Normal Mouse Cerebellum Research Articles

Abstract A46: Transcriptome analysis on tumor and its normal cerebellar counterpart from mouse models reveals molecular mechanisms underlying evolution of SHH and Group3 medulloblastoma

Abstract Human medulloblastoma (MB) consists of four molecular subgroups. In spite of enormous effort majority of current MB mouse models are SHH subgroup, represented by Patched heterozygous mice (designated as SHH-A). GTML MB is the only published spontaneous Group3 MB mouse model (designated as G3), which was induced by expressing MycN under the regulation of GLT1 promoter. By using Sleeping Beauty transposon system we have created and published two SHH mouse models: Math1SB11/T2Onc/Ptch+/- (designated as SHH-B) and Math1SB11/T2Onc/P53mut (designated as SHH-C). To dissect the molecular mechanism of SHH and Group3 MB during tumor evolution (initiation, maintenance and maturation), we profiled transcriptomes on SHH-A, SHH-B, SHH-C and G3 MBs along with normal mouse cerebellum (CB) of different developmental stage (E15, E18, P0, P60, P95 and P122). Fetal and/or adult human CBs are routinely used as normal controls for human pediatric MBs. One major advantage of profiling mouse tumor samples over human tumor samples is the availability of age-matched normal mouse CB counterparts. This is particularly true in case of developmental diseases such as pediatric MBs. Indeed, our study showed that transcriptomes of normal mouse CB from E15, E18 and P0 (designated as EdCB, representing early developmental CB), are similar to each other but distinct from mouse CB at P60, P95 and P122 (designated as LdCB, representing late developmental CB). Importantly P60 to P120 are the ages at which most of spontaneous MBs are harvested from a tumor-bearing mouse. We therefore analyzed global gene expression level of MBs and EdCB in comparison with LdCB, the mouse MB's normal counterparts. As a developmental disease, MB is presumably initiated from a single cell or a single cell clone that is transformed at an early developmental stage of a normal CB. In fact, it is well known that a SHH MB is initiated from transformation of granule cell precursors at EGL, the external granule layer. However, it is unclear about the initiation of Group3 MB. In this study we showed that compared to a normal LdCB, a particular MB transriptome harbors four major types of abnormally expressed genes: 1. Genes not only specific to one subgroup but also shared with EdCB; representing tumor initiation events. 2. Genes specific to a subgroup only; representing subgroup-specific tumor promotion events. 3. Genes shared not only by both subgroups but also by EdCB, representing non-subgroup specific tumor maintenance events; 4. Genes shared by both subgroups only; representing non-subgroup specific late tumor maturation events. We confirmed that SHH MBs from SHH-A, B, and C are all initiated at Math1/Atoh1 expressing cells, which are further transformed by up-regulation of Olig3 and MycN among other genes. Rxrg-expressing cells are identified as Group3 MB initiating cells, which are further transformed by over-expression of Crx (Otx3) along with other genes such as Otx2, and down-regulation of cerebellum-specific Zic1 and others such as Lgi1. Both subgroups of MBs may be maintained and mature as genes associated with cell cycle, mitosis and chromosome integrity, such as KIFs, CDK1, PLK1/4, AURKA/B, CENP family and MCMs are abnormally overexpressed. Our study reveals distinct molecular mechanisms underlying tumor evolution of SHH and Group3 MBs. We propose a MB evolution model, illustrating how a normal cell at specific cell of origin can be transformed to a fully developed subgroup specific MB. Furthermore, our results demonstrate that despite the fact that they are initiated from distinct cell of origin, different subgroup of MBs do share common abnormal molecular events for tumor maintenance and maturation, suggesting that subgroup specific target therapies alone may be not as effective as their combination with therapies targeting abnormalities common to different subgroup of MBs. Citation Format: Xiaochong Wu, Adrian Dubuc, William A. Weiss, Robert J. Wechsler-Reya, Michael D. Taylor. Transcriptome analysis on tumor and its normal cerebellar counterpart from mouse models reveals molecular mechanisms underlying evolution of SHH and Group3 medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A46.

