External Granule Cell Research Articles

Impact of varying maternal dietary folate intake on cerebellar cortex histomorphology and cell density in offspring rats.

The cerebellum has a long, protracted developmental period that spans from the embryonic to postnatal periods; as a result, it is more sensitive to intrauterine and postnatal insults like nutritional deficiencies. Folate is crucial for foetal and early postnatal brain development; however, its effects on cerebellar growth and development are unknown. The aim of this study was to examine the effects of maternal folate intake on the histomorphology and cell density of the developing cerebellum. Twelve adult female rats (rattus norvegicus) were randomly assigned to one of four premixed diet groups: standard (2mg/kg), folate-deficient (0mg/kg), folate-supplemented (8mg/kg) or folate supra-supplemented (40 mg/kg). The rats started their diets 14 days before mating and consumed them throughout pregnancy and lactation. On postnatal days 1, 7, 21 and 35, five pups from each group were sacrificed, and their brains were processed for light microscopic analysis. Histomorphology and cell density of the external granule, molecular, Purkinje and internal granule layers were obtained. The folate-deficient diet group had smaller, dysmorphic cells and significantly lower densities of external granule, molecular, Purkinje and internal granule cells. Although the folate-enriched groups had greater cell densities than the controls, the folate-supplemented group had considerably higher cell densities than the supra-supplemented group. The folate supra-supplemented group had ectopic Purkinje cells in the internal granule cell layer. These findings imply that a folate-deficient diet impairs cellular growth and reduces cell density in the cerebellar cortex. On the other hand, folate supplementation increases cell densities, but there appears to be an optimal dose of supplementation since excessive folate levels may be detrimental.

BIOL-23. LOSS OF MICRORNAS COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE MYC-DEPENDENT METASTATIC MEDULLOBLASTOMA

Abstract To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma (MB) instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and the cerebellar tumors were histologically indistinguishable from human MB. The minimum genetic mutations required for MB formation were Dicer1 and Trp53, while additional loss of Pten produced more invasive tumors with leptomeningeal metastasis. Analysis of tumor transcriptome and MB subtype-specific markers show that Dicer tumors most resemble human Sonic Hedgehog (SHH) MB. The presence of leptomeningeal metastasis, Trp53 loss and upregulation of Mycn and Gli2 in the setting of activated Shh signaling suggests that this model phenocopies extremely high risk p53-mutated SHH MB, which carries poor prognosis and is associated with the majority of treatment failures in SHH MB. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the external granule cell layer (EGL), suggesting that tumors may arise from cerebellar granule neuron precursors (CGNPs). However, targeting CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Dicer tumors exhibit reduced microRNAs and RNA-induced silencing complexes consistent with loss of Dicer. One of these downregulated microRNAs, miR-101, is a regulator of Mycn and its overexpression leads to Mycn downregulation and inhibition of MB growth. Likewise, genetic and pharmacologic inhibition of Myc leads to inhibition of MB growth. These studies demonstrate a role for microRNAs in Myc-dependent MB development and show an alternative pathway for SHH MB tumorigenesis.

Loss of CRMP1 and CRMP2 results in migration defects of Purkinje cells in the X lobule of the mouse cerebellum

The three-layered structure of the mammalian cerebellar cortex is generated through the coordinated migration of cerebellar neurons. Purkinje cells migrate and form a three- to four-cell-thick aggregate below the external granule cell layer during the embryonic stage, and align to form a monocellular arrangement in the Purkinje cell layer during the postnatal period. We previously reported the involvement of Cdk5-mediated CRMP2 phosphorylation in Purkinje cell migration and the synergistic roles of two other CRMPs, CRMP1 and CRMP4. In the present study, we investigated the loss of function of CRMP2 along with the synergistic function of CRMP1 in the migration and alignment of Purkinje cells. We found deficits in the migration and alignment of Purkinje cells in lobule X of the cerebella of CRMP1 and CRMP2 double knockout mice. Because lobule X, also called the flocculonodular lobe, is involved in the maintenance of balance equilibrium and muscle tone, we conducted balance beam and grip power tests in these mice and found impaired performance on the balance beam test and lower grip power in CRMP1 and CRMP2 double knockout mice, indicating the importance of these genes in proper cerebellar development.

