellar developmental disorder, thereby affecting motor function of mice.

Intrauterine hypoxia slowing the development of Purkinje cells and reducing the expression of cerebellin in mice / 解剖学报

Objective To investigate the effects of the development of Purkinje cells and expression of cerebellin in postnatal mice with intrauterine hypoxia. Methods Twenty healthy adult Kunming mice were randomly divided into two groups : control group and hypoxia group, with 10 mice in each group. Mice in the hypoxia group were placed in the hypoxia chamber of the animal since the 14th day of gestation to make an animal model of intrauterine hypoxia. After the mother gave birth, the experimental animals were divided into hypoxia group and control group. There were 6 age groups including postnatal day (P)0, P5, P9, P14, P21 and P30 in each group, and 5 mice in each age group. The cerebellum tissue was taken for vibrating sectioning. The developmental changes of calbindin-positive Purkinje cells were detected by immunofluorescence technique. The expression of cerebellin in Purkinje cell protuberances was detected by cerebellin (CBLN)l, CBLN4 and calbindin double labeling. Finally, Western blotting was used to semi-quantitatively analyze the protein expression of cerebellar peptide in cerebellum at each time point. Results Compared with the control group of the same age, the number of cerebellar Purkinje cells in the hypoxic group decreased, the dendritic branches decreased, and the arrangement was disordered, and the expression of CBLN1 and CBLN4 in the cortex were significantly reduced. Conclusion Intrauterine hypoxia leads to abnormal development of the cerebellar Purkinje cells and synaptic changes.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish / 神经科学通报·英文版

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. 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 Arl13b 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.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish / 神经科学通报·英文版

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. 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 Arl13b 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.

Neuronal Activity in the Cerebellum During the Sleep-Wakefulness Transition in Mice / 神经科学通报·英文版

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 low-frequency 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 non-rapid 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.

Neuroprotective effect of melatonin in cerebellums in Alzheimer's disease animal models via MAPK/ERK signaling pathway / 中国药理学通报

Aim: To investigate the effect of melatonin on neuroprotection in cerebellums of rats with Alzheimer' s disease via MAPK/ERK signaling pathway. Methods: Thirty-two male Sprague-Dawley rats were randomly divided into four groups: control group, Aβ1-42 lateral ventricle injection group (AD), and melatonin intraperitoneal injection group (MT), and Aβ1-42 lateral ventricle injection combined with melatonin intraperitoneal injection group (AD + MT). The pathological changes of rat cerebellar cortex were detected by HE staining; the expression of NeuN (neuronal marker), Calbindin(Purkinje cell marker) and p-ERK protein in each group was detected by immunofluorescence; the expression of ERK and p-ERK in each group was determined by Western blot. Results: The HE staining showed that the expression of neurons decreased, followed with the disordered arrangement and morphological alteration of cells in AD. Melatonin could significantly alleviate the pathological damage in cerebellum. Immunofluorescence results showed that compared with AD group, the expression of NeuN (neuronal marker) increased, the number of Purkinje cells marked by Calbindin significantly was up-regulated(P < 0. 01), and the expression of p-ERK was down-regulated in AD + MT group. Western blot showed that the expression of p-ERK was down-regulated by melatonin. Conclusions: Melatonin may exert the neuroprotective effect and relieve the pathological changes by inhibiting the activation of MAPK/ERK signaling pathway.

Screening and Verification of RNA-seq-based PBCRS for Treating Key Genes of Breast Cancer / 中国实验方剂学杂志

Objective: To study the inhibitory effect of the medicine group of promoting blood circulation and removing stasis (PBCRS) on breast cancer induced by 7, 12-dimethylbenz(a)anthracene (DMBA) in rats, and screen out and verify key genes based on RNA Sequencing (RNA-seq) technology and Ingenuity Pathway Analysis (IPA). Method: Totally 96 Sprague-Dawley (SD) rats were randomly divided into blank group, DMBA model control group, tamoxifen (TAM) group (1.9 mg·kg-1·d-1), high-dose, middle-dose and low-dose PBCRS groups (12.96, 6.48, 3.24 g·kg-1·d-1). One week after drug intervention, except for the blank group, the DMBA was used to induce the rat model of breast cancer (with an interval of a week, irrigation for two times at the dose of 100 mg·kg-1). After 10 weeks, the changes in tumor weight and tumor volume were observed. The total RNA was extracted by total RNA extraction kit, and three RNA samples were collected from the DMBA model control group and the middle-dose PBCRS group for genetic testing. Based on RNA-seq, key differential genes were screened out and verified by Real-time PCR. Result: Comparing with the DMBA model control group, the tumor volume and tumor weight in middle-dose PBCRS group were decreased significantly (PPConclusion: PBCRS may inhibit the occurrence of breast cancer by interfering with the expression of FBP1 in breast cancer tissue.

