Progressive restriction of cell fates in relation to neuroepithelial cell mingling in the mouse cerebellum

Neurogenesis in the cerebellum proceeds through a temporal series of cell production from two separate epithelia, the ventricular zone (VZ) and the external granule cell layer (EGL). Using the laacZ cell lineage tracer in transgenic mice, we describe cellular clones whose dates of birth span the entire period of cerebellar development and deduce a sequence of cell dispersion leading to the final allocation of cells in the cerebellum. Clones probably labeled early during neural tube formation show that individual progenitors can give rise to all cerebellar cell types. The distribution of clonally related granule cells in these clones indicates a mediolateral organization of EGL progenitors already established before the allocation of the EGL progenitors to the cerebellum. Clones restricted to the cerebellar VZ show that the VZ derives progenitors for deep nuclei and multipotent cortical progenitors, which lose their systematic lineage relationship when longitudinal cell intermingling in the cerebellar VZ becomes more limited. The small clones also show that cell dispersion is radial in the internal granule layer and tangential in the molecular layer. Together, the data demonstrate the broad maintenance of the relative order of cells from neural tube stages to the adult cerebellum.     

Generation of cerebellar neuron precursors from embryonic stem cells

Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.     

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N- CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.     

小熊猫小脑皮层的组织结构及RT-97、KGF和Bax蛋白在小脑皮层的表达

为了解小熊猫(Ailurus fulgens)小脑皮层的结构特征,观察神经丝蛋白抗体RT-97、角质细胞生长因子(KGF)及Bax蛋白在小脑皮层中的表达,利用组织学方法和免疫组织化学方法观察了小熊猫小脑皮层的显微结构,检测了RT-97、KGF和Bax蛋白的表达.结果表明,小脑皮层从外向内依次可分为分子层、Purkinje细胞层、颗粒层3层.RT-97在小熊猫小脑皮层Purkinje细胞层、颗粒层中神经细胞的轴突、分子层中颗粒细胞的轴突及小脑髓质中有阳性表达;KGF在小脑皮层分子层、Purkinje细胞层和颗粒细胞层及髓质中均有阳性表达;Bax蛋白在小脑皮层分子层、Purkinje细胞层和颗粒细胞层中有阳性表达.RT-97、KGF和Bax蛋白在小脑皮层神经结构的构筑中可能发挥着不同的功能.     

Relocalization of a microtubule-anchoring protein,ninein, from the centrosome to dendrites during differentiation of mouse neurons

Microtubules in typical cells form radial arrays with their plus-ends pointing toward the cell periphery. In contrast, microtubules in dendrites of neurons are free from centrosomes and have a unique arrangement in which about half have a polarity with a minus-end distal orientation. Mechanisms for generation and maintenance of the microtubule arrangement in dendrites are not well understood. Here, we examined dendritic localization of a centrosomal protein, ninein, which has microtubule-anchoring and stabilizing functions. Immunohistochemical analysis of developing mouse cerebral and cerebellar cortices showed that ninein is localized at the centrosome in undifferentiated neural precursors. In contrast, ninein was barely detected in migrating neurons, such as those in the intermediate layer of the cerebral cortex and the internal granular layer of the cerebellar cortex. High expression was observed in thick dendrite-bearing neurons such as pyramidal neurons of the cerebral cortex and Purkinje neurons in the cerebellar cortex. Ninein was not detected at the centrosome of these cells, but was diffusely present in cell soma and dendrites. In cultured cortical neurons, ninein formed granular structures in soma and dendrites, being not associated with γ-tubulin. About 60% of these structures showed resistance to detergent and association with microtubules. Our observations suggest that the minus-ends of microtubules may be anchored and stabilized by centrosomal proteins localized in dendrites.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers

We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-Ng-CAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers.     

Acute neonatal glucocorticoid exposure produces selective and rapid cerebellar neural progenitor cell apoptotic death

There has been a growing controversy regarding the continued use of glucocorticoid therapy to treat respiratory dysfunction associated with prematurity, as mounting clinical evidence has shown neonatal exposure produces permanent neuromotor and cognitive deficits. Here we report that, during a selective neonatal window of vulnerability, a single glucocorticoid injection in the mouse produces rapid and selective apoptotic cell death of the proliferating neural progenitor cells in the cerebellar external granule layer and permanent reductions in neuronal cell counts of their progeny, the cerebellar internal granule layer neurons. Our estimates suggest that this mouse window of vulnerability would correspond in the human to a period extending from approximately 20 weeks gestation to 6.5 weeks after birth. This death pathway is critically regulated by the proapoptotic Bcl-2 family member Puma and is independent of p53 expression. These rodent data indicate that there exists a previously unknown window of vulnerability during which a single glucocorticoid exposure at clinically relevant doses can produce neural progenitor cell apoptosis and permanent cerebellar pathology that may be responsible for some of the iatrogenically induced neurodevelopmental abnormalities seen in children exposed to this drug. This vulnerability may be related to the physiological role of glucocorticoids in regulating programmed cell death in the mammalian cerebellum.     