Ethanol modulation of cerebellar neuroinflammation in a postnatal mouse model of fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) are alarmingly common, result in significant personal and societal loss, and there is no effective treatment for these disorders. Cerebellar neuropathology is common in FASD and causes aberrant cognitive and motor function. Ethanol-induced neuroinflammation is believed to contribute to neuropathological sequelae of FASD, and was previously demonstrated in the cerebellum in animal models of FASD. We now demonstrate neuroinflammation persists in the cerebellum several days following cessation of ethanol treatment in an early postnatal mouse model, with meaningful implications for timing of therapeutic intervention in FASD. We also demonstrate by Sholl analysis that ethanol decreases ramification of microglia cell processes in cells located near the Purkinje cell layer but not those near the external granule cell layer. Ethanol did not alter the expression of anti-inflammatory molecules or molecules that constitute NLRP1 and NLRP3 inflammasomes. Interestingly, ethanol decreased the expression of IL-23a (P19) and IL-12Rβ1 suggesting that ethanol may suppress IL-12 and IL-23 signaling. Fractalkine-fractalkine receptor (CX3CL1-CX3CR1) signaling is believed to suppress microglial activation and our demonstration that ethanol decreases CX3CL1 expression suggests that ethanol modulation of CX3CL1-CX3CR1 signaling may contribute to cerebellar neuroinflammation and neuropathology. We demonstrate ethanol alters the expression of specific molecules in the cerebellum understudied in FASD, but crucial for immune responses. Ethanol increases the expression of NOX-2 and NGP and decreases the expression of RAG1, NOS1, CD59a, S1PR5, PTPN22, GPR37, and Serpinb1b. These molecules represent a new horizon as potential targets for development of FASD therapy.

Intrauterine growth restriction compromises cerebellar development by affecting radial migration of granule cells via the JamC/Pard3a molecular pathway

Intrauterine growth restriction (IUGR) affects ~10% of human pregnancies, results in infants born small for gestational age (SGA), and is associated with motor and cognitive deficits. Human studies suggest that some deficits in SGA patients originate in the cerebellum, a major motor-coordination and cognitive center, but the underlying mechanisms remain unknown. To identify the cerebellar developmental program affected by IUGR, we analyzed the pig as a translational animal model in which some fetuses spontaneously develop IUGR due to early-onset chronic placental insufficiency. Similar to humans, SGA pigs revealed small cerebella, which contained fewer mature granule cells (GCs) in the internal granule cell layer (IGL). Surprisingly, newborn SGA pigs had increased proliferation of GC precursors in the external granule cell layer (EGL), which was associated with an increased density of Purkinje cells, known to non-autonomously promote the proliferation of GCs. However, the GCs of SGA pigs did not properly initiate exit from the EGL to IGL, which was associated with a decreased density of guiding Bergmann glial fibers, reduced expression of pro-migratory genes Pard3a, JamC and Sema6a, and increased apoptosis. While proliferation spontaneously normalized during postnatal development, accumulation of pre-migratory GCs and apoptosis in the EGL were long-lasting consequences of IUGR. Using organotypic cerebellar slice cultures, we showed that normalizing expression of Pard3a and JamC, which operate in the same molecular pathway in GCs, was sufficient to rescue both migratory and, at a later time point, apoptotic defects of IUGR. Thus, a decreased exit of GCs from the EGL, due to disrupted Pard3a/JamC radial migration initiation pathway, is a major mechanism of IUGR-related cerebellar pathology.

Effects of Phosphatidylserine Source of Docosahexaenoic Acid on Cerebellar Development in Preterm Pigs.

