Internal Granule Cell Research Articles

Balanced effect of PACAP and FasL on granule cell death during cerebellar development: a morphological, functional and behavioural characterization

It is now established that the development of the CNS requires equilibrium between cell survival and apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a powerful protective effect on cerebellar granule cells by inhibiting the caspase 3. In contrast, Fas ligand (FasL) plays an essential role during ontogenesis in eliminating supernumerary neurons by apoptosis. To determine if PACAP and FasL interact during cerebellar development, we characterized the effects of these factors on cerebellar morphogenesis and caspase 3 activity in PACAP+/+ and PACAP-/- mice. First, we demonstrated in vivo that PACAP is able to reverse the diminution of internal granule cell layer thickness induced by FasL in PACAP+/+ and PACAP-/- mice. Second, ex vivo and immunohistochemical studies revealed that interaction between FasL and PACAP occurs through the caspase 3 activity. Third, behavioural study showed a significant difference for the PACAP + FasL group in the righting reflex test at P8 which does not persist at P60. Finally, a time course study revealed that the pro-apoptotic effect of FasL characterized at P8 was followed by a progressive compensatory mechanism in caspase 3 activity and bromodeoxyuridine incorporation. These data suggest that PACAP and FasL interact during cerebellar development to control apoptosis of granule cells and may affect some motor cerebellar functions.

Age-dependent Expression of GAP-43, Netrin-1, Collapsin-1, and Neuropilin-1 in Murine Cerebellum*

GAP-43, netrin-1, collapsin-1, and neuropilin-1 have been regarded to play crucial roles in the formation of patterned neural connections. The cerebellum consists of five distinct concentric layers: white matter, internal granule layer (IGL), Purkinje cell layer (PCL), molecular layer (ML), and external granule layer (EGL) in young rodents. Cells in EGL are generated after birth. In contrast Purkinje neurons are born before birth, which receive main innervations of climbing fibers from the inferior olivary nucleus and parallel fibers from the internal granule cells. These innervations are mostly established in the first three postnatal weeks, accompanying the sprouting and maturation of Purkinje cells. The potential roles of GAP-43, netrin-1, collapsin-1 and neuropilin-1 in the postnatal development of cerebellum remain unclear. To get insights into the above issue, the expression of GAP-43, netrin-1, collapsin-1, and neuropilin-1 mRNAs and proteins were examined in the cerebellum of mice at postnatal days (P) 5, P10, P20 and adulthood. The results showed that these four molecules were expressed in different temporal and spatial patterns in the postnatal cerebellum of mice, which was in match with axonal synaptogenesis, elongation and synapse formation during postnatal development and adulthood. By using double immunohistochemistry, it was found that the Purkinje cells stained for GAP-43 were also positive for either netrin-1 or collapsin-1 at P10, and cells stained for collapsin-1 were also positive for netrin-1 or neuropilin-1. It was suggested that the four molecules are involved in the postnatal development of cerebellum.

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

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

Targets of the nuclear factor I regulon involved in early and late development of postmitotic cerebellar granule neurons

Recent studies have shown that the nuclear factor I (NFI) family controls multiple stages of the postmitotic differentiation of cerebellar granule neurons (CGNs). Regulation of cell-cell signaling is an integral part of this NFI program, which involves expression of the cell adhesion molecules N cadherin and ephrin B1 throughout postmitotic CGN development. Here, we identify two additional downstream targets of NFI that are involved in extracellular CGN interactions. The cell adhesion molecule Tag-1 is highly enriched in CGNs undergoing parallel fiber formation and is down-regulated prior to onset of radial migration. We found that Tag-1 expression was strongly reduced by NFI dominant repression in immature primary CGNs and in the cerebella of E18 Nfib-null mice. Transient transfection and chromatin immunoprecipitation suggested that the Tag-1 gene is directly regulated by NFI. Furthermore, functional, Nfi knockout and chromatin immunoprecipitation studies implicated Wnt7a as a direct target of NFI in maturing CGNs. Wnt7a is secreted by developing CGNs and is required for maturation of mossy fiber-CGN synaptic rosettes. Consistent with this, synapsin I was greatly reduced within the internal granule cell layer of P17 Nfia-null mice. These findings indicated that NFI controls CGN postmitotic maturation through a combination of extracellular signaling molecules that operate either continuously to regulate multiple stages of development (N cadherin and ephrin B1) or primarily at early (Tag-1) or late (Wnt7a) maturation steps. They also illustrate the importance of NFI as a critical link between cell-intrinsic mechanisms and cell-cell interactions in the development of the mouse cerebellum.