Abstract 4774: LC-MS/MS detection of the dansyl-modified biologically active CDK inhibitor VMY-1-103 using an in vivo model system

Abstract The development of new small molecule kinase inhibitors as cancer therapeutic drugs has placed increased emphasis on determining the mechanism of action of the new compounds preclinically, such as in genetically engineered mice or using human tissue xenografts. The ability to accurately measure the pharmacokinetics and pharmacodynamics of new compounds is often hampered by the lack of suitable platforms to measure serum and tissue distribution with high sensitivity and specificity. We have recently developed a novel dansylated cyclin dependent kinase inhibitor called VMY-1-103 (VMY) that has purvalanol B as its base compound. We established that VMY was significantly more effective in blocking cell cycle progression and inducing apoptosis than purvalanol B. Since dansylation is frequently used to enhance mass spectrometry-based quantitative analyses, we hypothesized that this modality could similarly be used to evaluate the tissue distribution of VMY in mouse models. In the current study, we developed a rapid liquid phase-extraction of VMY and purvalanol B from mouse tissue and validated a sensitive liquid chromatography-tandem mass spectrometry based assay to quantify tissue delivery of these compounds in vivo. Comparisons of the standard curves for VMY versus Purvalanol B revealed a greater than 7.7-fold increase in sensitivity for VMY. Using both the ND-2 SmoA1, a genetically engineered mouse model of medulloblastoma, and normal mice, we quantified tissue delivery by mass spectrometry as well as Area Under the Curve (AUC) analysis by integrating the peak intensity normalized by an internal standard. Our analyses revealed that VMY delivered intraperitoneally at 20mg/kg was found at its highest concentration in serum 4 hours after injection. We further established that VMY was distributed in multiple tissues, including pancreas and prostate at varying levels and, importantly, we establish that VMY was able to traverse an intact blood-brain barrier as it was detected in the brain and cerebellum of normal mice. Purvalanol B delivered in the same manner as VMY was found at significantly lower levels in all tissues examined and unlike VMY, we failed to detect purvalanol B in brain or and cerebellar tissue. The significance of this study illustrates that a biologically active compound can be modified by dansylation and exhibit both increased sensitivity to mass spectrometry and enhanced bioavailability and tissue delivery, including the brain, in vivo. We also define a simple mass spectrometry-based tissue and serum assay. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4774. doi:1538-7445.AM2012-4774

The role of microRNAs during the genesis of medulloblastomas induced by the hedgehog pathway

Constitutive hedgehog (Hh) signaling is associated with the genesis of medulloblastomas (MB). The objective of this study is to identify special microRNAs (miRNAs) regulated by the Hh pathway, and to clarify the role of miRNAs during the genesis of MB induced by sustained Hh activation. In the primary screening, we used stem-loop RT-PCR to test the expression of 90 different miRNAs in the wildtype (WT) and Ptc−/− MEF cell lines. In the secondary screening, the miRNAs screened from the first screening were validated in the Sufu−/− MEF cell lines. We then verified the expression of miRNAs both in the normal cerebellar tissues and the MB induced by activated Hh pathway, and examined the expression of the other 21 miRNA members of the miR-154 cluster in the MB and normal cerebellum. In the first screening, 13 miRNAs showed significant differential expression in WT and Ptc−/− MEF cell lines, while 10 of them had significant difference in the Sufu−/− MEF cell line. Compared to the normal mouse cerebellum, only 2 miRNAs in 15 miRNAs were differentially expressed between the MB and normal cerebellar tissues. Among 21 members of the miR-154 cluster, 6 miRNAs were downregulated in the MB. Our study demonstrated that miR-154 may be regulated by the Hh pathway, and the activation of the Hh pathway led to the downregulation of the miR-154 cluster, resulting in the genesis of MB.