Preterm birth, a major contributor to infant mortality and morbidity, impairs development of the cerebellum, the brain region involved in cognitive processing and motor function. Previously, we showed that at term-equivalent age, preterm pigs that received formula supplemented with docosahexaenoic acid (DHA) esterified to phosphatidylserine (PS) had cerebellar weights similar to those of newborn term pigs and were heavier than control preterm pigs. However, whether PS-DHA promotes the development of specific cerebellar cell populations or enhances key developmental processes remains unknown. Here we investigated the effects of the PS-DHA on development of the cerebellum in preterm pigs delivered via caesarean section and reared for ten days on a milk replacer with either PS-DHA (experimental group) or sunflower oil (control group). Upon necropsy, key cerebellar populations were analyzed using immunohistochemistry. Consumption of PS-DHA was associated with the expansion of undifferentiated granule cell precursors and increased proliferation in the external granule cell layer (EGL). Preterm pigs that received PS-DHA also had significantly fewer apoptotic cells in the internal granule cell layer (IGL) that contains differentiated granule neurons. PS-DHA did not affect the number of differentiating granule cells in the inner EGL, thickness of the inner EGL, density of Purkinje cells, or Bergmann glial fibers, or diameter of Purkinje cells. Thus, PS-DHA may support cerebellar development in preterm subjects by enhancing proliferation of granule cells, a process specifically inhibited by preterm birth, and increasing the survival of granule cells in the IGL. These findings suggest that PS-DHA is a promising candidate for clinical studies directed at enhancing brain development.

Paradoxically Sparse Chemosensory Tuning in Broadly Integrating External Granule Cells in the Mouse Accessory Olfactory Bulb.

The accessory olfactory bulb (AOB), the first neural circuit in the mouse accessory olfactory system, is critical for interpreting social chemosignals. Despite its importance, AOB information processing is poorly understood compared with the main olfactory bulb (MOB). Here, we sought to fill gaps in the understanding of AOB interneuron function. We used 2-photon GCaMP6f Ca2+ imaging in an ex vivo preparation to study chemosensory tuning in AOB external granule cells (EGCs), interneurons hypothesized to broadly inhibit activity in excitatory mitral cells (MCs). In ex vivo preparations from mice of both sexes, we measured MC and EGC tuning to natural chemosignal blends and monomolecular ligands, finding that EGC tuning was sparser, not broader, than upstream MCs. Simultaneous electrophysiological recording and Ca2+ imaging showed no differences in GCaMP6f-to-spiking relationships in these cell types during simulated sensory stimulation, suggesting that measured EGC sparseness was not due to cell type-dependent variability in GCaMP6f performance. Ex vivo patch-clamp recordings revealed that EGC subthreshold responsivity was far broader than indicated by GCaMP6f Ca2+ imaging, and that monomolecular ligands rarely elicited EGC spiking. These results indicate that EGCs are selectively engaged by chemosensory blends, suggesting different roles for EGCs than analogous interneurons in the MOB.SIGNIFICANCE STATEMENT The mouse accessory olfactory system (AOS) interprets social chemosignals, but we poorly understand AOS information processing. Here, we investigate the functional properties of external granule cells (EGCs), a major class of interneurons in the accessory olfactory bulb (AOB). We hypothesized that EGCs, which are densely innervated by excitatory mitral cells (MCs), would show broad chemosensory tuning, suggesting a role in divisive normalization. Using ex vivo GCaMP6f imaging, we found that EGCs were instead more sparsely tuned than MCs. This was not due to weaker GCaMP6f signaling in EGCs than in MCs. Instead, we found that many MC-activating chemosignals caused only subthreshold EGC responses. This indicates a different role for AOB EGCs compared with analogous cells in the main olfactory bulb.

Deletion of Btg1 Induces Prmt1-Dependent Apoptosis and Increased Stemness in Shh-Type Medulloblastoma Cells Without Affecting Tumor Frequency.

