Impaired Structural and Functional Development of Cerebellum Following Gestational Exposure of Deltamethrin in Rats: Role of Reelin

Reelin is an extracellular matrix molecule that is involved in the normal development of the cerebellar lamination, Bergmann glial fibres alignment, Purkinje cell monolayer arrangement and granule cell migration. In this study, we have examined the effects of maternal exposure of deltamethrin (DLT), a type II pyrethroid insecticide, on the structural and functional development of rat cerebellum during postnatal life. DLT (0.75 mg/kg body weight, intraperitoneally dissolved in dimethylsulphoxide) was administered in timed pregnant rats during two different gestational time periods, i.e. gestational days of 7–10 and 11–14, respectively. In DLT exposed rats, a significant overexpression of reelin was observed in the cells of the external granule cell layer (EGL) and internal granule cell layer along with an ectopic expression of reelin in the EGL as well as in the migrating granule cells just below the EGL, revealing an arrest of granule cell migration in this zone. Mis-orientation and hypertrophy of the Bergmann glial fibres further hampered the journey of the granule cells to their final destination. Possibly reelin overexpression also caused misalignment of the Purkinje cells and inhibited the neurite growth leading to a significant decrease in the spine density, main dendritic length and width of the dendritic arbour. Thus, it is proposed that the DLT exerts its neurotoxic effects possibly via the intracellular accumulation and low release of reelin leading to an impaired granule cell and Purkinje cell migration, inhibition of neurite outgrowth and reduced spine density. Such impaired cerebellar development leads to motor coordination deficits.     

Cellular and molecular mechanisms of cerebellar granule cell migration

The real-time observation of cell movement in brain slice preparations reveals that in the developing brain, postmitotic neurons alter their shape concomitantly with changes in the mode, direction, tempo, and rate of migration as they traverse different cortical layers. Although it has been hypothesized that orchestrated activities of multiple external cues and cell-cell contact are essential for controlling the cortical-layer-specific changes in cell migration, signaling mechanisms and external guidance cues related to the alteration of neuronal cell migration remain to be determined. In this article, we will first review recent studies on position-specific changes in granule cell behavior through different migratory terrains of the developing cerebellar cortex. We will then present possible roles for the coordinated activity of Ca2+ channels, NMDA type of glutamate receptors, and intracellular Ca2+ fluctuations in controlling cerebellar granule cell movement. Furthermore, we will discuss the crucial roles of brain-derived neurotrophic factor (BDNF), neuregulin (NRG), stromal cell-derived factor 1alpha (SDF-1alpha), ephrin-B2, and EphB2 receptor in providing directional cues promoting granule cell migration from the external granular layer (EGL) to the internal granular layer (IGL). Finally, we will demonstrate that endogenous somatostatin controls the migration of granule cells in a cortical layer-specific manner: Endogenous somatostatin accelerates granule cell movement near the birthplace within the EGL, but significantly slows down the movement near their final destination within the IGL.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.     

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti‐CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive‐like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 463–484, 2015     

Cellular and molecular mechanisms of cerebellar granule cell migration

The real-time observation of cell movement in brain slice preparations reveals that in the developing brain, postmitotic neurons alter their shape concomitantly with changes in the mode, direction, tempo, and rate of migration as they traverse different cortical layers. Although it has been hypothesized that orchestrated activities of multiple external cues and cell-cell contact are essential for controlling the cortical-layer-specific changes in cell migration, signaling mechanisms and external guidance cues related to the alteration of neuronal cell migration remain to be determined. In this article, we will first review recent studies on position-specific changes in granule cell behavior through different migratory terrains of the developing cerebellar cortex. We will then present possible roles for the coordinated activity of Ca²⁺ channels, NMDA type of glutamate receptors, and intracellular Ca²⁺ fluctuations in controlling cerebellar granule cell movement. Furthermore, we will discuss the crucial roles of brain-derived neurotrophic factor (BDNF), neuregulin (NRG), stromal cell-derived factor 1α (SDF-1α), ephrin-B2, and EphB2 receptor in providing directional cues promoting granule cell migration from the external granular layer (EGL) to the internal granular layer (IGL). Finally, we will demonstrate that endogenous somatostatin controls the migration of granule cells in a cortical layer-specific manner: Endogenous somatostatin accelerates granule cell movement near the birthplace within the EGL, but significantly slows down the movement near their final destination within the IGL.     

Granule cell migration in developing rat cerebellum. Influence of neonatal hypo- and hyperthyroidism.

The rate of cerebellar granule cell migration is altered by neonatal hypo- and hyperthyroidism in a manner similar to previously reported effects on the growth of granule cell axons, the parallel fibers, suggesting that the two processes may be intimately linked. Altered rates of granule cell acquisition in these experimental animals reflect changes in germinal cell proliferation in the external granular layer (EGL), movement of postmitotic cells within the EGL, as well as the rate and time course of granule cell migration. Results of this study support the hypothesis that granule cells migrate to the internal granular layer by translocation of the cell body through the descending portion of the growing parallel fiber, rather than by amoeboid-like migration of the perikaryon trailing the elongating parallel fiber behind.     