Mapping the development of cerebellar Purkinje cells in zebrafish

The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1174–1188, 2015     

Cerebellum- and forebrain-derived stem cells possess intrinsic regional character

The existence of stem cells in the adult nervous system is well recognized; however, the potential of these cells is still widely debated. We demonstrate that neural stem cells exist within the embryonic and adult cerebellum. Comparing the potential of neural stem cells derived from the forebrain and cerebellum, we find that progeny derived from each of these brain regions retain regional character in vitro as well as after homotopic transplantation. However, when ectopically transplanted, neurosphere-derived cells from either region are largely unable to generate neurons. With regard specifically to embryonic and adult cerebellar stem cells, we observe that they are able to give rise to neurons that resemble different select classes of cerebellar subclasses when grafted into the perinatal host cerebellum. Most notably, upon transplantation to the perinatal cerebellum, cerebellar stem cells from all ages are able to acquire the position and mature electrophysiological properties of cerebellar granule cells.     

Contribution of the neural cell recognition molecule NB‐3 to synapse formation between parallel fibers and Purkinje cells in mouse

The neural cell recognition molecule NB‐3, also referred to as contactin‐6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB‐3 to cerebellar development, focusing on lobule 3 where NB‐3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG‐1, L1, and NB‐3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB‐3‐immunoreactive zone did not overlap with TAG‐1‐immunoreactive zone. By contrast, the L1‐immunoreactive zone overlapped with both the TAG‐1‐ and NB‐3‐immunoreactive zones. NB‐3‐positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB‐3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB‐3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB‐3 knockout mice relative to wild‐type mice at P5 to P10. In parallel with these findings, caspase‐dependent cell death was significantly increased in the NB‐ 3‐deficient cerebellum at P15. Collectively, our results indicate that NB‐3 deficiency affects synapse formation during postnatal cerebellar development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Murine numb regulates granule cell maturation in the cerebellum

Notch is a key regulator of vertebrate neurogenesis and the cytoplasmic adaptor protein Numb is a modulator of the Notch signaling pathway. To address the role of murine Numb in development of the central nervous system, we used a conditional gene ablation approach. We show that Numb is involved in the maturation of cerebellar granule cells. Although the specification of neural cell fates in the cerebellum is not affected in the absence of Numb, the transition from a mitotic progenitor to a mature granule cell is aberrant and migration of postmitotic granule cells to the internal granule cell layer is delayed. In some animals, this results in a complete agenesis of granule cells and a strong ataxia. We confirmed these findings in vitro and found that Numb-deficient cerebellar progenitor cells show a marked delay in granule cell maturation. Our results suggest that Numb plays a role in the transition of a mitotic progenitor to a fully differentiated granule cell in the cerebellum. In addition, the maturation of Purkinje cells is also delayed in Numb-deficient mice.     

人体小脑神经元发育的定量观察

目的:研究人体小脑神经元的发育过程。方法:应用体视学方法,对18例不同时期人体小脑组织Golgi染色后进行观察,观测小脑皮质分层出现的时间,观测并计算神经元的数密度、体密度和表面积密度。结果:6月龄时,小脑皮质出现较明显的分子层、蒲肯野细胞层和颗粒层;星形细胞、篮状细胞、蒲肯野细胞、颗粒细胞和高尔基细胞的的数密度随月龄/年龄的增长而减少,体密度和表面积密度随月龄/年龄的增长而增加,但这些减小和增大是不等速的,6-8月龄变化最明显。结论:人体小脑神经元的发育呈现快慢交替、不均速发展,6～8月是小脑神经元发育的重要时期。     

A neural network model of the cerebellar cortex performing dynamic associations

The present paper proposes a model which applies formal neural network modeling techniques to construct a theoretical representation of the cerebellar cortex and its performances in motor control. A schema that makes explicit use of propagation delays of neural signals, is introduced to describe the ability to store temporal sequences of patterns in the Golgi-granule cell system. A perceptron association is then performed on these sequences of patterns by the Purkinje cell layer. The model conforms with important biological constraints, such as the known excitatory or inhibitory nature of the various synapses. Also, as suggested by experimental evidence, the synaptic plasticity underlying the learning ability of the model, is confined to the parallel fiber — Purkinje cell synapses, and takes place under the control of the climbing fibers. The result is a neural network model, constructed according to the anatomy of the cerebellar cortex, and capable of learning and retrieval of temporal sequences of patterns. It provides a framework to represent and interpret properties of learning and control of movements by the cerebellum, and to assess the capacity of formal neural network techniques for modeling of real neural systems.     