Alteration of Brain Type N-Glycans in Neurological Mutant Mouse Brain

We have previously detected two brain-specific and development-dependent N-glycans [H. Shimizu, K. Ochiai, K. Ikenaka, K. Mikoshiba, and S. Hase (1993) J. Biochem. 114, 334-338]. In the present study we attempted to analyze specific N-glycans detected in neurological mutant mice. N-glycans in cerebrum and cerebellum obtained from 3-week-old neurological mutant mice (jimpy, staggerer, and shiverer) were compared with those obtained from normal mice. N-glycans liberated from the cerebrum and cerebellum by hydrazinolysis-N-acetylation were pyridylaminated, and pyridylamino derivatives of N-glycans thus obtained were separated into neutral and five acidic fractions by anion exchange chromatography. PA-N-glycans in each fraction were compared with those obtained from normal mice by reversed-phase HPLC, and the following results were obtained. The ratio of the two brain-type N-glycans, Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-1) to GlcNAcbetaManalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fuca1-6)GlcNAc (BA-2), was higher in staggerer mice than other mutant mice and normal mice. Sia-Gal-BA-2, triantennary N-glycans, and bisected biantennary N-glycans were found in the cerebellum of shiverer and staggerer mice but not in normal or jimpy mice. High-mannose type N-glycans were not altered in mutant mice brains. The amounts of disialylbiantennary N-glycans and disialylfucosylbiantennary N-glycans were lower in jimpy mouse cerebellum than in normal mouse cerebellum, but were higher in shiverer mouse. Some alterations of N-glycans specific to mutations were successfully identified, suggesting that expression of component(s) of the N-glycan biosynthetic pathway was specifically affected in neurological mutations.

Cell Death

Back to table of contents Previous article Next article Images in NeuroscienceFull AccessCell DeathMichael W. Vogel, Ph.D., Michael W. VogelSearch for more papers by this author, Ph.D., Baltimore, Md.Published Online:1 Aug 2005https://doi.org/10.1176/appi.ajp.162.8.1503AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail The top figures illustrate the magnitude of volume loss in the Lurcher cerebellum (B) relative to normal mouse cerebellum (A) due to the caspase-mediated apotosis that occurs in the Lurcher mouse Purkinje cells. In (C), a dying neuron is shown in bright green expressing significant amounts of activated caspase-3, one of the cell death proteins. In the same section, a Purkinje cell-specific calcium binding protein labels the healthy neurons (bright pink) but not the dying neuron (D).During human development, approximately half of the neurons that are originally laid down in the brain are estimated to die during the process of central nervous system formation. Many neurodegenerative diseases, including Huntington’s, Alzheimer’s, and Parkinson’s, are thought to involve cell death, a process called apoptosis. Moreover, the regulation of cell death continues to be an important physiological process throughout the lifespan of an animal as a mechanism to respond to disease and injury. It is not surprising that a complex cellular machinery has evolved to regulate cell death during development and after injury. Many of the genes and protein families involved in cell death and survival have been identified. One animal model involving regulated cell death is a mouse mutant called Lurcher; this model has been used to identify many of the molecular events involved in cell death. The Lurcher mutation is a single base-pair substitution in the gene for the delta-2 glutamate receptor (one of the glutamate receptor subtypes in the cerebellum) that aberrantly codes for a receptor with a continuously open sodium channel instead of a channel that opens and closes in responsive to local modulation. This delta-2 glutamate receptor is predominately expressed in cerebellar Purkinje cells; the Lurcher Purkinje cells start to die after the first week of postnatal development. Virtually all of the Purkinje cells and many of the neurons that connect to the Purkinje cells die within a month of birth (see Figure), greatly reducing the size of the cerebellum.The mechanisms of cell death in the Lurcher Purkinje cells are thought to model for some of the mechanisms involved in human neurodegenerative disorders like Huntington’s, Alzheimer’s, and Parkinson’s. Several important key molecules involved in the regulation of cell death belong to a family called Bcl-2 proteins. There are “good” Bcl-2 proteins: e.g., Bcl-2, Bcl-xL, Bcl-w, and Boo; these promote cell survival. And there are “bad” Bcl-2 proteins: e.g., Bax, Bak, and Bim; these promote cell death. Indeed, the ratio of Bcl-2 to Bax within a cell is thought to modulate the ultimate outcome of the cell. If there is more Bcl-2 then Bax, the cell will live; if more Bax than Bcl-2, then the cell will die. The cell death pathway is initiated by the activation of another class of proteins (suitably named “executioner” proteins) called caspases that are already present in most cells in an inactive form. A high ratio of Bax to Bcl-2 activates caspases family members to obliterate cellular proteins and DNA, leading to cell death. The Lurcher Purkinje cell in the Figure (bright green cell) is immunolabeled for activated caspase-3, one of the final caspases in the cell death pathway, indicating a cell slated for death. This dying Purkinje cell is surrounded by other Lurcher Purkinje cells that have not yet begun to die (bright pink cells). These same proteins (among others) regulate cell death in humans during development, life and death. Moreover, this same caspase-mediated cell death pathway can be initiated by any number of physiological stressors, including loss of trophic support, oxidative stress, drugs, and ionizing radiation. The Lurcher mouse is used in the study of these cell death pathways, informing mechanisms of neurodegeneration in humans.Address reprint requests to Dr. Tamminga, UT Southwestern Medical Center, Department of Psychiatry, 5323 Harry Hines Blvd., #NC5.914, Dallas, TX 75390-9070; [email protected] (e-mail). FiguresReferencesCited byDetailsCited byNone Volume 162Issue 8 August 2005Pages 1503-1503 Metrics PDF download History Published online 1 August 2005 Published in print 1 August 2005