About 30% of medulloblastomas (MBs), a tumor of the cerebellum, arise from cerebellar granule cell precursors (GCPs) undergoing transformation following activation of the Sonic hedgehog (Shh) pathway. To study this process, we generated a new MB model by crossing Patched1 heterozygous (Ptch1+/−) mice, which develop spontaneous Shh-type MBs, with mice lacking B-cell translocation gene 1 (Btg1), a regulator of cerebellar development. In MBs developing in Ptch1+/− mice, deletion of Btg1 does not alter tumor and lesion frequencies, nor affect the proliferation of neoplastic precursor cells. However, in both tumors and lesions arising in Ptch1+/− mice, ablation of Btg1 increases by about 25% the apoptotic neoplastic precursor cells, as judged by positivity to activated caspase-3. Moreover, although Btg1 ablation in early postnatal GCPs, developing in the external granule cell layer, leads to a significant increase of proliferation, and decrease of differentiation, relative to wild-type, no synergy occurs with the Ptch1+/− mutation. However, Btg1 deletion greatly increases apoptosis in postnatal GCPs, with strong synergy between Btg1-null and Ptch1+/− mutations. That pronounced increase of apoptosis observed in Ptch1+/−/Btg1 knockout young or neoplastic GCPs may be responsible for the lack of effect of Btg1 ablation on tumorigenesis. This increased apoptosis may be a consequence of increased expression of protein arginine methyltransferase 1 (Prmt1) protein that we observe in Btg1 knockout/Ptch1+/− MBs. In fact, apoptotic genes, such as BAD, are targets of Prmt1. Moreover, in Btg1-null MBs, we observed a two-fold increase of cells positive to CD15, which labels tumor stem cells, raising the possibility of activation of quiescent tumor cells, known for their role in long-term resistance to treatment and relapses. Thus, Btg1 appears to play a role in cerebellar tumorigenesis by regulating the balance between apoptosis and proliferation during MB development, also influencing the number of tumor stem cells.

Expression pattern of Nav2 in the murine CNS with development

Neuron navigator 2 (NAV2, RAINB1, POMFIL2, HELAD1, unc53H2) is essential for nervous system development. In the present study the spatial distribution of Nav2 transcript in mouse CNS during embryonic, postnatal and adult life is examined. Because multiple NAV2 proteins are predicted based on alternate promoter usage and RNA splicing, in situ hybridization was performed using probes designed to the 5′ and 3′ ends of the Nav2 transcript, and PCR products using primer sets spanning the length of the mRNA were also examined by real time PCR (qPCR). These studies support full-length Nav2 transcript as the predominant form in the wild-type mouse CNS. The developing cortex, hippocampus, thalamus, olfactory bulb, and granule cells (GC) within the cerebellum show the highest expression, with a similar staining pattern using either the 5′Nav2 or 3′Nav2 probe. Nav2 is expressed in GC precursors migrating over the cerebellar primordium as well as in the postmitotic premigratory cells of the external granule cell layer (EGL). It is expressed in the cornu ammonis (CA) and dentate gyrus (DG) throughout hippocampal development. In situ hybridization was combined with immunohistochemistry for Ki67, CTIP2 and Nissl staining to follow Nav2 transcript location during cortical development, where it is observed in neuroepithelial cells exiting the germinal compartments, as well as later in the cortical plate (CP) and developing cortical layers. The highest levels of Nav2 in all brain regions studied are observed in late gestation and early postnatal life which coincides with times when neurons are migrating and differentiating. A hypomorphic mouse that lacks the full-length transcript but expresses shorter transcript shows little staining in the CNS with either probe set except at the base of the cerebellum, where a shorter Nav2 transcript is detected. Using dual fluorescent probe in situ hybridization studies, these cells are identified as oligodendrocytes and are detected using both Olig1 and the 3′Nav2 probe. The identification of full-length Nav2 as the primary transcript in numerous brain regions suggests NAV2 could play a role in CNS development beyond that of its well-established role in the cerebellum.

