Immunohistochemical characterization of the orphan nuclear receptor ROR alpha in the mouse nervous system.

ROR alpha is an orphan nuclear receptor. A deletion mutation in the ROR alpha gene leads to severe cerebellar defects, known as the staggerer mutant mouse. Although previous in situ hybridization (ISH) studies have shown that ROR alpha is highly expressed in the cerebellum, especially in Purkinje cells, and in the thalamus, sufficient immunohistochemical (IHC) study has not yet been presented. I demonstrate here the IHC analysis of ROR alpha using a specific anti-ROR alpha antibody, in adult and developing mouse nervous system. ROR alpha immunoreactivity was observed in the Purkinje cell and molecular layers of the cerebellum. The co-localization of ROR alpha with calbindin D(28K) (CaBP) and parvalbumin indicates that ROR alpha-positive cells were Purkinje cells, stellate cells, and basket cells. In addition to the cerebellum, strong to medium ROR alpha immunoreactivity was found in the thalamus, cerebral cortex (mainly in the layer IV), dorsal cochlear nucleus (DCN), suprachiasmatic nucleus (SCN), superior colliculus, spinal trigeminal nucleus, and retina. The immunostaining was restricted in nuclei of neurons. Developmentally, ROR alpha immunoreactivity was observed in the cerebellum and thalamus from embryonal day 16 (E16). The distribution of ROR alpha immunoreactivity and ROR alpha mRNA hybridization signal was almost coincident. However, the intensity of hybridization signal was not always parallel to that of immunoreactivity.     

Ectopic parvalbumin-positive cells in the cerebellum of the adult mutant mouse 'nervous'

The basket and stellate cells of the 'nervous' mouse cerebellum lose 50% of their presynaptic parallel fibres and more than 90% of Purkinje cells, their major postsynaptic targets, after postnatal day 23. To study the fate of these molecular layer cells we examined the cerebellum of the mutant mouse 'nervous' with antisera against the 'marker protein' parvalbumin, which exclusively tags Purkinje, basket, and stellate cells. Ten homozygous 'nervous' mice, 12-14 weeks old, and 30 control animals of several inbred strains were examined. The number of basket and stellate cells decreased in affected areas of the molecular layer. In addition, parvalbumin-positive cells were detected in the granular layer, in the white matter of the cerebellum and in the area of the cerebellar peduncles of all 'nervous' mice, but not in those of the control animals. These cells mainly occurred in areas which still displayed degenerating Purkinje cell axons. Ectopic parvalbumin-positive cells could either represent a class of interneurons, which have changed their antigenic properties, and therefore happen to stain with antisera against parvalbumin, or stratum moleculare cells which have failed to recognise their proper position during ontogenesis. Alternatively these cells could represent postdevelopmental basket cells, which have acquired the ability to migrate.     

Cerebellar histogenesis is disturbed in mice lacking cyclin D2

Formation of brain requires deftly balancing primary genesis of neurons and glia, detection of when sufficient cells of each type have been produced, shutdown of proliferation and removal of excess cells. The region and cell type-specific expression of cell cycle regulatory proteins, such as demonstrated for cyclin D2, may contribute to these processes. If so, regional brain development should be affected by alteration of cyclin expression. To test this hypothesis, the representation of specific cell types was examined in the cerebellum of animals lacking cyclin D2. The loss of this cyclin primarily affected two neuronal populations: granule cell number was reduced and stellate interneurons were nearly absent. Differences between null and wild-type siblings were obvious by the second postnatal week. Decreases in granule cell number arose from both reduction in primary neurogenesis and increase in apoptosis of cells that fail to differentiate. The dearth of stellate cells in the molecular layer indicates that emergence of this subpopulation requires cyclin D2 expression. Surprisingly, Golgi and basket interneurons, thought to originate from the same precursor pool as stellate cells, appear unaffected. These results suggest that cyclin D2 is required in cerebellum not only for proliferation of the granule cell precursors but also for proper differentiation of granule and stellate interneurons.     

Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish.

Members of different vertebrate species share a number of developmental mechanisms and control genes, suggesting that they have similar genetic programs of development. We compared the expression patterns of the Pax-2 protein in Mus musculus and Brachydanio rerio to gain a better understanding of the evolution of developmental control genes. We found that the tissue specificity and the time course of Pax-2 expression relative to specific developmental processes are remarkably similar during the early development of the two organisms. The brain, the optic stalk, the auditory vesicle, the pronephros, and single cells in the spinal cord and the hindbrain express Pax-2 in both species. The Pax-2 expression domain in the prospective brain of E8 mouse embryos has not been described previously. Expression appears first during early neurulation at the junction between the midbrain and hindbrain. However, there are some differences in Pax-2 expression between the two species. Most notable, expression at the midbrain/hindbrain boundary is no longer detectable after E11 in the mouse. Using monoclonal antibodies, we could exclude that primary neurons express Pax-2 in the zebrafish spinal cord. Our results confirm that Pax genes are highly conserved both in sequences and in expression patterns, indicating that they may have a function during early development that has been conserved during vertebrate evolution.     

13-cis-retinoic acid causes patterning defects in the early embryonic rostral hindbrain and abnormal development of the cerebellum in the macaque

BACKGROUND: We have previously reported that exposure of embryos to 13-cis-retinoic acid (cRA) results in an abnormal phenotype of the fetal cerebellum. In this study, we analyzed early changes in the cerebellar anlagen (midbrain-hindbrain junction) as well as lesions of the fetal cerebellar vermis after a teratogenic dosing regimen of cRA in the macaque model. METHODS: We examined embryo coronal sections of the midbrain-hindbrain junction immunostained for Pax-2, Engrailed-2 (En-2) and Krox-20. To characterize the cerebellum foliation and fissure formation processes, we analyzed vermal cortical cell layer development and the number and depth of the major fissures on sagittal sections of fetal vermis. We also examined Purkinje cell development in vermal sections immunostained for CD3. RESULTS: Compared with controls, there was a consistent truncation of the midbrain-hindbrain region of early embryos exposed to cRA. The cRA-induced fetal vermis lesions included inhibition in its anteroposterior growth, altered folial patterning, a general loss of prominence of the fissures accompanied by a total loss of sublobular fissures, and changes in cortical cell layer development. CD3(+) Purkinje cells were abnormally dispersed deep into the molecular layer in the vermis. CONCLUSIONS: Our findings indicate that the effects of cRA on the developing cerebellum involve interference with the hierarchy of complex cellular and genetic interactions that lead to the growth and subdivision of the cerebellum into smaller units. The regional vermal defects may be related to early postnatal functional deficits.     

Widespread contribution of Gdf7 lineage to cerebellar cell types and implications for hedgehog-driven medulloblastoma formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.     

The isthmic neuroepithelium is essential for cerebellar midline fusion

The cerebellum comprises a medial domain, called the vermis, flanked by two lateral subdivisions, the cerebellar hemispheres. Normal development of the vermis involves fusion of two lateral primordia on the dorsal midline. We investigated how the cerebellum fuses on the midline by combining a study of mid/hindbrain cell movements in avian embryos with the analysis of cerebellar fusion in normal and mutant mouse embryos. We found that, in avian embryos, divergent cell movements originating from a restricted medial domain located at the mid/hindbrain boundary produce the roof plate of the mid/hindbrain domain. Cells migrating anteriorly from this region populate the caudal midbrain roof plate whereas cells migrating posteriorly populate the cerebellar roof plate. In addition, the adjacent paramedial isthmic neuroepithelium also migrates caudalward and participates in the formation of the cerebellar midline region. We also found that the paramedial isthmic territory produces two distinct structures. First, the late developing velum medullaris that intervenes between the vermis and the midbrain, and second, a midline domain upon which the cerebellum fuses. Elimination or overgrowth of this isthmic domain in Wnt1(sw/sw) and En1(+/Otx2lacZ) mutant mice, respectively, impair cerebellar midline fusion. Because the isthmus-derived midline cerebellar domain displays a distinct expression pattern of genes involved in BMP signaling, we propose that the isthmus-derived cells provide both a substratum and signals that are essential for cerebellar fusion.     