A murine model for neuropsychiatric disorders associated with group A beta-hemolytic streptococcal infection.

A syndrome of motoric and neuropsychiatric symptoms comprising various elements, including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, can occur in association with group A beta-hemolytic streptococcal (GABHS) infection. We tested the hypothesis that an immune response to GABHS can result in behavioral abnormalities. Female SJL/J mice were immunized and boosted with a GABHS homogenate in Freund's adjuvant, whereas controls received Freund's adjuvant alone. When sera from GABHS-immunized mice were tested for immunoreactivity to mouse brain, a subset was found to be immunoreactive to several brain regions, including deep cerebellar nuclei (DCN), globus pallidus, and thalamus. GABHS-immunized mice having serum immunoreactivity to DCN also had increased IgG deposits in DCN and exhibited increased rearing behavior in open-field and hole-board tests compared with controls and with GABHS-immunized mice lacking serum anti-DCN antibodies. Rearing and ambulatory behavior were correlated with IgG deposits in the DCN and with serum immunoreactivity to GABHS proteins in Western blot. In addition, serum from a GABHS mouse reacted with normal mouse cerebellum in nondenaturing Western blots and immunoprecipitated C4 complement protein and alpha-2-macroglobulin. These results are consistent with the hypothesis that immune response to GABHS can result in motoric and behavioral disturbances and suggest that anti-GABHS antibodies cross-reactive with brain components may play a role in their pathophysiology.

Activation of the IGF-IR system contributes to malignant growth of human and mouse medulloblastomas.

Insulin-like growth factor I receptor (IGF-IR) has been implicated in the normal and malignant growth of many cell types including cells from the central nervous system. In the cerebellar cortex IGF-IR mRNA is found in granular cells and IGF-I stimulation is mitogenic and protects cells from low-potassium-induced apoptosis. Since primitive neuroectodermal tumors/medulloblastomas (PNETs/medulloblastomas) are suspected to originate from the external cerebellar granular layer, it is reasonable to postulate that IGF-IR and/or its signaling molecules may contribute to the transformation of these poorly differentiated cells. To study activation of the IGF-IR system in medulloblastomas, we have utilized an antibody (anti-pY1316) that specifically recognizes the phosphorylated (active) form of the IGF-IR. Medulloblastoma biopsy specimens were positive when examined immunohistochemically with anti-Y1316 antibody. Further analysis of the IGF-IR system was performed in three human (Daoy, TE-671, D283 Med) and four mouse (BsB8, BsB13, Bs-1b, Bs-1c) medulloblastoma cell lines. All the murine cell lines examined express IGF-IR and PI3-kinase at relatively normal levels, and grossly overexpress IRS-1, when compared with normal mouse cerebellum. Within 15 min following IGF-I stimulation both mouse and human cell lines phosphorylate the beta subunit of the IGF-IR, IRS-1, Akt, and MAP kinases. They respond with cell proliferation when stimulated solely with IGF-I and are strongly inhibited when challenged with a dominant negative mutant of the IGF-IR (486/STOP), or with antisense oligonucleotides against the IGF-IR mRNA.