BDNF-Mediated Cerebellar Granule Cell Development Is Impaired in Mice Null for CaMKK2 or CaMKIV

The Ca(2+)/calmodulin-activated kinases CaMKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca(2+). To address the biological functions of Ca(2+) signaling via these kinases during brain development, we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here, we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of cAMP response element-binding protein (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Furthermore, loss of CaMKK2 or CaMKIV results in decreased CREB phosphorylation (pCREB), Bdnf exon I and IV-containing mRNAs, and brain-derived neurotrophic factor (BDNF) protein in cerebellar granule cell neurons. Reexpression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild-type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca(2+)-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process.

Splicing variations in the ligand-binding domain of ApoER2 results in functional differences in the binding properties to Reelin

Reelin plays critical roles in brain formation by binding to apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor. Several isoforms and fragments of Reelin are generated by alternative splicing and proteolytic cleavage. In addition, two splice variants of ApoER2 have been recognized, namely, LA1237 and LA12378, that differ in the number of ligand-binding type A (LA) repeats. Here, we quantitatively investigated the affinity between various isoforms/fragments of Reelin and the ApoER2 splice variants. ApoER2-LA1237 bound rather strongly to the Reelin central fragment than to the fragment bearing Reelin repeat 8 (RR8). ApoER2-LA12378 bound comparably to all Reelin fragments without the C-terminal region. These findings suggest that LA8 of ApoER2 and RR8 interfere with the interaction between the Reelin central fragment and ApoER2. Using a monoclonal antibody that only recognizes ApoER2-LA12378, we found that this variant of ApoER2 was expressed in the cerebral cortical wall and in the internal granule cells of the cerebellum during development. Primary-cultured cortical neurons did not express ApoER2-LA12378, and the extent of signal activation by Reelin fragments was well correlated with their affinity for ApoER2-LA1237. Therefore, proteolytic cleavage of Reelin and alternative splicing of ApoER2 may be involved in the fine regulation of Reelin signaling.

CDG Type Ia and Congenital Cytomegalovirus Infection: Two Coexisting Conditions

Congenital disorders of glycosylation are a heterogeneous group of disorders with multisystemic involvement. The most common form is phosphomannomutase deficiency or congenital disorders of glycosylation type Ia with an autosomal recessive inheritance and incidence estimated at 1/20000-1/50000 live born. Congenital disorders of glycosylation Ia can manifest as severe multisystemic disease of infancy or milder disorder with only neurological problems including ataxia, hypotonia, and psychomotor retardation. The brain pathological findings in congenital disorders of glycosylation type Ia patients corroborate with cerebellar dysfunction. Usually the most affected part is the anterior lobe of the vermis. Microscopic analysis demonstrates the prominent Purkinje cell loss and subtotal loss of the external and internal granule cell layers. The authors present clinical and pathological picture of a 4-month-old girl with congenital disorders of glycosylation type Ia, additionally complicated by congenital cytomegalovirus infection. The diagnosis was confirmed by low phosphomannomutase activity in patient's fibroblasts and mutations on both alleles of phosphomannomutase 2 gene.

Continuous exposure to low concentrations of methylmercury impairs cerebellar granule cell migration in organotypic slice culture

Chronic, low-level perinatal exposure to methylmercury (MeHg) is associated with neurological and motor deficits that appear to result from cerebellar dysfunction. Neuropathological studies suggest that these deficits are due to impaired cerebellar granule cell (CGC) migration. Although neuronal migration in vivo and in vitro has been shown to be impaired during acute and/or high level exposure to MeHg, the cellular effects of chronic exposure to submicromolar and micromolar levels of MeHg during development are not clear. The majority of CGC migration in rats occurs between postnatal days 8 and 14 (P8 and 14); migration peaks on P10 and 11. Organotypic cultures of parasagittal slices of cerebellum from P8 rats were exposed to low levels of MeHg (0.2–5.0 μM) for 3 or 7 days, and CGC viability and migration were assessed. MeHg-induced cell death was time- and concentration-dependent. After 3 days of exposure CGC viability decreased in 3 μM MeHg and declined to 42.7% in 5 μM MeHg. Cultures treated with MeHg for 7 days showed decreased CGC viability in 1 μM MeHg, which declined to 62.8% in 3 μM MeHg. CGC migration was assessed by BrdU pulse-chase labeling. Migration into the internal granule cell layer (IGL) was impaired in cultures exposed to ≥1 μM MeHg for 3 days or ≥0.5 μM for 7 days. CGCs failed to initiate migration from the external germinal cell layer at the same level of exposure. For those cells which initiated migration, MeHg reduced the number that migrated into the IGL. This implied a slowing of migration once it had begun. These effects occurred with no overall change in cerebellar cortical structure, or loss of granule cell viability. Thus, chronic exposure to low micromolar concentrations of MeHg impairs development of the cerebellar cortex in a slice culture model.