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum

Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2-4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5-bromo-2'-deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5-trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.     

Growth arrest specific gene 1 is a positive growth regulator for the cerebellum.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595--607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated.     

A subpial, transitory germinal zone forms chains of neuronal precursors in the rabbit cerebellum

Protracted neurogenesis occurs at different postnatal stages in different brain locations, whereby leading to site-specific adult neurogenesis in some cases. No spontaneous genesis of neurons occurs in the cerebellum after the postnatal genesis of granule cells from the external germinal layer (EGL), a transitory actively proliferating zone which is thought to be exhausted before puberty. Here, we show the protracted genesis of newly generated neuronal precursors in the cerebellar cortex of young rabbits, persisting beyond puberty. Neuroblasts generated within an actively proliferating subpial layer thus extending the postnatal EGL are arranged to form thousands of tangential chains reminiscent of those responsible for cell migration in the forebrain subventricular zone. These subpial chains cover the whole cerebellar surface from the 2nd to the 5th month of life, then disappearing after puberty. In addition, we describe the appearance of similar groups of cells at the end of granule cell genesis in the mouse cerebellum, here limited to the short period of EGL exhaustion (4-5 days). These results show common features do exist in the postnatal reorganization of secondary germinal layers of brain and cerebellum at specific stages, parallel to differences in the slowing down of cerebellar neurogenesis among mammalian species.     

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.     

Reduction of BDNF expression in Fmr1 knockout mice worsens cognitive deficits but improves hyperactivity and sensorimotor deficits

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and a well-characterized form of autism spectrum disorder. As brain-derived neurotrophic factor (BDNF) is implicated in the pathophysiology of FXS we examined the effects of reduced BDNF expression on the behavioral phenotype of an animal model of FXS, Fmr1 knockout (KO) mice, crossed with mice carrying a deletion of one copy of the Bdnf gene (Bdnf(+/-)). Fmr1 KO mice showed age-dependent alterations in hippocampal BDNF expression that declined after the age of 4 months compared to wild-type controls. Mild deficits in water maze learning in Bdnf(+/-) and Fmr1 KO mice were exaggerated and contextual fear learning significantly impaired in double transgenics. Reduced BDNF expression did not alter basal nociceptive responses or central hypersensitivity in Fmr1 KO mice. Paradoxically, the locomotor hyperactivity and deficits in sensorimotor learning and startle responses characteristic of Fmr1 KO mice were ameliorated by reducing BNDF, suggesting changes in simultaneously and in parallel working hippocampus-dependent and striatum-dependent systems. Furthermore, the obesity normally seen in Bdnf(+/-) mice was eliminated by the absence of fragile X mental retardation protein 1 (FMRP). Reduced BDNF decreased the survival of newborn cells in the ventral part of the hippocampus both in the presence and absence of FMRP. Since a short neurite phenotype characteristic of newborn cells lacking FMRP was not found in cells derived from double mutant mice, changes in neuronal maturation likely contributed to the behavioral phenotype. Our results show that the absence of FMRP modifies the diverse effects of BDNF on the FXS phenotype.     

Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum.

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats (rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics by Pax6 is independent of the ROCK-mediated Rho small GTPase pathway. We propose that in addition to its roles during early patterning of the CNS, Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons.     

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.     

Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.     

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.     

Nurr1 protein is required for N-methyl-D-aspartic acid (NMDA) receptor-mediated neuronal survival

NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and mature during its migration from the external granular layer to the internal granular layer, and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5 mm) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole genome expression profiles after 3 and 8 h of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR stimulation. ChIP assay confirmed CREB binding to Nurr1 promoter, whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Moreover, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in brain-derived neurotrophic factor (BDNF) protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and that it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons.     

Type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors promote the differentiation of granule cell precursors in the postnatal cerebellum

During postnatal development of the cerebellum, granule cell precursors (GCPs) proliferate in the external granular layer (EGL), exit the cell cycle, differentiate, and migrate from the EGL to the internal granular layer. In the present study, we report that type 2 and 3 inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R2 and IP₃R3) regulate the differentiation of GCPs after postnatal day 12 (P12). 5-Bromodeoxyuridine labeling experiments revealed that in mutant mice lacking both of these receptors (double mutants) a greater number of GCPs remain undifferentiated after P12. Consequently, the EGL of the double mutants is thicker than that of control mice at this age and thereafter. In addition, granule cells remain in the EGL of the double mutants at P21, an age when migration has concluded in wild-type mice. Whereas differentiation of GCPs was reduced in the double mutants, the absence of IP₃R2 and IP₃R3 did not affect the doubling time of GCPs. We conclude that intracellular calcium release via IP₃R2s and IP₃R3s promotes the differentiation of GCPs within a specific interval of postnatal development in the cerebellum.