Modulation of the dynamics of cerebellar Purkinje cells through the interaction of excitatory and inhibitory feedforward pathways

The dynamics of cerebellar neuronal networks is controlled by the underlying building blocks of neurons and synapses between them. For which, the computation of Purkinje cells (PCs), the only output cells of the cerebellar cortex, is implemented through various types of neural pathways interactively routing excitation and inhibition converged to PCs. Such tuning of excitation and inhibition, coming from the gating of specific pathways as well as short-term plasticity (STP) of the synapses, plays a dominant role in controlling the PC dynamics in terms of firing rate and spike timing. PCs receive cascade feedforward inputs from two major neural pathways: the first one is the feedforward excitatory pathway from granule cells (GCs) to PCs; the second one is the feedforward inhibition pathway from GCs, via molecular layer interneurons (MLIs), to PCs. The GC-PC pathway, together with short-term dynamics of excitatory synapses, has been a focus over past decades, whereas recent experimental evidence shows that MLIs also greatly contribute to controlling PC activity. Therefore, it is expected that the diversity of excitation gated by STP of GC-PC synapses, modulated by strong inhibition from MLI-PC synapses, can promote the computation performed by PCs. However, it remains unclear how these two neural pathways are interacted to modulate PC dynamics. Here using a computational model of PC network installed with these two neural pathways, we addressed this question to investigate the change of PC firing dynamics at the level of single cell and network. We show that the nonlinear characteristics of excitatory STP dynamics can significantly modulate PC spiking dynamics mediated by inhibition. The changes in PC firing rate, firing phase, and temporal spike pattern, are strongly modulated by these two factors in different ways. MLIs mainly contribute to variable delays in the postsynaptic action potentials of PCs while modulated by excitation STP. Notably, the diversity of synchronization and pause response in the PC network is governed not only by the balance of excitation and inhibition, but also by the synaptic STP, depending on input burst patterns. Especially, the pause response shown in the PC network can only emerge with the interaction of both pathways. Together with other recent findings, our results show that the interaction of feedforward pathways of excitation and inhibition, incorporated with synaptic short-term dynamics, can dramatically regulate the PC activities that consequently change the network dynamics of the cerebellar circuit.     

Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been found in the cerebellum of many vertebrates and in the gastrointestinal tract of African ostrich chicks, but little is known about its distribution in the cerebellum of the African ostrich. In the present study, the distribution and morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich were investigated using immunohistochemistry. The results indicate that the cerebellum is divided into two sections: the outer cerebellar cortex and the inner medulla of cerebellum. The cerebellar cortex comprises a molecular layer, a Purkinje cell layer and a granular layer; ghrelin-immunopositive (ghrelin-ip) cells were localized throughout the entire cerebellum, but sparsely in the medulla. The greatest number of ghrelin-ip cells was found in the stratum granulosum, and the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex. The ghrelin-ip cells were fusiform or irregular polygons and their cytoplasm was stained intensely. These results clearly demonstrate the presence of ghrelin-ip cells in the cerebellum of the African ostrich. It is speculated that ghrelin may have a physiological function in the cerebellum.     

Adult stem cells in the knifefish cerebellum

Adult neurogenesis has been described in dozens of brain regions in teleost fish, with the largest number of new neurons being generated in the cerebellum. Here, we characterized the cerebellar neural stem/progenitor cells (NSPCs) in the brown ghost knifefish (Apteronotus leptorhynchus), an established model system of adult neurogenesis. The majority of the new cerebellar cells arise from neurogenic niches located medially, at the interface of the dorsal/ventral molecular layers and the granular layer. NSPCs within these niches give rise to transit‐amplifying progenitors which populate the molecular layer, where they continue to proliferate during their migration toward target areas in the granular layer. At any given time, the majority of proliferating cells are located in the molecular layer. Immunohistochemical staining revealed that the stem cell markers Sox2, Meis1/2/3, Islet1, and, to a lesser extent, Pax6, are widely expressed in all regions of the adult cerebellum. A large subpopulation of these NSPCs coexpress S100, GFAP, and/or vimentin, indicating astrocytic identity. This is further supported by the specific effect of the gliotoxin l ‐methionine sulfoximine, which leads to a targeted decrease in the number of GFAP+ cells that coexpress Sox2 or the proliferation marker PCNA. Pulse‐chase analysis of the label size associated with new cells after administration of 5‐bromo‐2′‐deoxyuridine demonstrated that, on average, two additional cell divisions occur after completion of the initial mitotic cycle. Overall numbers of NSPCs in the cerebellum niches increase consistently over time, presumably in parallel with the continuous growth of the brain. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 39–65, 2015     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.