Isoform-dependent immunolocalization of 14-3-3 proteins in developing rat cerebellum

We investigated the expression of 14-3-3 protein and its 7 isoforms during postnatal development of rat cerebellum with immunoblot and immunohistochemistry with isoform-specific antibodies. The relative amounts of total 14-3-3 protein, probed by an antibody (14-3-3 COM) recognizing a sequence shared among its isoforms, exhibited no significant changes from postnatal day 2 (P2) to P100. 14-3-3 COM-like immunoreactivity (IR), initially in the apical portion of Purkinje cells at P2, extended to Purkinje cell bodies at P14 and to their dendrites (P100) with increasing intensity. Molecular layer (after P7) and cerebellar nucleus neurons (after P14) were also immunolabeled with this antibody. These chronological changes were shared with those obtained with beta, gamma, and eta isoforms. In contrast, epsilon isoform-like IR was initially identified in processes of radial and Bergmann glia at P2 prior to its appearance in the molecular layer at P7 with subsequent intensification also in Purkinje cells after P14. Zeta and tau isoform-like IR was identified in the white matter and/or in oligodendroglial cells. The sigma isoform was the only isoform exhibiting a significant quantitative change with a peak at P14. Immunolocalization of sigma isoform was initially restricted in several cells in Purkinje cell layer at P2 and shifted to nuclei of external and internal granule cells and Purkinje cells after P14, whereas its immunolabeling was markedly weaker at P100. Different immunolocalizations of the 7 isoforms suggest that 14-3-3 protein isoforms individually associate with the neuronal and glial proliferation, differentiation, migration and development during postnatal formation of rat cerebellum.

The accessory olfactory bulb in the adult rat: A cytological study of its cell types, neuropil, neuronal modules, and interactions with the main olfactory system

The accessory olfactory bulb (AOB) in the adult rat is organized into external (ECL) and internal (ICL) cellular layers separated by the lateral olfactory tract (LOT). The most superficial layer, or vomeronasal nerve layer, is composed of two fiber contingents that distribute in rostral and caudal halves. The second layer, or glomerular layer, is also divided by a conspicuous invagination of the neuropil of the ECL at the junction of the rostral and caudal halves. The ECL contains eight cell types distributed in three areas: a subglomerular area containing juxtaglomerular and superficial short-axon neurons, an intermediate area harboring large principal cells (LPC), or mitral and tufted cells, and a deep area containing dwarf, external granule, polygonal, and round projecting cells. The ICL contains two neuron types: internal granule (IGC) and main accessory cells (MACs). The dendrites and axons of LPCs in the two AOB halves are organized symmetrically with respect to an anatomical plane called linea alba. The LPC axon collaterals may recruit adjacent intrinsic, possibly gamma-aminobutyric acid (GABA)-ergic, neurons that, in turn, interact with the dendrites of the adjacent LPCs. These modules may underlie the process of decoding pheromonal clues. The most rostral ICL contains another neuron group termed interstitial neurons of the bulbi (INBs) that includes both intrinsic and projecting neurons. MACs and INBs share inputs from fiber efferents arising in the main olfactory bulb (MOB) and AOB and send axons to IGCs. Because IGCs are a well-known source of modulatory inputs to LPCs, both MACs and INBs represent a site of convergence of the MOB with the AOB.

Role of complement anaphylatoxin receptors (C3aR, C5aR) in the development of the rat cerebellum

There is now strong evidence for non-immune or inflammatory functions of complement, notably in the central nervous system. In particular, it has been recently reported that the anaphylatoxin receptors C3aR and C5aR are transiently expressed in the cerebellar cortex of newborn rat, suggesting that anaphylatoxins are involved in the histogenesis of the cerebellum. In the present study, we have investigated the effects of C3aR and C5aR agonists and antagonists on the development of the cerebellum of 11–12-day-old rats in vivo and in vitro. Sub-dural injection of C3aR and C5aR agonists at the surface of the cerebellum transiently modified the thickness of the cortical layers. The C5aR agonist provoked an enlargement of the external granule cell layer (EGL) that was due to increased proliferation of immature granule neurons. Conversely, the C3aR agonist decreased the thickness of the EGL and increased the thickness of the internal granule cell layer (IGL), suggesting that C3a accelerates the migration process of granule cells from the EGL to the IGL. Video-microscopy examination of cultured granule neurons confirmed the role of C3aR in cell motility. These results provide clear evidence for the involvement of anaphylatoxin receptors in the histogenesis of the cerebellar cortex.