Cerebellar excitatory amino acid binding sites in normal, granuloprival, and purkinje cell-deficient mice

Using quantitative autoradiography, the cellular localization and characterization of cerebellar excitatory amino acid binding sites in normal, Purkinje cell-deficient and granuloprival (granule cell-deficient) mouse cerebella were investigated. In the molecular layer of normal mouse cerebellum, the quisqualate subtype of excitatory amino acid receptor (assayed by[ 3H](RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive l-[ 3H]glutamate, and [ 3H]6-cyano-7-nitroquinoxaline-2,3-dione binding) predominated. In the granule cell layer of the cerebellum,N-methyl- d-aspartate-sensitive l-[ 3H]glutamate and [ 3H]glycine binding sites were predominant. In the molecular layer of Purkinje cell-deficient mutant mice,[ 3H](RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites and [ 3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding were reduced to 24% (P < 0.01) and 36% (P < 0.001) of control, respectively, while quisqualate-sensitive [ 3H]glutamate binding sites were reduced to 54% of control (P < 0.01).N-Methyl- d-aspartate-sensitive [ 3H]glutamate and [ 3H]glycine binding were unchanged. In the granule cell layer of these mouse cerebella, there was no change in excitatory amino acid receptor binding. In the molecular layer of granuloprival mouse cerebella,[ 3H](RS)-α-amino-3-hydroxy-5-methylisoxa-zole-4-propionate binding was increased to 205% of control (P < 0.01), [ 3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding was increased to 136% of control (P < 0.02), and quisqualate-sensitive [ 3H]glutamate binding was increased to 152% of control (P < 0.01).N-Methyl- d-aspartate-sensitive [ 3H]glutamate and [ 3H]glycine binding were unchanged. In areas of granule cell depletionN-methyl- d-aspartate-sensitive [ 3H]glutamate and [ 3H]glycine binding were reduced to 68% (P <0.01) and 59% (P < 0.01) of control, respectively. In the granule cell layer, binding to quisqualate receptors was not significantly different from binding in controls with any of the ligands tested. These results suggest that three different receptor assays:[ 3H](RS)-α-amino-3-hydroxy-5-methylisoxa-zole-4-propionate, quisqualate-sensitive l-[ 3H]glutamate, and [ 3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding can be used to demonstrate that quisqualate receptor specific binding sites are located on Purkinje cell dendrites in the molecular layer of cerebellum, and that these binding sites apparently up-regulate in response to granule cell ablation and Purkinje cell deafferentation.

Developmental expression of polypeptide PEP-19 in cerebellar cell suspensions transplanted into the cerebellum of pcd mutant mice.

Cerebellar cell suspensions were prepared from normal mouse embryos and implanted into the cerebellum of Purkinje cell degeneration (pcd) mutant mice, which are characterized by a virtually complete degeneration of Purkinje cells between postnatal day (P) 17 and P45. The expression of immunoreactivity for PEP-19, a developmentally-regulated brain-specific polypeptide, was analyzed in normal mouse cerebellum, as well as in pcd mutants with or without grafts. In the normal cerebellum, PEP-19 immunoreactivity was present in Purkinje cells. In unoperated mutants, 45 days of age or older, Purkinje cells were absent. In grafted pcd mice, numerous PEP-19 immunoreactive, neuroblast-like cells were seen in the graft at 5 days after transplantation. By 9 days, large PEP-19 immunoreactive neurons were found in the host molecular layer; by 17 days after transplantation, such neurons displayed an extensive dendritic tree and resembled differentiated Purkinje cells. The vast majority of PEP-19 immunoreactive cells was located in the molecular layer of the host at 9 days after transplantation and beyond; nonetheless, the same cells extended axonal processes toward the graft, indicating an affinity for co-grafted (possibly deep nuclei) neurons. These results point to the ability of donor Purkinje cells for survival, migration into the host brain and morphological and chemical differentiation following transplantation to the degenerated cerebellar cortex of the recipient mutants.