Effect of intracerebellar microinjection of cdc2-siRNA on the coordination of mice with Niemann-Pick disease type C / 中华行为医学与脑科学杂志

Objective To observe the effect of inhibiting the abnormal activation of cdc2 gene on the coordination of mice with Niemann-Pick disease type C(NPC).Methods Recombinant adeno-associated virus(rAAV) encoding cdc2-siRNA was packaged,and then was injected into the cerebellum of 2 weeks old npc-/-mice.Footprint test and vertical screen test were performed to assess the coordination of mice at the age of 8 weeks.Purkinje cells visualized by HE staining in cerebellum were counted,and the phosphorylation of microtubule-associated protein Tau recognized by PHF-1 antibody was detected by immunoblotting technology.Results (1) Footprint test showed that the stride length in cdc2-siRNA npc-/-group((4.92±0.31)cm) was markedly longer than that in empty vector npc-/-group((4.05 ± 0.19) cm) (P＜ 0.05).(2) Vertical screen test showed that the latency to turn head upwards or reach the upper edge of the screen in cdc2-siRNA npc-/-group((26.01± 1.82) s,(50.93±1.98) s) was significantly shorter than that in the empty vector npc-/-group ((31.96± 3.47) s,(56.89 ± 2.97) s),respectively (P＜ 0.05 for all comparisons).(3) The number of Purkinje cells in cerebellum was dramatically increased in cdc2-siRNA npc-/-group(11.0±2.5) compared with the empty vector npc-/-group (5.1 ± 2.2) (P＜0.05).(4)The relative optical densities of cdc2 and phosphorylated Tau immunoreactive bands in cdc2-siRNA npc-/-group(1.42±0.22,0.95±0.31)were significantly lower than those in the empty vector npc-/-group(2.11±0.29,2.61±0.62),respectively (P＜0.05 for all comparisons).Conclusion Inhibiting the abnormal activation of cdc2 gene can improve the coordination of npc-/-mice by ameliorating Purkinje cell's loss and reducing the hyperphosphorylation of Tau in cerebellum.

Acid sensing ion channel 1a involving in development of cerebella Purkinje cells / 中华神经医学杂志

Objective To investigate the mechanism of acid sensing ion channel (ASIC) 1a in the development of Purkinje cells in the cerebellum.Methods Newborn C57BL/6 mice were chosen and Purkinje cells were obtained from these mice by in vitro culture.Purkinje cells were divided into experimental group and control group,and shRNA-ASIC1a or shRNA-control sequence was used to construct recombinant plasmid lentivirus infections at the early and late stages of cell developments.The morphological structures of Purkinje cells were detected by immunofluorescent staining and the dendritic branches were counted at the early and late stages of cell developments.Western blotting was employed to detect the calcium binding protein D-28K,glial fibrillary acidic protein,Zic,parvalbumin and N-methyl-D-aspartate receptor (NMDAR) expressions.Real time (RT)-PCR was used to detect the expressions of endoplasmic reticulum stress related factor CCAAT/enhancer binding protein homologous protein CHOP and protein kinase R-like ER kinase (PERK).Results Immunofluorescence indicated that Purkinje cells showed obvious morphological differences between the experimental group and control group;the dendrite growth and development in the control group were significantly better than those in the experimental group (P＜0.05);the number of 2nd and 3nd stage dendritic branches of Purkinje cells in the control group was significantly larger than that in the experimental group (P＜0.05).Western blotting showed that D-28K expression in the Purkinje cells of experimental group was significantly decreased as compared with that in the control group at the early stage (P＜0.05);NMDAR expression in the Purkinje cells of experimental group was significantly increased as compared with that in the control group at the early and late stages (P＜0.05).RT-PCR results showed that CHOP and PERK expressions in the Purkinje cells of experimental group were significantly higher than those in the control group (P＜0.05).Conclusion ASIC1a has an important role in the development of cerebella Purkinje cells.