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

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

Distribution and corticosteroid regulation of glucocorticoid receptor in the brain of Xenopus laevis

Glucocorticoids (GCs) play essential roles in physiology, development, and behavior that are mediated largely by the glucocorticoid receptor (GR). Although the GR has been intensively studied in mammals, very little is known about the GR in nonmammalian tetrapods. We analyzed the distribution and GC regulation of GR in the brain of the frog Xenopus laevis by immunohistochemistry. GR-immunoreactive (GR-ir) cells were widely distributed, with the highest densities in the medial pallium (mp; homolog of the mammalian hippocampus), accumbens, anterior preoptic area (POA; homolog of the mammalian paraventricular nucleus), Purkinje cell layer of the cerebellum, and rostral anterior pituitary gland (location of corticotropes). Lower but distinct GR-ir was observed in the internal granule cell layer of the olfactory bulbs, dorsal and lateral pallium, striatum, various subfields of the amygdala, bed nucleus of the stria terminalis (BNST), optic tectum, various tegmental nuclei, locus coeruleus, raphe nuclei, reticular nuclei, and the nuclei of the trigeminal motor nerves. Treatment with corticosterone (CORT) for 4 days significantly decreased GR-ir in the POA, mp, medial amygdala (MeA), BNST, and rostral pars distalis. Treatment with the corticosteroid synthesis inhibitor metyrapone (MTP) also significantly reduced GR-ir in the POA, mp, MeA and BNST, but not in the rostral pars distalis. Replacement with a low dose of CORT in MTP-treated animals reversed these effects in brain. Thus, chronic increase or decrease in circulating corticosteroids reduces GR-ir in regions of the frog brain. Our results show that the central distribution of GR-ir and regulation by corticosteroids are highly conserved among vertebrates.

The NR2B subunit in NMDA receptors is functionally important during cerebellar granule cell migration

Migration of cerebellar granule cells (CGCs) from the external germinal cell layer (EGL) to the internal granule cell layer (IGL) within the cerebellar cortex is a crucial developmental process. Antagonists to NMDA receptors impair CGC migration significantly, but studies to determine which subunit subtypes control or affect migration have been controversial. Migrating CGCs transiently express NMDA receptor subunit subtypes NR1a plus NR2B. Grafted NR1-/- subunit knockout cells continue to migrate, indicating that the NR1 subunit is not necessary for migration. In the present study, the functional importance of the NR2B subtype in developing cerebellum was investigated using organotypic slice cultures prepared from postnatal day 8 (P8) rats. Slice cultures were labeled with bromodeoxyuridine (BrdU) during the first 20h and then continuously treated with the NR2B-subtype-specific NMDA antagonist, ifenprodil, or the non-specific NMDA antagonist, APV, for 7 days. Cultures were incubated with fluorescently tagged anti-BrdU IgG and the percent of BrdU-labeled CGCs that migrated from the EGL to the IGL during treatment was analyzed using laser confocal microscopy. Migration into the IGL was significantly impaired by treatment with 0.5 and 1.0 microM ifenprodil. Fewer cells had migrated to the IGL in 1.0 microM ifenprodil than in 0.5 microM ifenprodil; there was no significant difference between the percent impairment caused by 1.0 microM ifenprodil and 50 microM APV. Untreated controls had few, if any, CGCs in the EGL at DIV 8. The percent of CGCs remaining in the EGL following treatment with antagonists significantly increased, indicating impairment of migration. In conclusion, the NR2B subunit appears to be necessary for CGC migration.