Expression of transcription factors and signaling molecules in the cerebellar granule cell development

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) constitute a good model system to investigate proliferation of neural precursors and differentiation of neurons. During development, GCPs proliferate in the outer external granule cell layer (outer EGL) and then exit the cell cycle in the inner EGL to become GCs, which inwardly migrate to the inner granule cell layer (IGL). Misregulation of GCP proliferation or GC differentiation leads to maldevelopment of the cerebellum and the formation of a cerebellar tumor, medulloblastoma. Despite many efforts in this field, the mechanisms underlying GC development remain elusive. In this study, we performed detailed immunostaining in the developing cerebellum, with particular focus on GCPs and GCs, looking at several transcription factors, signaling molecules, cell cycle regulators, some of which are known to regulate neural development. Interestingly, we found distinct distribution patterns of certain proteins within the outer and inner EGL, suggesting the existence of subpopulations of GCPs and GCs in those layers. This study provides a basis for future studies on the cerebellar GC development and medulloblastoma.

C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

BackgroundConditional ablation of the Smarca5 gene in mice severely impairs the postnatal growth of the cerebellum and causes an ataxic phenotype. Comparative gene expression studies indicated that complement-related proteins were upregulated in the cerebellum of Smarca5 mutant mice. Complement proteins play critical roles within innate immune signaling pathways and, in the brain, are produced by glial cells under both normal and pathological conditions. The C3 complement protein-derived signaling peptide, C3a, has been implicated in contributing to both tissue damage and repair in conditions such as multiple sclerosis and stroke. Here, we investigated whether C3a receptor (C3aR) signaling promoted damage or repair in the developing cerebellum of Smarca5 mutant mice.MethodsBrain and cerebellum lysates from single Smarca5 conditional knockout (Smarca5 cKO) mice, C3aR1 KO mice, or double mutant mice were used for qRT-PCR and immunoblotting to assess the contribution of C3aR to the Smarca5 cKO brain pathology. Immunohistochemistry was used to characterize alterations to astroglia and phagocyte cells in the developing cerebellum of each of the genotypes.ResultsC3aR signaling was observed to limit gliosis and promote granule neuron survival during postnatal cerebellar development. In Smarca5 cKO mice, disorganized astroglia with increased GFAP expression develops concurrently with cerebellar granule neuron loss and phagocyte invasion over the first 10 days following birth. Potential ligand precursors of C3aR—VGF and C3—were found to have upregulated expression and/or altered processing during this time. Phagocytes (microglia and macrophages) in both the control and Smarca5 mutant mice were the only cells observed to express C3aR. Loss of C3aR in the Smarca5 cKO cerebellum resulted in increased numbers of apoptotic cells and early phagocyte invasion into the external granule cell layer, as well as an exacerbated disorganization of the Bergmann glia. The loss of C3aR expression also attenuated an increase in the expression of the efferocytosis-related protein, MerTK, whose transcript was upregulated ~ 2.5-fold in the Smarca5 mutant cerebellum at P10.ConclusionsThis data indicates that C3aR can play an important role in limiting astrogliosis and regulating phagocyte phenotypes following developmental cell loss in the brain.

Interneuron Functional Diversity in the Mouse Accessory Olfactory Bulb.

In the mouse accessory olfactory bulb (AOB), inhibitory interneurons play an essential role in gating behaviors elicited by sensory exposure to social odors. Several morphological classes have been described, but the full complement of interneurons remains incomplete. In order to develop a more comprehensive view of interneuron function in the AOB, we performed targeted patch clamp recordings from partially overlapping subsets of genetically labeled and morphologically defined interneuron types. Gad2 (GAD65), Calb2 (calretinin), and Cort (cortistatin)-cre mouse lines were used to drive selective expression of tdTomato in AOB interneurons. Gad2 and Calb2-labeled interneurons were found in the internal, external, and glomerular (GL) layers, whereas Cort-labeled interneurons were enriched within the lateral olfactory tract (LOT) and external cellular layer (ECL). We found that external granule cells (EGCs) from all genetically labeled subpopulations possessed intrinsic functional differences that allowed them to be readily distinguished from internal granule cells (IGCs). EGCs showed stronger voltage-gated Na+ and non-inactivating voltage-gated K+ currents, decreased IH currents, and robust excitatory synaptic input. These specific intrinsic properties did not correspond to any genetically labeled type, suggesting that transcriptional heterogeneity among EGCs and IGCs is not correlated with expression of these particular marker genes. Intrinsic heterogeneity was also seen among AOB juxtaglomerular cells (JGCs), with a major subset of Calb2-labeled JGCs exhibiting spontaneous and depolarization-evoked plateau potentials. These data identify specific physiological features of AOB interneurons types that will assist in future studies of AOB function.