Effect of gestational diabetes on neuronal cells in rat cerebellum in early postnatal life / Efecto de la diabetes gestacional sobre las células neuronales del cerebelo de ratas en el período postnatal

Previous study has shown the adverse effects of gestational diabetes on hippocampal neuronal density in animal model. This study was conducted to determine the effect of gestational diabetes on rat cerebellum in early postnatal life. In this experimental study, 10 dams randomly allocated into control and diabetic groups on day 1 of gestation. Five dams in diabetic group were administered 40 mg/kg/BW (intraperitoneally) of streptozotocin and control animals received normal saline. Six offspring of each gestational diabetes mellitus and controls were randomly selected at day 7 of postnatal life. Offspring were sacrificed and coronal sections were taken from the cerebellum and stained with cresyl violet. The number of Purkinje and granular cells and thickness of layers of cerebellum were evaluated by quantitative computer-assisted morphometric method. The Purkinje cells density at apex and depth of cerebellar lobules in the experimental group (14.40±0.7, 14.86±0.6) significantly reduced in comparison with the control group (16.72±0.3, 17.85±0.7) (P<0.05). The granular cell density at apex and depth of cerebellar lobules in the experimental group (23.94±0.6, 22.81±0.5) significantly reduced in comparison with the control group (29.20±0.8, 28.1±0.8) (P<0.05). The thickness of the Purkinje and internal granular and molecular layers at apex and depth of cerebellar cortex significantly reduced in diabetics group compared to controls (P<0.05). This study revealed that gestational diabetes induces loss of number and size of the Purkinje cells and the granular cells and reduction of thickness of the Purkinje and internal granular layer of the cerebellar cortex in neonatal mice.

Estudios previos han demostrado los efectos adversos de la diabetes gestacional sobre la densidad neuronal del hipocampo en modelos animales. Este estudio se realizó para determinar el efecto de la diabetes gestacional en el cerebelo de ratas durante la edad temprana postnatal. Fueron asignadas 10 ratas hembras al azar en grupos control y diabético en el primer día de gestación. Cinco en el grupo diabético recibieron una dosis de 40 mg/kg/Peso corporal de estreptozotocina (intraperitoneal) y los control una solución salina normal. Seis crías de cada una de las hembras del grupo diabetes mellitus gestacional y del grupo controles fueron seleccionados al azar el día 7 de vida postnatal. Fueron sacrificadas y se obtuvieron secciones coronales desde el cerebelo que fueron teñidas con violeta de cresilo. El número de células granulares de Purkinje y espesor de las capas de cerebelo fueron evaluadas por método morfométrico y ordenador cuantitativo. La densidad de células de Purkinje en el ápice y profundidad de los lóbulos del cerebelo en el grupo experimental (14,40±0,7 y 14,86±0,6) se redujeron significativamente en comparación con el grupo control (16,72±0,3 y 17,85±0,7) (P<0,05). La densidad de células granulares en el ápice y profundidad de los lóbulos del cerebelo en el grupo experimental (23,94±0,6 y 22,81±0,5) se redujo significativamente en comparación con el grupo control (29,20±0,8 y 28,1±0,8) (P<0,05). En el espesor de células Purkinje, las capas moleculares y granulares internas en el ápice y profundidad de la corteza del cerebelo, se observó una reducción significativa en el grupo diabéticos en comparación con los controles (P<0,05). Se observó que la diabetes gestacional induce la pérdida del número y tamaño de las células Purkinje y de células granulares, así como la reducción del espesor de las capas de Purkinje y granular interna de la corteza del cerebelo en ratones neonatos.

Characterization of Membrane Excitability of Purkinje Cells in Vestibulocerebellum Using Patch Clamp Techniques / 대한이비인후과학회지

Cerebellum is known as a center for sensory/motor coordination and memory storage in motor learning. The vestibular nuclei have extensive afferent and efferent connections with posterior cerebellum which can be referred to as vestibulocerebellum. While secondary vestibular afferents are distributed bilaterally in the vestibulocerebellum, primary afferents may directly project to ipsilateral vestibulocerebellum. The Purkinje cells which are the only output neurons from the cerebellar cortex receive vestibular information via parallel and climbing fibers. That information is integrated and encoded in the Purkinje cells and then conveyed into the vestibular nucleus or deep cerebellar nucleus, which permits adaptive guidance of vestibular function by the vestibulocerebellum.