NMDA receptor NR2B subunit over‐expression increases cerebellar granule cell migratory activity

Glutamate acting on NMDA receptors (NMDARs) is known to influence cerebellar granule cell migration. Subunit composition of NMDARs in granule cells changes characteristically during development: NR2B subunit containing receptors are abundant during migration towards the internal granule cell layer but are gradually replaced by NR2A and/or NR2C subunits once the final position is reached. Cerebellar granule cell migration was investigated using mutant mouse lines either with a deletion of the NR2C gene (NR2C(-/-) mice) or expressing NR2B instead of the NR2C subunit (NR2C-2B mice). BrdU-labeling revealed that over-expression of NR2B increased granule cell translocation in vivo, while the lack of NR2C subunit did not have any detectable effects on cell migration. Cellular composition of wild-type and mutant dissociated cerebellar granule cell cultures isolated from 10-day-old cerebella were similar, but NR2C-2B cultures had elevated level of NR2B subunits and intracellular Ca2+ imaging revealed higher sensitivity towards the addition of NR2B-selective antagonist in vitro. Time-lapse videomicroscopic observations revealed that average migratory velocity and the proportion of translocating cell bodies were significantly higher in NR2C-2B than in wild-type cultures. Our results provide evidence that NR2B-containing NMDARs can have specialized roles during granule cell migration and can increase migratory speed.

Heterogeneity of parvalbumin-containing neurons in the mouse main olfactory bulb, with special reference to short-axon cells and βIV-spectrin positive dendritic segments

The structural features of parvalbumin-positive neurons were studied in the mouse main olfactory bulb (MOB). Parvalbumin-positive neurons were heterogeneous, including numerous medium-sized interneurons in the external plexiform layer (EPL), some few large short-axon cells and a few periglomerular cells. Their overall distribution pattern and structural features resembled those of the rat MOB. However, large short-axon cells were frequently encountered in the internal plexiform and granule cell layers, which were rare in the rat MOB. In addition a few large short-axon cells were also encountered throughout the EPL. These short-axon cells extended their axons mainly in the EPL, usually making columnar axonal fields. Most parvalbumin-positive cells except periglomerular cells were confirmed to be glutamic acid decarboxylase positive. We examined the immuno-localization of the markers for the axon initial segments (AISs), βIV-spectrin and sodium channels, to determine whether or not heterogeneous parvalbumin-positive neurons have axons. We confirmed their localization on the AISs of the large short-axon cells and periglomerular cells. However, these markers were encountered on some patch-like segments on the dendritic processes instead of the thin axon-like processes of the medium-sized EPL interneurons. The present study revealed the diversity of parvalbumin-positive neurons in the mouse MOB and their particular structural properties hitherto unknown.

GABAergic synaptogenesis marks the onset of differentiation of basket and stellate cells in mouse cerebellum

Type 2 glycine transporter (GlyT2) mediates intracellular glycine transport and is expressed selectively in glycinergic neurons. Expression of GlyT2 gene promoter-driven enhanced green fluorescent protein (eGFP) in BAC transgenic mice allows selective visualization of glycinergic neurons by fluorescence microscopy. Here, we show that cerebellar interneuron precursors identified by the transcription factor Pax2, including gamma-aminobutyric acid (GABA)ergic interneurons of the molecular layer (ML; basket and stellate cells), transiently express GlyT2-eGFP during development. In contrast, expression of endogenous GlyT2 is restricted to glycinergic Golgi cells. Comparison with knock-in mice expressing eGFP in GABAergic neurons [glutamic acid decarboxylase (GAD)67-eGFP] revealed that GlyT2-eGFP expression often precedes GAD67-eGFP and is therefore a marker of immature GABAergic interneurons. In the internal granule cell layer, GABAergic Golgi cells differentiated shortly after birth, prior to glycinergic Golgi cells. In the ML, GlyT2-eGFP-positive precursor cells migrated until the boundary with the external granule cell layer, forming an inside-out maturation gradient that determined the final position of interneurons in the ML. After migration, GlyT2-eGFP gradually disappeared, while interneurons differentiated morphologically and became immunoreactive for parvalbumin, the GABA(A) receptor alpha1 subunit, and the K(+)Cl(-) exchanger KCC2 (K(+)Cl(-) cotransporter type 2). Numerous presumptive GABAergic synaptic terminals were seen on immature ML interneurons as early as P4, preceding the expression of these neurochemical markers. These results suggest that GABAergic synaptogenesis marks the onset of differentiation of basket and stellate cells in the mouse cerebellum, and that GABAergic synaptic function might contribute to the differentiation of interneurons in the cerebellar cortex.