Histochemical localization of 5'-nucleotidase in the reeler mutant mouse.

In the normal mouse cerebellum, 5'-nucleotidase localization exhibits a parasagittal organization with bands of positive and negative staining within the molecular layer. The reeler cerebellum, which possesses an abnormal cytoarchitecture with numerous ectopically located Purkinje cells, was stained histochemically for the presence of 5'-nucleotidase. The resulting staining pattern suggests that 5'-nucleotidase is found in association with areas containing subpopulations of Purkinje cells, independent of whether they are in normal or ectopic positions.

Autoradiographic localization of cerebellar excitatory amino acid binding sites in the mouse

We have investigated the cellular localization of cerebellar excitatory amino acid binding sites in normal mice, in mice deficient in granule cells and, perhaps, stellate, basket and Golgi cells (granuloprival mice) and in mice lacking Purkinje cells. In the molecular layer of normal mouse cerebellum, the quisqualate-sensitive binding sites were the predominant type of excitatory amino acid receptor and there were relatively few N-methyl- d-aspartate or kainate-sensitive binding sites. The granule cell layer of normal mice contained a mixture of all 3 types, the N-methyl- d-aspartate-sensitive binding sites being predominant. In the molecular layer of granuloprival mice, the number of quisqualate-sensitive binding sites was increased to 214% of control ( P < 0.01), whereas N-methyl- d-aspartate-sensitive binding sites were decreased to 62% of control ( P < 0.001) and kainate-sensitive binding sites were unchanged. In the granule cell layer of these mice, quisqualate-sensitive binding sites were increased to 200% ( P < 0.01), N-methyl- d-aspartate-sensitive binding sites were decreased to 47% ( P < 0.001) and kainate-sensitive binding sites were decreased to 49% ( P < 0.01) of their respective control values. In the molecular layer of mice lacking Purkinje cells, quisqualate-sensitive binding sites were reduced to 29% ( P < 0.001) of control and N-methyl- d-aspartate-sensitive binding sites were unchanged. In the granule cell layer of these mice, neither quisqualate nor N-methyl- d-aspartate-sensitive binding sites were changed. These results suggest that (1) quisqualate-sensitive binding sites are located principally on dendrites of Purkinje cells and that they up-regulate after deafferentation; (2) N-methyl- d-aspartate-sensitive binding sites are located on granule cells and, perhaps, stellate, basket and Golgi cells, and (3) kainate binding sites are located on cell bodies of granule and, perhaps, Golgi cells.

Cerebellar structure and function in the murine mutant “jolting”

The morphology of the cerebellar cortex of the murine mutant “jolting” and its phenotypically normal littermate was studied. The general organisation of the cerebellum of normal mice was similar in all respects to that described for other vertebrate species. In “jolting” mice aged 4 months or more there was a marked loss of Purkinje cells and spheroids were present on Purkinje cell axons. An ultrastructural examination showed that the spheroids contained randomly orientated neurofilaments, degenerating mitochondria and autophagic vacuoles. The abnormalities in the cerebellum appeared confined to the Purkinje cells. Extracellular recordings were made of the electrical activity of the cerebellar cortex of anaesthetised mice. In the cerebellar cortex of “jolting” animals, Purkinje cells generated little spontaneous activity, but complex discharges resulting from climbing fibre inputs were indistinguishable from normal. The abnormal electrical activity of Purkinje cells in “jolting” mice could be seen in animals as young as 3 weeks. It was concluded that the “jolting” mouse suffers from a cerebellar ataxia.