Diferenciação de células-tronco embrionárias murinas (mESCs) em células produtoras de insulina (IPCs) e caracterização funcional do gene Purkinje cell protein 4 (Pcp4) neste processo / Differentiation of murine embryonic stem cells (mESCs) into insulin-producing cells (IPCs) and functional characterization of the Purkinje Cell Protein 4 (Pcp4) gene in this process

Fontes alternativas de células ß têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células ß-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células ß. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células ß e no processo de diferenciação ß-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoß-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células ß-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células

New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic ß-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to ß-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in ß-cells and in ß-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to ß-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic ß-cells and possibly being related to maintenance of cell viability and ß-cell maturation

TRPC-Mediated Current Is Not Involved in Endocannabinoid-Induced Short-Term Depression in Cerebellum

It has been reported that activation of metabotropic glutamate receptor 1 (mGluR1) can mediate endocannabinoid-induced short-term depression of synaptic transmission in cerebellar parallel fiber (PF)-Purkinje cell (PC) synapse. mGluR1 has signaling pathways involved in intracellular calcium increase which may contribute to endocannabinoid release. Two major mGluR1-evoked calcium signaling pathways are known: (1) slow-kinetic inward current carried by transient receptor potential canonical (TRPC) channel which is permeable to Ca2+; (2) IP3-induced calcium release from intracellular calcium store. However, it is unclear how much each calcium source contributes to endocannabinoid signaling. Here, we investigated whether calcium influx through mGluR1-evoked TRPC channel contributes to endocannabinoid signaling in cerebellar Purkinje cells. At first, we applied SKF96365 to inhibit TRPC, which blocked endocannabinoid-induced short-term depression completely. However, an alternative TRP channel inhibitor, BTP2 did not affect endocannabinoid-induced short-term depression although it blocked mGluR1-evoked TRPC currents. Endocannabinoid signaling occurred normally even though the TRPC current was mostly blocked by BTP2. Our data imply that TRPC current does not play an important role in endocannabinoid signaling. We also suggest precaution in applying SKF96365 to inhibit TRP channels and propose BTP2 as an alternative TRPC inhibitor.

Numbers of Purkinje Cell with Increasing Age-A Post Mortem Study.

The change in the number of Purkinje cells with increasing age is evident especially in disorders of fine movement, equilibrium, hypotonia, postural changes, and disturbances of voluntary movement. The present study was done to see the changes in the number of Purkinje cells per square mm in different age groups of Bangladeshi people. This cross sectional descriptive type of study was designed and done in the Department of Anatomy, Dhaka Medical College, Dhaka, from January to December 2010, which was performed on the cerebellum of 28 Bangladeshi people, collected during autopsy examination of unclaimed dead bodies from Department of Forensic Medicine. Paraffin blocks of cerebellum were cut at 5mm thickness and stained with routine Harris' Haematoxylin and Eosin (H & E) stain. Estimation of number of Purkinje cell was done by using the counting circle and examined under the light microscope. The mean ± SD of number of Purkinje cell was 160.71 ± 24.47 in group A (Age 20-29 years) and 152.20 ± 6.49 in group D (age> 50 years), the mean reduction was 2.5% per decade. Histological studies revealed the number of Purkinje cell per square mm decreased with age which was statistically significant and further cytological study of Purkinje cell with larger sample size is recommended.

