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Altered intracellular localization of the glutamate receptor channel δ2 subunit in weaver and reeler Purkinje cells
Brain Research 745 Ž1997. 231–242
Research report
Altered intracellular localization of the glutamate receptor channel d 2 subunit in weaver and reeler Purkinje cells Chitoshi Takayama a
a, )
, Shin Nakagawa a , Masahiko Watanabe a , Hideo Kurihara a , Masayoshi Mishina b , Yoshiro Inoue a
Department of Anatomy, Hokkaido UniÕersity School of Medicine, Kita-15 Nishi-7, Kita-ku, Sapporo 060, Japan b Department of Pharmacology, Faculty of Medicine, UniÕersity of Tokyo, Tokyo 113, Japan Accepted 17 September 1996
Abstract The glutamate receptor ŽGluR. channel d 2 subunit is expressed abundantly and specifically in cerebellar Purkinje cells. Our previous study demonstrated that the GluR d 2 mRNA is expressed as early as embryonic day 15 prior to Purkinje cell synaptogenesis, and its protein product accumulates in dendritic spines during normal Purkinje cell maturation. In this study, we examined expression and distribution of the GluR d 2 in the weaver and reeler mutant cerebelli, which show abnormal cytoarchitecture and neural circuitry. In situ hybridization analysis showed that GluR d 2 mRNA was expressed in entire Purkinje cells in both mutant mice. Immunohistochemical analysis revealed that intracellular localization of GluR d 2 was altered in some region of mutant cerebelli. In the cortical surface where Purkinje cells form synapses with parallel fibers, GluR d 2-immunoreactivity was restricted to dendritic spines of Purkinje cells as observed in normal mice. In contrast, in the subcortical region where granule cells and parallel fibers are absent, the immunoreactivity was found widely in Purkinje dendrites. Thus, the GluR d 2 protein did not accumulate to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers. These results suggest that the expression of both GluR d 2 mRNA and protein is independent of abnormalities in the mutant cerebelli, but relocalization of the GluR d 2 protein might depend on the formation of synapses between Purkinje cells and parallel fibers. Keywords: Glutamate receptor channel d 2 subunit; Purkinje cell; Granule cell; Parallel fiber; Weaver mouse; Reeler mouse; In situ hybridization; Immunohistochemistry
1. Introduction The glutamate receptor ŽGluR. channel mediates fast excitatory synaptic transmission, and is involved in various types of synaptic plasticity and development of the central nervous system w5,27,33,34x. In the past decade, more than 17 types of GluR channel subunits were identified by molecular cloning w4,22,35,37,51x. Among them, the GluR d 2 subunit has not been assigned to any of the three major classes of functional GluR channels, i.e., a-amino-3-hydroxy-5-methyl-4-isoxazole propionate ŽAMPA., kainate and N-methyl-D-aspartate ŽNMDA. receptor channels, since no GluR channel activity was detected in Xenopus oocytes or mammalian cells after the expression of the GluR d 2 subunit w1,28x. However, the GluR d 2 has been considered to be related to specific functions of Purkinje
) Corresponding author. Fax: q81 Ž11. 706-7863. E-mail: [email protected]
cells, based on its restricted expression in Purkinje cells w1,28,32,57x. Recently, Mishina and his colleagues reported that the subunit is a functional molecule playing roles in motor coordination, formation of parallel fiberPurkinje cell synapses and climbing fiber-Purkinje cell synapses, and long-term depression ŽLTD. of parallel fiber synaptic transmission by analyses of the GluR d 2-knockout mice w20,24x. We previously demonstrated that the GluR d 2 protein is expressed and localized widely in Purkinje dendrites at early postnatal stages, and subsequently accumulates in the synaptic membrane of the dendritic spines during postnatal development of the cerebellum w58x. To elucidate possible cellular mechanisms underlying expression and relocalization of the GluR d 2 protein, two cerebellar mutant mice, reeler and weaver, were employed in this study. First, abnormality of weaver cerebellum is characterized by a serious loss of granule cells w48x. Although granule cells are produced in the external granule cell layer, as occurs in normal mice, the majority of them die there and do not
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achieve their final position in the internal granule cell layer w15,16,19,44,45,48,54x. In the adult weaver cerebellum, the granule cell layer is not present except in a few folia in the hemisphere, and a few granule cells are scattered between and under Purkinje cells w18x. Accordingly, parallel fibers are few in the molecular and Purkinje cell layers, and full synaptic complexes between dendritic spines and parallel fiber varicosities are rare. Dendritic spines without synaptic contact are frequently found in processes of Bergmann glial cells, and some spines form heterologous synapses with terminals of mossy fibers and Golgi cells w19,44,45,53–55x. In contrast, reeler cerebellum is characterized by the malposition of neurons w13,31,52x. In the surface of the cerebellum, molecular and granule cell layers are present, but the cortical structure is poorly developed beneath the pia matter. Many neurons are localized and intermingled in the subcortical region, and a central cerebellar mass ŽCCM. is formed under the white matter w56x. A few Purkinje cells are found between the molecular and granule cell layers, whereas majority of them remain within the CCM w11,17,23x. Granule cells, in contrast, are localized exclusively in the granule cell layer above the white matter, and absent in the CCM w3,30x. In the cortical surface of the cerebellum, neural circuitry around Purkinje cells is preserved, and parallel fibers form synaptic contact with Purkinje dendrites normally. In the CCM, however, synapses between parallel fibers and Purkinje cells are absent, and spines of Purkinje dendrites form heterologous synapses with mossy fibers and axon terminals of Golgi and stellate cells w3,30,46,55,56,61x. In addition, multiple innervation from climbing fibers is detected in the reeler CCM, even at the adult stage w30x. In the present study, to elucidate possible cellular mechanisms underlying expression and relocalization of the GluR d 2, we examined expression of the GluR d 2 mRNA and distribution of its protein product in these two mutant cerebelli by in situ hybridization and immunohistochemistry and determined whether abnormal cytoarchitecture and changed neural circuitry in the mutant mice affect the expression and relocalization.
