Development Of Cerebellar Cortex Research Articles

Constitutive activation of β-Catenin in neural progenitors results in disrupted proliferation and migration of neurons within the central nervous system

Wnt signaling is known to play crucial roles in the development of multiple organs as well as in cancer. In particular, constitutive activation of Wnt/β-Catenin signaling in distinct populations of forebrain or brainstem precursor cells has previously been shown to result in dramatic brain enlargement during embryonic stages of development as well as in the formation of medulloblastoma, a malignant brain tumor in childhood. In order to extend this knowledge to postnatal stages of both cerebral and cerebellar cortex development, we conditionally activated Wnt signaling by introducing a dominant active form of β-catenin in hGFAP-positive neural precursors. Such mutant mice survived up to 21 days postnatally. While the mice revealed enlarged ventricles and an initial expansion of the Pax6-positive ventricular zone, Pax6 expression and proliferative activity in the ventricular zone was virtually lost by embryonic day 16.5. Loss of Pax6 expression was not followed by expression of the subventricular zone marker Tbr2, indicating insufficient neuronal differentiation. In support of this finding, cortical thickness was severely diminished in all analyzed stages from embryonic day 14.5 to postnatal day 12, and appropriate layering was not detectable. Similarly, cerebella of hGFAP-cre::Ctnnb1(ex3)Fl/+ mice were hypoplastic and displayed severe lamination defects. Constitutively active β-Catenin induced inappropriate proliferation of granule neurons and inadequate development of Bergmann glia, thereby preventing regular migration of granule cells and normal cortical layering. We conclude that Wnt signaling has divergent roles in the central nervous system and that Wnt needs to be tightly controlled in a time- and cell type-specific manner.

Expression of IFN-γ, IL-1α, NGF-β and TNF-α during the development of cerebellar cortex of Western Anhui white goose

The strep avidin-biotin-peroxidase complex (SABC) immunohistochemical methods were applied to investigate the localization and semi-quantitative distribution of IFN-γ, IL-1α, NGF-β and TNF-α-immunoreactive cells in the cerebellar cortex of Western Anhui white goose at embryonic day 13, 19, 24, 28 (E13, E19, E24, E28) and postnatal day 7, 15 (P7, P15). The possible roles of IFN-γ,IL-1α,NGF-β and TNF-α in the development of cerebellar cortex were discussed. The results indicated that in the external granular layer, there were IFN-γ and TNF-α positive cells at E13, E19, E24, E28, P7, IL-1α positive cells at E13, E19, E24, E28 and NGF-β positive cells at E13, E19 , E24. The expression levels of these four cytokines all reached peaks at E19 of the six tested periods in this study. In the Purkinje cell layer, there were IFN-γ, IL-1α and TNF-α positive cells at E13, E19, E24, E28, P7, P15 and NGF-β positive cells at E13, E19, E24, E28, P7. In the internal granular layer, there were IFN-γ positive cells at E13, E19, E24, E28, P7, P15, IL-1α and TNF-α positive cells at E13, E19, E24, E28, P7 and NGF-β positive cells at E13, E19, E24, E28. These results showed that E19 might be the "critical stage"in the cerebellar cortex development of Western Anhui white goose. IFN-γ, IL-1α and TNF-α might be synthesized by cerebellar cortex itself, and NGF-β could be transported from regions which project to Purkinje cells. IFN-γ may interfer the transfer of granular cells, and NGF-β may have neurotrophic functions that are beneficial to the growth and development of Purkinje cells.

Radial glial cells and the lamination of the cerebellar cortex

Radial glial cells are stem cells that play an important role in neuronal migration and proliferation in the developing brain. However, how radial glial cells contribute to the lamination of the cerebellar cortex is not well understood. We therefore used immunohistochemistry and BrdU labeling to follow radial glial cell differentiation, cell migration and cerebellar cortex development in mice from embryonic day 8 to postnatal day 180. We report that radial glial cells represent the stem cell population of the neuroepithelium of the neural tube, and act as progenitors for both neurons and neuroglia. In addition, radial glial cells not only give rise to the principal cells of the cerebellar cortex, the Purkinje and granule cells, but they also provide a scaffold for the migration of these cells. We conclude that radial glial cells play a pivotal role in establishing the laminar structure of the cerebellar cortex.