Neuronal Migration Generates New Populations of Neurons That Develop Unique Connections, Physiological Properties and Pathologies.

Central nervous system neurons become postmitotic when radial glia cells divide to form neuroblasts. Neuroblasts may migrate away from the ventricle radially along glia fibers, in various directions or even across the midline. We present four cases of unusual migration that are variably connected to either pathology or formation of new populations of neurons with new connectivities. One of the best-known cases of radial migration involves granule cells that migrate from the external granule cell layer along radial Bergman glia fibers to become mature internal granule cells. In various medulloblastoma cases this migration does not occur and transforms the external granule cell layer into a rapidly growing tumor. Among the ocular motor neurons is one unique population that undergoes a contralateral migration and uniquely innervates the superior rectus and levator palpebrae muscles. In humans, a mutation of a single gene ubiquitously expressed in all cells, induces innervation defects only in this unique motor neuron population, leading to inability to elevate eyes or upper eyelids. One of the best-known cases for longitudinal migration is the facial branchial motor (FBM) neurons and the overlapping inner ear efferent population. We describe here molecular cues that are needed for the caudal migration of FBM to segregate these motor neurons from the differently migrating inner ear efferent population. Finally, we describe unusual migration of inner ear spiral ganglion neurons that result in aberrant connections with disruption of frequency presentation. Combined, these data identify unique migratory properties of various neuronal populations that allow them to adopt new connections but also sets them up for unique pathologies.

Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors

BackgroundThe cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known.MethodsWe used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) Our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atoh1-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion.ResultsMosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved.ConclusionWe demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of Atoh1-Cre conditional mutants affecting GCPs.

Preterm birth disrupts cerebellar development by affecting granule cell proliferation program and Bergmann glia

Preterm birth is a leading cause of long-term motor and cognitive deficits. Clinical studies suggest that some of these deficits result from disruption of cerebellar development, but the mechanisms that mediate cerebellar abnormalities in preterm infants are largely unknown. Furthermore, it remains unclear whether preterm birth and precocious exposure to the ex-utero environment directly disrupt cerebellar development or indirectly by increasing the probability of cerebellar injury, including that resulting from clinical interventions and protocols associated with the care of preterm infants. In this study, we analyzed the cerebellum of preterm pigs delivered via c-section at 91% term and raised for 10 days, until term-equivalent age. The pigs did not receive any treatments known or suspected to affect cerebellar development and had no evidence of brain damage. Term pigs sacrificed at birth were used as controls. Immunohistochemical analysis revealed that preterm birth did not affect either size or numbers of Purkinje cells or molecular layer interneurons at term-equivalent age. The number of granule cell precursors and Bergmann glial fibers, however, were reduced in preterm pigs. Preterm pigs had reduced proliferation but not differentiation of granule cells. qRT-PCR analysis of laser capture microdissected external granule cell layer showed that preterm pigs had a reduced expression of Ccnd1 (Cyclin D1), Ccnb1 (Cyclin B1), granule cell master regulatory transcription factor Atoh1, and signaling molecule Jag1. In vitro rescue experiments identified Jag1 as a central granule cell gene affected by preterm birth. Thus, preterm birth and precocious exposure to the ex-utero environment disrupt cerebellum by modulating expression of key cerebellar developmental genes, predominantly affecting development of granule precursors and Bergmann glia.