Evidence that mouse astrocytes may be derived from the radial glia: An immunohistochemical study of the cerebellum in the normal and reeler mouse

Astrocytic cells of unusual aspect can be detected in the cerebellum of normal mice during the first 4 weeks of life. They are visualized with anti-GFAP (glial fibrillary acidic protein), anti-S100 and anti-vimentin immune sera. Their perikaryons, located in the white matter or in the granular layer, extend long processes which are inserted onto the pial surface. These cells may be transitional forms between the radial glial cells and some of the differentiated astroglial elements. These unusual astrocytes are more numerous and heavily stained in the reeler mutant than in the normal mouse and it is suggested that our observations signify some degree of glial immaturity in the cerebellum of the mutant.

Effects of 6-hydroxydopamine-induced norepinephrine depletion on cerebellar development.

In order to investigate the possible influence of norepinephrine (NE) on neuronal development, the effects of neonatal NE depletion on postnatally developing cell populations were characterized in (1) the normal mouse cerebellum, and (2) a cerebellar model system with delayed germinal cell development produced by neonatal methylazoxymethanol acetate (MAM) administration. Reduction in NE content was achieved by neonatal 6-hydroxydopamine (6-OHDA) injections. In the MAM-treated groups, 6-OHDA did not produce significant changes in external germinal layer (EGL) reconstitution. In the other groups, 6-OHDA treatment produced a reduction in the vermis area, alterations in foliation, and changes in the schedule of EGL development. These results support a role for NE in regulation of neuronal development in the cerebellum.

Net sulfatide synthesis, galactosylceramide sulfotransferase and arylsulfatase a activity in the developing cerebrum and cerebellum of normal mice and myelin-deficient jimpy mice

Net sulfatide synthesis, galactosylceramide sulfotransferase (EC 2.8.2.11) and arylsulfatase A (EC 3.1.6.1) activities were measured in two brain regions, cerebrum and cerebellum, of normal and jimpy mice during postnatal development. In normally myelinating mice, two phases of increasing rates of net sulfatide synthesis were observed, the first coinciding with oligodendrocyte proliferation and the second with myelination. Net sulfatide synthesis was quantitatively higher in the cerebellum than in the cerebrum. In both brain regions, the developmental patterns of net sulfatide synthesis were related to the activity patterns of both galactosylceramide sulfotransferase and arlysulfatase A. In jimpy mice, a neurological mutant showing hypomyelination in brain, the first phase of net sulfatide synthesis was preserved in both brain regions and galactosylceramide sulfotransferase and arylsulfatase A activities were normal up to 12 days. However, during the phase in which myelination occurred in controls, the net sulfatide synthesis in both brain regions of jimpy mice was zero or even negative. The sulfatide deficit was larger in the cerebellum than in the cerebrum. In both mutant brain parts, galactosylceramide sulfotransferase activity increased up to 12 days showing about 50% of the maximal activities observed in normal brain regions. Thereafter up to 15 days, enzyme activity decreased to about 25% of that of controls and remained low in both brain regions. The developmental patterns and the activities of arylsulfatase A were, however, normal in the cerebrum and cerebellum of jimpy mice. These results suggest that the enzyme activities and the developmental patterns of galactosylceramide sulfotransferase and arylsulfatase A as measured in vitro reflect to a high degree their functional activity in vivo. Furthermore, sulfatide degradation by arylsulfatase A seems to be important in regulating net sulfatide synthesis during normal and impaired myelination.

Estradiol and testosterone binding in normal and mutant mouse cerebellum: Biochemical and cellular specificity

Estradiol and testosterone binding macromolecules are demonstrated in normal mouse cerebellum. Identity of the estradiol binder as the ‘receptor’ is provided by its binding to DNA—cellulose, which does not occur for other estradiol binding proteins. The androgen binder is identified as ‘receptor’ by its specific deficiency (85% reduced) in the androgen-insensitive mutant mouse, testicular feminization ( Tfm). The relative amounts of these two components are reversed in cerebellum compared to hypothalamus-preoptic area. Neurological mouse mutants, which lack specified neurons, are examined to test for the cells in which these hormone binding proteins are located. Experiments with Purkinje cell degeneration ( pcd) weaver ( wv) and staggerr ( sg), suggest that the majority of these receptors are present in granule cells.

Glial cells and the central myelin sheath.

Glial cells and the central myelin sheath.