Prenatal blockade of Ang II receptors affects neonatal rat hindbrain structure and receptor localization

The development of knock-out mice for Angiotensin II (Ang II) AT 2 receptors, which exhibited altered exploratory behavior, prompted us to investigate the cerebellum and brainstem. We evaluated the effect of stimulation/inhibition of Ang II receptors on hindbrain development, in offspring (postnatal days P0, P8) of pregnant rats treated during late pregnancy (Ang II, Losartan or PD123319, 1 mg/kg/day). Receptor localization by autoradiography showed in P0 and P8 hindbrains, that most structures expressed AT 2 subtype: cerebellar cortex, cerebellar nuclei, genu facial nucleus, inferior colicullus, inferior olive. In the cerebellar cortex, [ 125I]Ang II AT 2 binding was predominant, while low AT 1 binding was observed in adjacent layers of the cerebellar cortex. Blockade of AT 2 receptors with PD123319 increased binding in cerebellar nuclei ( p < 0.05) and brainstem nuclei at P0, P8, in correlation with increased AT 2 receptor expression by RT-PCR. The enlarged external granular layer (EGL) in PD123319-treated P0 pups contrast with the significant decrease in Ang II binding ( p < 0.001) in the cerebellar cortex. Blockade of AT 2 receptors during late pregnancy seems to arrest cerebellar cortex development in P0 animals. On the contrary, increased AT 2 binding was observed in cerebellar cortex and DTg nucleus in PD123319-treated P8 animals ( p < 0.001). Ang II treatment leads to increased binding in the brainstem. In spite of the low doses of Ang II antagonists used, treatments were performed during a time-frame critical for hindbrain development, leading to remarkable effects. The present study makes a contribution to understand the role of Ang II receptors during hindbrain development.

Temporal release of GABA from anion channel on Bergmann glia (BG) accelerates granule cell precursors (GCPs) proliferation during rat cerebellar cortex development

Temporal release of GABA from anion channel on Bergmann glia (BG) accelerates granule cell precursors (GCPs) proliferation during rat cerebellar cortex development

Effects of an anti-thyroid drug, methimazole, administration to rat dams on the cerebellar cortex development in their pups

In the present study, the effects of methimazole (a major anti-thyroid drug) administration to rat dams on the development of cerebellum of their pups were investigated with morphological, morphometrical and functional procedures. A motor performance in the pups was evaluated by a rota-rod test. Brains removed on 6, 9, 12, 15, 25, and 30 postnatal days were analyzed using the sagittal sections of the cerebellum. Results showed that orally administered methimazole to dams produced a congenital hypothyroid model accompanied with an impaired motor coordination assured by the reduced thyroid hormones. The prominent anomaly was found in the internal granular layer in that there were excess bulges or branching and formation of excess sublobules although the normal lobulation pattern was kept. Three dimensional reconstruction imaging revealed the complex morphological pattern of internal granular layer of the cerebellar hemispheres as well as of the vermis, in which bulges and branches were viewed stereoscopically as the smooth ridges rather than irregular or nodal. In addition, the external granular layer in hypothyroidism survived another several days than that in controls. It is suggested that the complex internal granular layer resulted from the overproduced internal granular cells, which originate in the prolonged external granular layer.

Temporal release of GABA in the external granular layer during rat cerebellar cortex development.

Temporal release of GABA in the external granular layer during rat cerebellar cortex development.

Histological and biological developmental characterization of the human cerebellar cortex

The aim of this study was to investigate the histological and biological features of the human cerebellar cortex development and differentiation. We analyzed 52 brains of fetal and infant death victims, aged from 17 gestational weeks to 12th postnatal month. In particular, in the cerebellar cortex at different ages we evaluated, besides the structural aspects, the expression of several biomarkers implicated in proliferative processes (c-fos, PCNA and apoptosis). We observed morphological patterns progressively evolving every month, from the indefinite structure of the second gestational trimester to the four-layered structure (external granular layer, molecular layer, Purkinje cell layer, internal granular layer) of the late fetal cortex and subsequently to the three-layered postnatal definitive morphology, due to involution of the external granular layer. The evaluation of the biological features of the cerebellar cortex showed high proliferative activity mainly confined to the transient external granular layer in prenatal life, and high apoptotic index after birth. Thus, the histological examination, better with the support of biomarker investigations, allows with accuracy to describe the dynamic sequence of steps that occur in human cerebellar cortex development and to establish in each case the age, namely the pre- or postnatal month of life. Consequently, we can diagnose delayed or altered processes of differentiation during the development of the human cerebellar cortex.