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal X-irradiation.

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilpateral forelimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 +/- 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal x-irradiation

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilateral forlimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 ± 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Functional origins of the vertebrate cerebellum from a sensory processing antecedent

The structure of the cerebellar cortex is remarkably similar across vertebrate phylogeny. It is well developed in basaljawed fishes, such as sharks and rays with many of the same cell types and organizational features found in other vertebrategroups, including mammals. In particular, the lattice-like organization of cerebellar cortex (with a molecular layer of parallel fibres,interneurons, spiny Purkinje cell dendrites, and climbing fires) is a common defining characteristic. In addition to the cerebell...     

Tinnitus,Unipolar Brush Cells,and Cerebellar Glutamatergic Function in an Animal Model

Unipolar brush cells (UBCs) are excitatory interneurons found in the dorsal cochlear nucleus (DCN) and the granule cell layer of cerebellar cortex, being particularly evident in the paraflocculus (PFL) and flocculus (FL). UBCs receive glutamatergic inputs and make glutamatergic synapses with granule cells and other UBCs. It has been hypothesized that UBCs comprise local networks of tunable feed-forward amplifiers. In the DCN they might also participate in feed-back amplification of signals from higher auditory centers. Recently it has been shown that UBCs, in the vestibulocerebellum and DCN of adult rats, express doublecortin (DCX), previously considered a marker of newborn and migrating neurons. In an animal model, both the DCN, and more recently the PFL, have been implicated in contributing to the sensation of acoustic-exposure-induced tinnitus. These studies support the working hypothesis that tinnitus emerges after loss of peripheral sensitivity because inhibitory processes homeostatically down regulate, and excitatory processes up regulate. Here we report the results of two sequential experiments that examine the potential role of DCN and cerebellar UBCs in tinnitus, and the contribution of glutamatergic transmission in the PFL. In Experiment 1 it was shown that adult rats with psychophysical evidence of tinnitus induced by a single unilateral high-level noise exposure, had elevated DCX in the DCN and ventral PFL. In Experiment 2 it was shown that micro-quantities of glutamatergic antagonists, delivered directly to the PFL, reversibly reduced chronically established tinnitus, while similarly applied glutamatergic agonists induced tinnitus-like behavior in non-tinnitus controls. These results are consistent with the hypothesis that UBC up regulation and enhanced glutamatergic transmission in the cerebellum contribute to the pathophysiology of tinnitus.     

Quantitative Organization of GABAergic Synapses in the Molecular Layer of the Mouse Cerebellar Cortex

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABA_A receptor α1 subunit (GABA_ARα1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the α1 subunit resulted in a complete loss of GABA_AR aggregates from Purkinje cells, allowing us to determine the density of GABA_AR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using α-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.     

RBP-J promotes the maturation of neuronal progenitors

During brain development, neurons and glias are generated from neural stem cells and more limited intermediate neural progenitors (INPs). Numerous studies have revealed the mechanisms of development of neural stem cells. However, the signaling pathways that govern the development of INPs are largely unknown. The cerebellum is suitable for examining this issue because cerebellar cortical inhibitory neurons such as basket and stellate cells are derived from small Pax2⁺ interneuronal progenitors. Here, we show that Sox2⁻/Pax2⁺ and Sox2⁺/Pax2⁻ progenitors, 2 types of interneuronal progenitors of basket and stellate cells, exist in the cerebellar white matter (WM) and that the former arise from the latter during the first postnatal week. Moreover, RBP-J promotes the neurogenesis of stellate and basket cells by converting Sox2⁺/Pax2⁻ interneuronal progenitors to more mature Sox2⁻/Pax2⁺ interneuronal progenitors. This study shows a novel RBP-J function that promotes INP differentiation.     