Constraints and Catalysts in Cerebellar Evolution

The cerebellum comprises a remarkably stereotyped neural circuit, elements of which, such as the granule cell‐Purkinje cell synapse, are highly conserved across vertebrates. It functions as an adaptive filter that can be employed in regulating feed forward control of movement, but has a wider range of functions including in mammals, a demonstrable role in cognitive development. The range of neural functions modulated by cerebellar activity is a product of its output connections. In many species these are mediated by cerebellar nuclei whose number and projections vary systematically across major classes of vertebrates. Notably, the connection of cerebellar nuclei to the thalamus and hence the ability of the cerebellum to participate in forebrain activity is a mammalian innovation. Cerebellum size is also a key adaptive variable amongst different species with some species of both fish and mammals displaying relatively large, elaborately foliated cerebellum. Different strategies for generating large numbers of cells are adopted by different classes of vertebrates. In particular, the appearance of a transient, external granule cell layer (EGL) in cerebellar development is a tetrapod innovation. In some reptiles, birds and mammals, the EGL becomes a proliferative layer, which leads to a rapid clonal expansion of granule cells and the potential for foliation. Both the diversification of connectivity (cerebellar nuclei) and transit amplification (in the EGL) are adaptive properties within derivatives of a single defined pool of progenitors at the embryonic “rhombic lip”. Different derivatives are generated within distinct cohorts. Within these cohorts the heterochronic shift in the expression of a small number of genes appears to be responsible for generating the key changes in cerebellar development in vertebrates.Support or Funding InformationMedical Research Council, UKBBSRCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient vimentin expression during the embryonic development of the chicken cerebellum

Complex morphogenetic events, critical for the development of normal cerebellum foliation and layering, are known to involve type III intermediate filament protein such as vimentin expressed by Bergmann glia. The present study aimed to determine aspects of intermediate and late embryonic pattern of vimentin expression during the corticogenesis of chicken cerebellum at embryonic days 10–19 (E10-E19), using single and double immunohistochemistry/immunofluorescence. Vimentin expression showed partial co-localization with the glial markers GFAP and BLBP. Within cerebellar cortex, vimentin+ fibers were first found within lobules I and X (E10) and gradually extended to all folia (E15-E17), located within the external granule (EGL) the molecular cell layer, showing a radial orientation towards the inner granular layer and the cerebellar white matter oriented longitudinally. Interestingly, within the immature fissures base of most lobules, vimentin+ fibers radiate in a fan shape. Short-term BrdU experiments revealed that EGL cell proliferation was higher in the fissure base compared to folia apex. In addition, following 24-h survival, BrdU+ cells were found in close association to vimentin+ fibers in the EGL pre-migratory zone and within immature molecular layer. Paralleling cerebellum development, vimentin expression gradually extended to all folia sub-regions (base, wall, apex), but, at day E19, it was mainly confined to the folia apex and secondary fissure base. Taken together our data further support the possible role of vimentin+ fibers in the structural events of cerebellum corticogenesis, suggesting the participation of radial/Bergmann glia in chicken cerebellum foliation, similarly to that described for mammalian cerebellum morphogenesis.

Induction of the UDP-Glucuronosyltransferase 1A1 during the Perinatal Period Can Cause Neurodevelopmental Toxicity.

Anticonvulsants can increase the risk of developing neurotoxicity in infants; however, the underlying mechanism has not been elucidated to date. Thyroxine [3,5,3',5'-l-tetraiodothyronine (T4)] plays crucial roles in the development of the central nervous system. In this study, we hypothesized that induction of UDP-glucuronosyltransferase 1A1 (UGT1A1)-an enzyme involved in the metabolism of T4-by anticonvulsants would reduce serum T4 levels and cause neurodevelopmental toxicity. Exposure of mice to phenytoin during both the prenatal and postnatal periods significantly induced UGT1A1 and decreased serum T4 levels on postnatal day 14. In the phenytoin-treated mice, the mRNA levels of synaptophysin and synapsin I in the hippocampus were lower than those in the control mice. The thickness of the external granule cell layer was greater in phenytoin-treated mice, indicating that induction of UGT1A1 during the perinatal period caused neurodevelopmental disorders. Exposure to phenytoin during only the postnatal period also caused these neurodevelopmental disorders. A T4 replacement attenuated the increase in thickness of the external granule cell layer, indicating that the reduced T4 was specifically associated with the phenytoin-induced neurodevelopmental disorder. In addition, these neurodevelopmental disorders were also found in the carbamazepine- and pregnenolone-16-α-carbonitrile-treated mice. Our study is the first to indicate that UGT1A1 can control neurodevelopment by regulating serum T4 levels.