Differential regulation of Purkinje cell dendritic spines in rolling mouse Nagoya (tg(rol)/tg(rol)), P/Q type calcium channel (alpha1(A)/Ca(v)2.1) mutant / 대한해부학회지

Voltage dependent calcium channels (VDCC) participate in regulation of neuronal Ca2+. The Rolling mouse Nagoya (Cacna1a(tg-rol) ) is a spontaneous P/Q type VDCC mutant, which has been suggested as an animal model for some human neurological diseases such as autosomal dominant cerebellar ataxia (SCA6), familial hemiplegic migraine and episodic ataxia type-2. Morphology of Purkinje cell (PC) dendritic spine is suggested to be regulated by signal molecules such as Ca2+ and by interactions with afferent inputs. The amplitude of excitatory postsynaptic current was decreased in parallel fiber (PF) to PC synapses, whereas apparently increased in climbing fiber (CF) to PC synapses in rolling mice Nagoya. We have studied synaptic morphology changes in cerebella of this mutant strain. We previously found altered synapses between PF varicosity and PC dendritic spines. To study dendritic spine plasticity of PC in the condition of insufficient P/Q type VDCC function, we used high voltage electron microscopy (HVEM). We measured the density and length of PC dendritic spines at tertiary braches. We observed statistically a significant decrease in spine density as well as shorter spine length in rolling mice compared to wild type mice at tertiary dendritic braches. In proximal PC dendrites, however, there were more numerous dendritic spines in rolling mice Nagoya. The differential regulation of rolling PC spines at tertiary and proximal dendrites in rolling mice Nagoya suggests that two major excitatory afferent systems may be regulated reciprocally in the cerebellum of rolling mouse Nagoya.

Effects of Fragile Mental Retardation Protein on Cerebellar Neuron Development and Migration in Mouse Model / 中国医科大学学报

Objective To investigate the effects of fragile X mental retardation protein（FMRP）on the development and migration of cerebellar neurons in mouse model.Methods Plasmids containing FMRPmutant-EGFP or EGFP were established and transfected into the lateral ventricle of the embryo mouse.Fragile X syndrome（FXS）genotype of the mouse model was identified.Nissl staining and immunofluorescence staining were conducted to assess the changes in neuron development and migration.Results In the experimental group,Nissl staining showed that the deep cerebellar neuclei contracted and divided by white matter,and the non-polarized Purkinje cells retained in internal granular layer;while immunofluorescence staining showed that Tbr2-positive unipolar brush cells changed the migration pathway and accumulated in the ventricular zone.Conclusion Cerebellar neurons showed abnormal formation and migration with the absence of FMRP.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 micrometer filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.

Expression of HSP27 and alphaB-crystallin in Avian Cerebellum during Development / 대한해부학회지

It is well known that small heat shock proteins play a role as molecular chaperone. However, during normal development of the cerebellum, expression and distribution of HSP27 and alphaB-crystallin (alphaBC) which are small heat shock proteins have not been reported. To verify the protective role of HSP27 and alphaBC in neurons and glial cells, we examined the expression and distribution of HSP27 and alphaBC in the developing chick cerebellum using immunoblot, immunohistochemical and double immunofluorescence staining. Expression of both HSP27 and alphaBC was first identified in the cerebellum of the embryonic day 14 (E14) embryo, and was increased at E18. Double immunofluorescence analysis with myelin-basic protein (MBP) demonstrated that alphaBC positive (+) cells were mature myelinating oligodendrocytes. alphaBC+ cells were observed in the white matter of the E14 cerebellum. At E18, there were a number of alphaBC+ cells in the white matter and a few cells in the granular layer of the gray matter. On the other hand, HSP27+ cells were observed in the white matter and the Purkinje cell layer at E14. At E18, HSP27+ signals were observed in Purkinje cells and neurons of cerebellar nucleus as well as oligodendrocytes in the white matter and the granular layer. The results that HSP27 and alphaBC were expressed in specific neurons and glial cells in the developing cerebellum suggest that HSP27 and alphaBC may be involved in the protective mechanism for the apoptosis of neurons and the physiological stress occurred in oligodendrocyts during cell maturation.

Neurodegeneration-related Aminoacyl-tRNA Synthetases / 生物化学与生物物理进展

Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of their tRNAs for protein biosynthesis. As belong to one of the most ancient and conserved enzyme family their additional functions in mammalian cells were focused recently. Mutations in tyrosyl-tRNA synthetase, glycyl-tRNA synthetase and alanyl-tRNA synthetase from patients and mice models were identified to cause two subtypes of Charcot-Marie-Tooth disease and cerebellar Purkinje cell loss, respectively. These mutations affect different functions of the three enzymes including aminoacylation, editing and unknown functions. These results combined AARSs with neurodegeneration and gave new sights into neuropathy.