Caytaxin deficiency causes generalized dystonia in rats

The genetically dystonic rat (SD-dt:JFL) is an autosomal recessive model of generalized dystonia. Without cerebellectomy, the dt rat dies prior to Postnatal Day 40. The dt locus was mapped to a 4.2 Mb region on Chr 7q11 and candidate genes were screened with semi-quantitative RT-PCR. Then, Southern blotting and genomic DNA sequencing identified the 3′-long terminal repeat portion of an intracisternal A particle element inserted into Intron 1 of Atcay, the gene which encodes caytaxin. Northern and Western blotting and quantitative real-time RT-PCR defined the Atcay allele in dt rats ( Atcay dt ) as hypomorphic. To establish a framework for functional studies of caytaxin, the developmental expression of rat Atcay transcript was analyzed with Northern blotting, relative quantitative multiplex real-time RT-PCR (QRT-PCR) and in situ hybridization. With a multiple tissue Northern blot, three Atcay transcripts were identified in brain but none were present in heart, spleen, lung, liver, muscle, kidney or testis. With a multiple time-point Northern blot, the same three transcripts were present in cerebellum at Embryonic Day (E15), Postnatal Day 1 (P1), P7, P14, P36 and 8 months. During early development (E15 to P14), the relative proportion of the smallest transcript was increased. QRT-PCR was performed with total RNA from cerebral cortex, striatum, thalamus, hippocampus and cerebellum. Transcript levels peaked at P7 in hippocampus, increased linearly from P1 to P36 in cerebellum, and showed minimal developmental regulation in cerebral cortex. Radioactive in situ hybridization localized Atcay transcript to seemingly all neuronal populations in brain. In cerebellum, Atcay transcript was present in the molecular, Purkinje and granular layers; transcript density in the molecular layer peaked at P14. In the background of previous biochemical, behavioral and electrophysiological studies in the dt rat, our data are compatible with a vital role for caytaxin in the development and neurophysiology of cerebellar cortex.

Expression of Mcl-1 in cerebellar granule neurons is regulated by IGF-I in a developmentally specific fashion

Transgenic (Tg) mice that overexpress IGF-I during postnatal brain development exhibit remarkable cerebellar overgrowth characterized by significant increases in granule cell number that is predominantly due to IGF-I anti-apoptotic actions. Using these mice as a model to define the gene expression profile underlying the pro-survival actions of IGF-I, we screened 243 apoptosis-related genes by cDNA arrays and found that Mcl-1 was down-regulated in cerebella of IGF-I Tg mice. Contrary to the results obtained by cDNA array, Northern blot analyses showed that the Mcl-1 mRNA abundance in the cerebella of IGF-I Tg mice at postnatal day 14 (P14) was five times more than that of wild-type (Wt) controls. The increase in Mcl-1 mRNA expression in IGF-I Tg mice was detected as early as P8, peaked at P14, and remained detectable at P20. Both IGF-I Tg and Wt mice showed a similar expression pattern of Mcl-1 mRNA which coincided with the post-mitotic migration and the post-migratory maturation of granule cells. We measured the relative abundance of Mcl-1 protein in the cerebellum by immunoblots and found that anti-apoptotic Mcl-1 L was the predominant form, while pro-apoptotic Mcl-1 S was minimally detectable. Cerebellar Mcl-1 L was 2.6-fold more abundant in IGF-I Tg mice compared with that in their Wt littermates. Using laser capture microdissection followed by RT-PCR, we determined that Mcl-1 mRNA was expressed in granule cells, but not in Purkinje cells. In summary, these findings show that the anti-apoptotic Mcl-1 isoform is expressed in cerebellar granule neurons, which undergo apoptosis during postnatal cerebellar cortical lamination, and Mcl-1 expression is up-regulated by IGF-I overexpression in a developmentally specific manner. These data suggest that anti-apoptotic Mcl-1 may mediate IGF-I pro-survival actions on granule neurons during the development of cerebellar cortex. They also point out pitfalls of cDNA array analyses.

Molecular morphology of neuronal apoptosis: Analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.

Normal Development and Malformations of the Cerebellar Cortex

The cerebellum is one of the first structures of the central nervous system that begins differentiation and one of the last to reach maturity. Neurogenesis of granular neurons is largely postnatal and cellular organization continues several months after birth. This protracted maturation causes susceptibility to developmental abnormalities. Such abnormalities encompass structural malformations and even cerebellar tumors. Current knowledge on the development of the cerebellar cortex is increasing. Cerebellar cortex development includes proliferation, migration, neuronal differentiation, guided axonal growth, neuronal map formation and synaptogenesis. These steps follow different timing and modalities compared to neocortex. The high resolution Magnetic Resonance (MR) improved the in vivo depiction of the cerebellar cortex and its developmental malformations. Correlation between neuroimaging and clinical findings as well as neurogenetic allowed practical classification of cerebellar malformations. The proposed morphology-based classifications appear incomplete and need continuous revision. In this review, we have followed a neuroradiological schema in order to reconcile previous classifications. Sviluppo normale e malformazioni della corteccia cerebellare