Morphopathological features in tissues of α‐mannosidosis guinea pigs at different gestational ages

Alpha-mannosidosis is an inherited metabolic disorder characterized by a reduction in alpha-D-mannosidase and intralysosomal accumulation of undegraded mannose-containing oligosaccharides. The alpha-mannosidosis guinea pig exhibits pathological similarities to its human counterpart, which make it a valuable animal model. To trace the progression of alpha-mannosidosis during foetal development, brain and visceral organs from affected and unaffected guinea pigs at 30, 36, 38, 51 and 65 days of gestation (dg) were examined by light and electron microscopy (term: approximately 68 dg). In the affected brain, distended lysosomes (vacuoles) were scarce up to 38 dg and were seen in few differentiating neuronal cells but mostly in macrophages, pericytes and endothelial cells. At 51 and 65 dg, several vacuoles were observed in some neurones, in many Purkinje cells, pericytes, endothelial and microglial cells, and in few cerebellar internal granule cells. Myelination had started by 51 dg. Non-myelinated axonal spheroids were detected in the brainstem at 65 dg. In the kidney cortex and liver, an increase in vacuolation was noticed between 36 and 65 dg. Some vacuolated cells were also noticed in the lungs and spleen at 51 and 65 dg. Altogether, these histological observations suggest that alpha-mannosidosis is unlikely to affect ontogenesis before the second half of gestation in guinea pigs; however, the morphopathological features recorded during the last quarter of gestation (which may roughly correspond to the period covering near term to 1-2 years of age in human) were clearly noticeable and may have had some impact.

Differentiation and developmental origin of cerebellar granule neuron ectopia in protein O-mannose UDP-N-acetylglucosaminyl transferase 1 knockout mice

The cerebellar cortex of protein O-mannose UDP-N-acetylglucosaminyl transferase 1 (POMGnT1) knockout mice contains discrete clusters of granule neurons that fail to migrate from the external germinal layer (EGL) to the internal granule cell layer (IGL). To test the hypothesis that the breaches in the pial basement membrane and glia limitans contribute to the formation of such heterotopias, POMGnT1 deficient mice were used to examine the mechanisms underlying these migration defects. The basement membrane, glia limitans, and granule neuron development were assessed with protein markers and immunofluorescent microscopy. Further, the integrity of the pial basement membrane, and granule neuron differentiation state were assessed by electron microscopy. Localized breaches in pial basement membrane and disruptions in the glia limitans were strongly associated with ectopia of EGL cells. In such ectopias, Bergmann glia fibers were retracted and disorganized with very few protruded into the ectopic area. Thus, migration failure was correlated with a compromised Bergmann glia scaffold. Nevertheless, the ectopic EGL cells showed characteristics of differentiated granule neurons and formed synapses with mossy fibers. Altogether, these results suggest that pial basement membrane breaches and glia limitans disruptions are the underlying causes of cerebellar granule neuron ectopia in POMGnT1 knockout mice. Moreover, migration into the IGL is not required for granule cell acquisition of certain differentiated characteristics.

Functions of Chondroitin Sulfate/Dermatan Sulfate Chains in Brain Development: CRITICAL ROLES OF E AND iE DISACCHARIDE UNITS RECOGNIZED BY A SINGLE CHAIN ANTIBODY GD3G7

Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the specificity of GD3G7 toward various glycosaminoglycan preparations showed that this antibody specifically reacted with squid CS-E (rich in the GlcUAbeta1-3GalNAc(4,6-O-sulfate) disaccharide unit E), hagfish CS-H (rich in the IdoUAalpha1-3GalNAc(4,6-O-sulfate) unit iE), and shark skin DS (rich in both E and iE units). In situ hybridization for the expression of N-acetylgalac-tosamine-4-sulfate 6-O-sulfotransferase in the postnatal mouse brain, which is involved in the biosynthesis of CS/DS-E, showed a widespread expression of the transcript in the developing brain except at postnatal day 7, where strong expression was observed in the external granule cell layer in the cerebellum. The expression switched from the external to internal granule cell layer with development. Immunohistochemical localization of GD3G7 in the mouse brain showed that the epitope was relatively abundant in the cerebellum, hippocampus, and olfactory bulb. GD3G7 suppressed the growth of neurites in embryonic hippocampal neurons mediated by CS-E, suggesting that the epitope is embedded in the neurite outgrowth-promoting motif of CS-E. In addition, a CS-E decasaccharide fraction was found to be the critical minimal structure needed for recognition by GD3G7. Four discrete decasaccharide epitopic sequences were identified. The antibody GD3G7 has broad applications in investigations of CS/DS chains during the central nervous system's development and under various pathological conditions.