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。     

Cell type-specific subunit composition of G protein-gated potassium channels in the cerebellum

G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels regulate cellular excitability and neurotransmission. In this study, we used biochemical and morphological techniques to analyze the cellular and subcellular distributions of GIRK channel subunits, as well as their interactions, in the mouse cerebellum. We found that GIRK1, GIRK2, and GIRK3 subunits co-precipitated with one another in the cerebellum and that GIRK subunit ablation was correlated with reduced expression levels of residual subunits. Using quantitative RT-PCR and immunohistochemical approaches, we found that GIRK subunits exhibit overlapping but distinct expression patterns in various cerebellar neuron subtypes. GIRK1 and GIRK2 exhibited the most widespread and robust labeling in the cerebellum, with labeling particularly prominent in granule cells. A high degree of molecular diversity in the cerebellar GIRK channel repertoire is suggested by labeling seen in less abundant neuron populations, including Purkinje neurons (GIRK1/GIRK2/GIRK3), basket cells (GIRK1/GIRK3), Golgi cells (GIRK2/GIRK4), stellate cells (GIRK3), and unipolar brush cells (GIRK2/GIRK3). Double-labeling immunofluorescence and electron microscopies showed that GIRK subunits were mainly found at post-synaptic sites. Altogether, our data support the existence of rich GIRK molecular and cellular diversity, and provide a necessary framework for functional studies aimed at delineating the contribution of GIRK channels to synaptic inhibition in the cerebellum.     

Postnatal expression of Doublecortin (Dcx) in the developing cerebellar cortex of mouse

We have investigated the expression of Doublecortin (Dcx) protein in the developing cerebellum of mouse from postnatal 2nd day to postnatal 22nd day and in young adults by immunohistochemistry. Strong expression of Dcx was present in the inner zone of the external granule cell layer, and remained strong while postmitotic granule cell precursors were present in this transitory layer. Descending granule cell precursors exhibited Dcx immunostaining not only while migrating but for a short time also after their settlement. Dcx-immunostained cells appeared in deep cerebellocortical territories and in the cerebellar white matter during the first postnatal week. These bipolar cells were arranged in the sagittal plane and built up transitory migratory streams during the second postnatal week and their number gradually decreased during the third postnatal week. Upward migration of bipolar cells was observed while leaving the migratory streams, penetrating the internal granule cell layer and the molecular layer. These cells were considered as precursors of late migrating molecular layer interneurons. However, a proportion of Dcx-immunostained cells underwent a bipolar-to-multipolar dendritic remodellation and - on the basis of strong morphological similarities - was taken for "multipotent progenitor cells", described recently in the neocortex of adult rat.     

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO₂. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.     

Four distinct phases of basket/stellate cell migration after entering their final destination (the molecular layer) in the developing cerebellum

In the adult cerebellum, basket/stellate cells are scattered throughout the ML, but little is known about the process underlying the cell dispersion. To determine the allocation of stellate/basket cells within the ML, we examined their migration in the early postnatal mouse cerebellum. We found that after entering the ML, basket/stellate cells sequentially exhibit four distinct phases of migration. First, the cells migrated radially from the bottom to the top while exhibiting saltatory movement with a single leading process (Phase I). Second, the cells turned at the top and migrated tangentially in a rostro-caudal direction, with an occasional reversal of the direction of migration (Phase II). Third, the cells turned and migrated radially within the ML at a significantly reduced speed while repeatedly extending and withdrawing the leading processes (Phase III). Fourth, the cells turned at the middle and migrated tangentially at their slowest speed, while extending several dendrite-like processes after having completely withdrawn the leading process (Phase IV). Finally, the cells stopped and completed their migration. These results suggest that the dispersion of basket/stellate cells in the ML is controlled by the orchestrated activity of external guidance cues, cell-cell contact and intrinsic programs in a position- and time-dependent manner.