Tobacco Nitrosamine Exposures Contribute to Fetal Alcohol Spectrum Disorder Associated Cerebellar Dysgenesis.

Variability in the phenotypic features and severity of fetal alcohol spectrum disorder (FASD) is not fully linked to alcohol dose. We hypothesize that FASD-type neurodevelopmental abnormalities may be caused by exposures to the tobacco-specific nitrosamine, NNK, since a high percentage of pregnant women who drink also smoke. In vitro experiments using PNET2 human cerebellar neuronal cultures examined ethanol and NNK effects on viability and mitochondrial function. Early postnatal rat cerebellar slice cultures were used to examine effects of ethanol and NNK on cerebellar histology and neuroglial and stress protein expression. Ethanol (50 mM) decreased viability and ATP content and increased mitochondrial mass, while NNK (100 μM or higher) selectively inhibited mitochondrial function. The slice culture studies demonstrated striking adverse effects of ethanol, NNK and ethanol+NNK exposures manifested by architectural disorganization of the cortex with relative reductions of internal granule cells, increases in external granule cells, and loss of Purkinje cells. Ethanol, NNK, and ethanol+NNK inhibited expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and increased levels of 4-hydroxynonenal (HNE). In addition, ethanol increased activated Caspase 3, NNK decreased tau and phospho-tau, and ethanol+NNK inhibited expression of Aspartyl-β-hydroxylase (ASPH), which mediates neuronal migration. In conclusion, ethanol and NNK were shown to exert independent but overlapping adverse effects on cerebellar cortical development, neuronal viability, function, and neuroglial protein expression. These findings support our hypothesis that NNK exposures via tobacco smoking in pregnancy can contribute to FASD-associated neurodevelopmental abnormalities.

Overexpression of Lin28b in Neural Stem Cells is Insufficient for Brain Tumor Formation, but Induces Pathological Lobulation of the Developing Cerebellum.

LIN28B is a homologue of the RNA-binding protein LIN28A and regulates gene expression during development and carcinogenesis. It is strongly upregulated in a variety of brain tumors, such as medulloblastoma, embryonal tumor with multilayered rosettes (ETMR), atypical teratoid/rhabdoid tumor (AT/RT), or glioblastoma, but the effect of an in vivo overexpression of LIN28B on the developing central nervous system is unknown. We generated transgenic mice that either overexpressed Lin28b in Math1-positive cerebellar granule neuron precursors or in a broad range of Nestin-positive neural precursors. Sections of the cerebellar vermis from adult Math1-Cre::lsl-Lin28b mice had an additional subfissure in lobule IV. Vermes from p0 and p7 Nestin-Cre::lsl-Lin28b mice appeared normal, but we found a pronounced vermal hypersublobulation at p15 and p21 in these mice. Also, the external granule cell layer (EGL) was thicker at p15 than in controls, contained more proliferating cells, and persisted up to p21. Consistently, some Pax6- and NeuN-positive cells were present in the EGL of Nestin-Cre::lsl-Lin28b mice even at p21, and we detected more NeuN-positive granule neuron precursors in the molecular layer (ML) as compared to control. Finally, we found some residual Pax2-positive precursors of inhibitory interneurons in the ML of Nestin-Cre::lsl-Lin28b mice at p21, which have already disappeared in controls. We conclude that while overexpression of LIN28B in Nestin-positive cells does not lead to tumor formation, it results in a protracted development of granule cells and inhibitory interneurons and leads to a hypersublobulation of the cerebellar vermis.