Effects of Early Hippocampal Lesions on Trace, Delay, and Long-Delay Eyeblink Conditioning in Developing Rats

The effects of bilateral hippocampal aspiration lesions on later acquisition of eyeblink conditioning were examined in developing Long-Evans rat pups. Lesions on postnatal day (PND) 10 were followed by evaluation of trace eyeblink conditioning (Experiment 1) and delay eyeblink conditioning (Experiment 2) on PND 25. Pairings of a tone conditioned stimulus (CS) and periocular shock unconditioned stimulus (US, 100 ms) were presented in one of three conditioning paradigms: trace (380 ms CS, 500 ms trace interval, 880 ms interstimulus interval [ISI]), standard delay (380 ms CS, 280 ms ISI), or long delay (980 ms CS, 880 ms ISI). The results of two experiments indicated that hippocampal lesions impaired trace eyeblink conditioning more than either type of delay conditioning. In light of our previous work on the ontogeny of trace, delay, and long-delay eyeblink conditioning (Ivkovich, Paczkowski, & Stanton, 2000) showing that trace and long-delay eyeblink conditioning had similar ontogenetic profiles, the current data suggest that during ontogeny hippocampal maturation may be more important for the short-term memory component than for the long-ISI component of trace eyeblink conditioning. The late development of conditioning over long ISIs may depend on a separate process such as protracted development of cerebellar cortex.

Involvement of pontocerebellar mossy fibers in the development of cerebellar cortex revealed by analyzing weaver mutant mouse

Involvement of pontocerebellar mossy fibers in the development of cerebellar cortex revealed by analyzing weaver mutant mouse

Apoptosis of undifferentiated progenitors and granule cell precursors in the postnatal human cerebellar cortex correlates with expression of BCL‐2, ICE, and CPP32 proteins

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621-632; Krueger et al. [1995] J. Neurosci. 15:3366-3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing 31-35% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5-7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and interleukin-1beta-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for alpha-internexin or type-III beta tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates.

Apoptosis of undifferentiated progenitors and granule cell precursors in the postnatal human cerebellar cortex correlates with expression of BCL‐2, ICE, and CPP32 proteins

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621–632; Krueger et al. [1995] J. Neurosci. 15:3366–3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing 31–35% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5–7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and interleukin-1β-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for α-internexin or type-III β tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates. J. Comp. Neurol. 399:359–372, 1998. © 1998 Wiley-Liss, Inc.

Morphological changes of cerebellar granule cells — Mossy fibers during development of cerebellar cortex

Morphological changes of cerebellar granule cells — Mossy fibers during development of cerebellar cortex

330 Morphological changes of cerebellar granule cells — Mossy fibers during development of cerebellar cortex

330 Morphological changes of cerebellar granule cells — Mossy fibers during development of cerebellar cortex

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo.

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.

Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex: Comparison to activity staining

Distribution patterns of activity and immunohistochemical staining for glutamate dehydrogenase were compared during the postnatal development of rat hippocampal formation and cerebellar cortex. On postnatal day 5, dendritic layers of the hippocampal formation showed only a very weak enzyme activity. Similarly, when studied at the same age, the external granule cell layer and Purkinje cells of the cerebellar cortex exhibited a very faint and moderate staining, respectively. With advancing age, in both brain regions a marked postnatal increase in glutamate dehydrogenase activity occurred in neuropil area as glutamatergic structures matured. However, compared to activity staining, both brain regions of early postnatal stages showed a relatively high level of glutamate dehydrogenase-like immunoreactivity. In this case, the immunohistochemical staining of hippocampal dendritic layers and of the molecular layer of the cerebellar cortex was rather diffuse, being not very similar to parameters of the maturation of the respective glutamatergic structures. In contrast to the activity staining for the enzyme, the immunohistochemical labelling in adult rats revealed a selective predominance of immunoreactivity in astroglial cells from postnatal day 5 onwards. The Bergmann glia in the cerebellar cortex exhibited the strongest intensity of immunoreactivity. Generally, the patterns of immunoreactivity were found to depend on the fixation procedure adopted. Concluding from our results, glutamate dehydrogenase is demonstrable in glial and in neuronal cell elements as well. Therefore, it is recommended that activity staining and the immunohistochemical procedure be combined to study qualitative and quantitative aspects of glutamate dehydrogenase in nervous tissues.