2. Materials and methods 2.1. Animals The reeler and weaver mice were obtained from Jackson Laboratories ŽBar Harbor, Maine, USA. and maintained in our laboratory. Two-month-old weaver and reeler mice were used in the present study.
mounted on glass slides precoated with 4% 3-aminopropyltriethoxysilane in acetone. The oligonucleotide probe ŽD2B. used for the detection of the GluR d 2 mRNA is complementary to the nucleotide residues 2901–2945 of the GluR d 2 cDNA w1x and the specificity of the probe was verified in previous papers w1,24,58x. In addition, 45-mer antisense oligonucleotide probe for the mouse inositol 1,4,5-triphosphate receptor ŽIP3 R. mRNA was used to detect the Purkinje cells in the weaver and reeler cerebelli w23,29x. The probe sequence is complementary to nucleotide residues 1079–1123 of the IP3 R cDNA w8x. In situ hybridization analysis was carried out as described in a previous paper w59x. Briefly, following fixation with 4% paraformaldehyde and prehybridization, hybridization was performed at 428C for 10 h in buffer containing 50% formamide, 0.1 M Tris-HCl ŽpH. 7.5., 4 = SSC Ž1 = SSC; 150 mM NaCl and 15 mM sodium citrate., 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2% sarkosyl, 250 mgrml of salmon sperm DNA, 10% dextran sulfate, 0.1 M dithiothreitol, and 10 4 dpmrml of 35 S-labeled oligonucleotide. The glass slides were washed at room temperature for 40 min in 2 = SSC containing 0.1% sarkosyl and were washed twice at 558C for 40 min in 0.1 = SSC containing 0.1% sarkosyl. The sections were dipped in nuclear track emulsion ŽNTB2, Kodak., and exposed for one month for the IP3 R and two months for the GluR d 2. 2.3. Immunohistochemistry Under deep ether anesthesia, brains were fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB, pH 7.4., removed from skulls, cryoprotected by immersion in 30% sucrose in PB overnight, and cut sagitally at a thickness of 20 mm. Sections were mounted on gelatin-coated glass slides. Immunohistochemistry for GluR d 2 was performed as described in previous papers w57,58x. For the electron microscopic analysis, the cryostat sections stained as above were postfixed in 0.2% OsO4 in PB for 2 h, followed by staining with 2% uranyl acetate aqueous solution and embedded in epoxy resin. After they were detached from glass slides, immunopositive areas were cut into ultrathin sections. These experiments were permitted by the Animal Care and Use committee of Hokkaido University School of Medicine.
3. Results
2.2. In situ hybridization
3.1. Expression of the GluR d 2 mRNA in the weaÕer and reeler cerebelli
Under deep ether anesthesia, brains were removed from skulls and frozen in powdered dry ice. Sagittal sections at a thickness of 20 mm were prepared by a cryostat and
In weaver cerebellum, hybridizing signal representing GluR d 2 mRNA was detected in the entire Purkinje cell layer ŽFig. 1A–C.. The distribution was almost identical to
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Fig. 1. Expression of the GluR d 2 ŽA–C, G–I. and IP3 R ŽD–F, J–L. mRNAs in the weaver ŽA–F. and reeler ŽG–L. cerebelli. In both types of mutant cerebelli, GluR d 2-expression was identical to that of IP3 R detected in serial sections in lateral ŽA,D,G,J., intermediate ŽB,E,H,K. and medial ŽC,F,I,L. regions. Scale bars: 500 mm.
