Localization of Hexokinase in Neural Tissue: Immunofluorescence Studies on the Developing Cerebellum and Retina of the Rat

Abstract: Antiserum against purified rat brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been used in a study of the distribution of hexokinase during the postnatal development of rat cerebellum and retina. The cells of the external germinal layer of the cerebellum exhibit little or no fluorescence. The Purkinje cells exhibit a transient increase in hexokinase levels between 2 and 8 days postnatally, followed by a precipitous decrease (8–12 days) to the relatively low levels found in the mature Purkinje cell. Development of the intensely fluorescent cerebellar glomeruli in the granule cell layer is readily followed during the 3rd and 4th weeks postnatally. With respect to postnatal changes in hexokinase distribution of the retina, perhaps most notable is the observation that even the cytoplasmic protrusions which represent the precursors of the photoreceptor segments are richly endowed with hexokinase. Biochemical differentiation of the photoreceptor segments into hexokinase-rich inner segments and hexokinase-poor outer segments is readily observed long before the growth of the photoreceptor segments has been completed.     

A Histochemical Study of the Distribution of β-Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum

The distribution of beta-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8-11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) the transient external germinal layer of the cerebellum did not show appreciable staining. Since beta-hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.     

Secretogranin II and its Derivative Peptide,Manserin, are Differentially Localized in Purkinje Cells and Unipolar Brush Cells in the Rat Cerebellum

The cerebellum has long been recognized as the primary center of motor coordination in the central nervous system. Cerebellar neuropeptides have been postulated to be involved in such motor coordination, though this role is not fully understood. We herein investigated the localization of novel neuropeptide, “manserin” in the adult rat cerebellum. Punctate signals of manserin immunoreactivity were observed in the granular layer of the rat cerebellum. Manserin signals were also observed in the fibers and fiber terminals in the granular layer as well as the molecular layer. Manserin did not localize in Purkinje cells. Interestingly, cerebellar manserin was preferentially colocalized with unipolar brush cells, a class of excitatory granular layer interneuron, which are known to be involved in vestibullocerebellar functions. These results indicate that manserin plays pivotal roles in the cerebellar functions.     

THE DEVELOPMENT OF D-AMINO ACID OXIDASE IN RAT CEREBELLUM

D-Amino acid oxidase (D-amino acid: O₂ oxidoreductase (deaminating), EC 1.4.3.3; D-AAO) activity is biochemically undetected in rat brain stem, cerebellum and forebrain until 14 days after birth. Adult levels are attained by day 30 in the brain stem, and by day 36 in the cerebellum. At adulthood, forebrain D-AAO activity per g wet weight of tissue is less than 2% that of the cerebellum. In contrast to the pattern in the CNS, substantial D-AAO activity is present in both liver and kidney 2 days before birth and adult levels are approached within 2 weeks of birth. Nonetheless, D-AAO activities in rat liver, kidney, brain stem and cerebellum are likely to be due to a single enzyme which has properties very similar to the purified hog D-AAO. The late ontogenesis of D-AAO activity in cerebellum and brain stem relative to that in liver and kidney parallels reported phylogenetic data. Histochemical staining for D-AAO in rat cerebellar cortex is absent until 15 days after birth when activity is first observed in some cells of the external germinal zone and adjacent molecular layer. These cells appear to migrate to a final destination around the Purkinje cell soma and leave processes at the pial surface. By 21 days of age an adult pattern of staining is manifest throughout the cerebellum but it is of weak intensity. The adult pattern includes some staining in the granular layer which seems to be associated with mossy fibers and certain cerebellar glomeruli, and strong staining at the pial surface, in the molecular layer, and in cells surrounding, but not within, the Purkinje cell soma. The data suggest that the biochemical appearance of D-AAO in developing cerebellum derives from two sources: one associated with differentiation of one of the last cell types to form from the external germinal zone, and the other with maturation of mossy fibers and their synapses (cerebellar glomeruli).     

Expression of FSH and its co-localization with FSH receptor and GnRH receptor in rat cerebellar cortex

The expression of follicle-stimulating hormone (FSH) and its receptor in extrapituitary and non-HPG axis tissues has been demonstrated and their non-reproductive functions in these tissues have been found. However, there have been no reports concerning the expression and function of FSH and its receptor in the cerebellum. In our study, immunofluorescence staining and in situ hybridization were used to detect the expression of FSH, double-labeled immunofluorescence staining was used to detect co-localization of FSH and its receptor and co-localization of FSH and gonadotropin-releasing hormone (GnRH) receptor in the rat cerebellar cortex. Results showed that some cells of the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex showed both FSH immunoreactivity and FSH mRNA positive signals; not only for FSH and FSH receptor, but also for FSH and GnRH receptor co-localized in some cells throughout the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex. These suggested that rat cerebellum could express FSH; cerebellum is a target tissue of FSH; FSH may exert certain functions through FSH receptor in a paracrine or autocrine manner; GnRH may regulate FSH positive cells through GnRH receptor in the cerebellum. Our study provides morphological evidence for further functional research on FSH and related hormones in the cerebellum.     

The differential distribution of beta tubulin mRNAs in individual mammalian brain cells

It has been shown by in vitro translation of polyadenylated messenger RNAs (poly(A)+ mRNAs) that the mRNAs encoding both alpha and beta tubulin isotypes are present at much higher relative levels in the developing rat brain than they are in the adult, suggesting that the requirements for tubulin subunits vary with cell type and/or with the developmental stages of a particular cell type. The postnatally developing rat cerebellum, with its readily identifiable cell populations that perform the gamut of developmental tasks, is a suitable model for analyzing specific cellular mRNA distributions during development. In this report, by in situ hybridization techniques it is shown that, by comparison to total cellular poly(A)+ mRNA levels, there is relatively more of the total beta tubulin mRNAs in mitotically active external granule layer cells than in those in the internal granule layer. These results show that migration and differentiation of these granule cells is accompanied by a decrease in their beta tubulin mRNA levels relative to the levels in granule cells of the external granule cell layer. Furthermore, the relative levels of beta tubulin mRNA both in the prenatally formed Purkinje cells and the postnatally formed stellate cells are two to fourfold less than in the granule cells of the internal granule cell layer.     

Disturbed Purkinje cell migration due to reduced expression of Reelin by X-irradiation in developing rat cerebellum.

The major histogenetic events of the rat cerebellum take place in the early postnatal days. During this period, precursors of microneurons, such as granule cells, form the external granular layer (EGL), extend over the surface of the primordial cerebellum, and actively proliferate. Postmitotic granule cells leave the EOL and migrate to the internal granular layer (IGL). On the other hand, guided by radial glial fibers, immature Purkinje cells migrate from the ventricular zone of the fourth ventricle and settle in the Purkinje cell plate with thickness of several cells. Various cell adhesion molecules are involved in the interaction between the migratory immature Purkinje cells and processes of the radial glia as the basis for contact guidance. The second process is the formation of immature Purkinje cells to the monolayer. This process takes place at the first week after birth of the rat and cell adhesion molecules such as neural cell adhesion molecule (NCAM), fibronectin, tenascin and Reelin are also suggested to play an important role for the cell patterning. When rat fetuses are exposed to X-radiation in the last gestation period, abnormal foliation of the cerebellum develops with ectopic Purkinje cells. The molecular mechanism that contributes to abnormal migration of Purkinje cells and foliar malformation induced by X-irradiation in the cerebellum are not yet clear. This study was undertaken to elucidate the mechanisms of ectopic Purkinje cell formation by examining the expression of cell adhesion molecules.     

Differential expression of alpha-tubulin mRNA in rat cerebellum as revealed by in situ hybridization

Nucleotide sequence analysis of two rat alpha-tubulin cDNA clones showed a marked divergence in their 3'-untranslated regions. However, each of the alpha-tubulin isotypes shows a high interspecies homology in this region, when compared with an isotubulin sequence from human and Chinese hamster. In situ hybridization of rat cerebellum with alpha-tubulin cDNA revealed differential expression in various cell layers. The mitotically active cells in the external granular layer show the highest level of alpha-tubulin mRNA, while lower levels are observed in the migrating cells in the molecular layer and in the differentiating cells in the internal granular layer. Very low levels of the mRNA are observed in the prenatally differentiated Purkinje cells.     

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.     

Regulation and developmental expression of the divalent metal-ion transporter in the rat brain.

Divalent metal ion transporter 1 (DMT1) is a recently identified metal-ion transporter that appears to mediate the absorption of iron in the intestine. DMT1 mRNA is also present in discrete areas of the brain. In this study, we examined the expression of DMT1 mRNA in developing rat brain. DMT1 mRNA was found by in situ hybridization in the striatum, cortex, hippocampus and cerebellum. During development, DMT1 mRNA was found in Purkinje and granule cells in the cerebellum at post-natal day (PND) 14 and PND 30. DMT1 mRNA was also expressed in the external granular layer of the cerebellum at PND 14. No change in the level of DMT1 mRNA was observed by Northern analysis in the cerebellum at different ages between PND 1 and 21. DMT1 was found by Northern analysis in cultures of rat astrocytes. Activation of protein kinase C increased the expression of DMT1 in kidney epithelial cells but not astrocytes from newborn rats. Because DMT1 is expressed in a wide variety of types of cells, we suggest that it plays an important role in metal homeostasis in the brain.     

Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.     

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.     

Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been found in the cerebellum of many vertebrates and in the gastrointestinal tract of African ostrich chicks, but little is known about its distribution in the cerebellum of the African ostrich. In the present study, the distribution and morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich were investigated using immunohistochemistry. The results indicate that the cerebellum is divided into two sections: the outer cerebellar cortex and the inner medulla of cerebellum. The cerebellar cortex comprises a molecular layer, a Purkinje cell layer and a granular layer; ghrelin-immunopositive (ghrelin-ip) cells were localized throughout the entire cerebellum, but sparsely in the medulla. The greatest number of ghrelin-ip cells was found in the stratum granulosum, and the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex. The ghrelin-ip cells were fusiform or irregular polygons and their cytoplasm was stained intensely. These results clearly demonstrate the presence of ghrelin-ip cells in the cerebellum of the African ostrich. It is speculated that ghrelin may have a physiological function in the cerebellum.     

Changes in neurotrophin responsiveness during the development of cerebellar granule neurons.

Neurotrophins and their receptors are widespread in the developing and mature CNS. Identifying the differentiation state of neurotrophin-responsive cells provides a basis for understanding the developmental functions of these factors. Studies using dissociated and organotypic cultures of rat cerebellum demonstrated that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) affect developing granule cells at distinct stages in differentiation. While early granule neurons in the external germinal layer responded to BDNF, more mature granule cells responded to NT-3. BDNF, but not NT-3, enhanced survival of granule cells in cultures of embryonic cerebella. Thus, BDNF and NT-3 have distinct sequential functions that are likely to be critical in the development of the cerebellum. BDNF may promote the initial commitment, while NT-3 may direct the subsequent maturation of granule cells.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.     

Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Abstract: Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13–day-old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [¹²⁵I-Tyr⁰,D-Trp⁸]SRIF-14 ([¹²⁵I-Tyr⁰,d -Trp⁸]S14) and ^I25I-SMS 204–090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13–day-old rat cerebellum showed that the A'_D values for [¹²⁵I-Tyr⁰,D-Trp⁸]S14 and ¹²⁵I-SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding B_max values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SSI). Competition studies showed that different D-Trp-sub-stituted analogs displaced [¹²⁵I-Tyr⁰,d -Trp⁸]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr⁰,d -Trp⁸]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day-old cerebellum and adult cerebral cortex. The higher-affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower-affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn²⁺ was the most efficient cation for promoting binding of [¹²⁵I-Tyr⁰,d -Trp⁸]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF-Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same K_D and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF-binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF-Rs. The extremely high density of SRIF-Rs found in the external granule cell layer in 13–day-old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.     

Cerebellar Hypoplasia in the Gunn Rat with Hereditary Hyperbilirubinemia: Immunohistochemical and Neurochemical Studies

Immunohistochemical reactions were conducted, using the antibodies against GFA and S-100 proteins on sections of cerebellum from the homozygous (jj) and the heterozygous (Jj) Gunn rats. Hypertrophy of the fibrous astrocytes was observed but hyperplasia of the glial cells was not. Although the molecular layer was very thin, the Bergmann fibre appeared normal. Among the free amino acids in the cerebellum from the jj rat, glutamate concentration decreased to two-thirds of the control level. The protein profile of the cerebellum from the jj rat obtained by SDS-polyacrylamide gel electrophoresis revealed that the amount of P400 protein that is characteristic of Purkinje cells decreased considerably and there were also some changes of the other unidentified proteins. By two-dimensional electrophoresis, it was observed that in the supernatant from the jj rat cerebellum one protein spot diminished and in the particulate fraction from the jj rat one spot was enormously increased. The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the cerebellum from the jj rat did not differ significantly from that of the control; however, activities of choline acetyltransferase and acetylcholinesterase of the jj rat were about twice as high as those of the control. 2-Deoxyglucose incorporation was maximum in the granular layer from both the jj and the Jj rat cerebella. However, the incorporation in the jj cerebellum was not higher than in the Jj control and even lower in some parts of the jj cerebellum than in the control Jj cerebellum.