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that of the IP3 R mRNA which is a marker molecule expressed abundantly and selectively in Purkinje cells in the normal cerebellum ŽFig. 1D–F.. In reeler cerebellum, signal of the GluR d 2 mRNA was found widely ŽFig. 1G–I.. Only a few neurons expressed the GluR d 2 beneath the molecular layer, whereas the mRNA was expressed in the majority of neurons in subcortical regions
under the white matter except for the deep cerebellar nuclei ŽFig. 1G–I.. This distribution in the reeler cerebellum, however, was identical to that of IP3 R mRNA detected in serial sections ŽFig. 1J–L.. These results demonstrated that abundant GluR d 2-expression is restricted to Purkinje cells and all Purkinje cells express the GluR d 2 in both mutant cerebelli.
Fig. 2.
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3.2. Light microscopic localization of the GluR d 2 in the mutant cerebelli 3.2.1. WeaÕer cerebellum The GluR d 2-immunoreactivity was found in throughout the molecular and Purkinje cell layers, whereas the white matter and cerebellar nuclei were negative ŽFig. 2A–C.. In the cortex of the vermis, Purkinje dendrites and cell bodies were discernible in the Purkinje cell layer where few granule cells were scattered ŽFig. 2D,E.. In the para-medial cerebellum, the GluR d 2-immunoreactivity was detected in the subcortical region as well as the cortex. Purkinje cells, remaining under the Purkinje cell layer, formed a large cluster with other neurons at the dorso-posterior portion ŽFig. 2B,F.. Within the cluster, moderate immunoreactivity of the GluR d 2 was detected in Purkinje dendrites and cell bodies ŽFig. 2F.. In the white matter, heavily stained Purkinje dendrites extended towards the deep cerebellar nuclei ŽFig. 2G.. In the hemisphere, Purkinje dendrites and cell bodies were stained similarly to those in the vermis, even in a folium where an incomplete granule cell layer was discernible ŽFig. 2H.. 3.2.2. Reeler cerebellum The GluR d 2-immunoreactivity was widely detected in the cerebellum except for cerebellar nuclei ŽFig. 3A,B.. The molecular layer underlying the pial surface was occupied by punctate immunoreaction product, whereas dendrites were not clearly discernible ŽFig. 3C.. In the granule cell layer, dendrites and cell bodies of malpositioned Purkinje cells were labeled with the antibody ŽFig. 3C.. In the white matter of the vermis and hemisphere, densely stained
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Purkinje dendrites extended from CCM towards the pia matter ŽFig. 3D,F.. In the CCM, GluR d 2-immunoreactivity was also detected in cell bodies and dendrites of malpositioned Purkinje cells localized near the ventricular wall ŽFig. 3E. and cerebellar nuclei ŽFig. 3G.. 3.3. Electron microscopic localization of the GluR d 2 in the mutant cerebelli We employed an electron microscopic analysis to clarify the intracellular localization in the mutant Purkinje dendrites. 3.3.1. WeaÕer cerebellum In the molecular and Purkinje cell layers, immunoreaction product of GluR d 2 was found in dendritic spines of Purkinje cells, but was absent within the dendritic shafts ŽFig. 4A–D.. The GluR d 2-immunopositive spines often formed synapses with axon terminals containing numerous synaptic vesicles, some of which were considered to be parallel fiber varicosities. However, some labeled spines did not formed synapses with any terminals in processes of Bergmann glia Žarrows in Fig. 4B,C.. By light microscopy, the GluR d 2-immunoreactivity was also detected in subcortical regions, such as a cluster formed under the normal Purkinje cell layer ŽFig. 2B circle F and Fig. 2F. and dendrites extending in the white matter ŽFig. 2B circle G, Fig. 2G.. In the cluster, immunoreaction product was detected in both dendritic spines and shafts ŽFig. 4E.. The immunoreactivity in the dendritic shafts was faint and restricted beneath plasma membrane Žthick arrows in Fig. 4E.. In the white matter, both dendritic shafts
Fig. 2. Light microscopic localization of the GluR d 2 in the weaver cerebellum. A–C: sagittal sections of the vermis ŽA., intermittent region ŽB. and hemisphere ŽC. at low magnification. The GluR d 2-immunoreactivity was detected in molecular and Purkinje cell layers, whereas white matter and cerebellar nuclei were negative except for small subcortical regions Žcircle F and G.. D: cerebellar cortex in the vermis at higher magnification of circle D in the panel A. Dense immunoreactivity was found in the molecular layer ŽMo.. In the Purkinje cell layer ŽPu., Purkinje dendrites Žarrowheads. and cell bodies were moderately labeled. E: Nissl-stained section at the similar region to panel D in the serial section. Granule cell layer was not detected, and granule-like cells were scattered between Purkinje cells. F: a cluster of Purkinje cells formed under the normal Purkinje cell layer at higher magnification of circle F in panel B. Inset: Nissl-stained section of the similar region. Purkinje cell bodies and dendrites Žarrowheads. extending randomly were discernible in a cluster consisting of Purkinje cells and other neurons. G: cerebellar cortex at higher magnification of circle G in the panel B. Inset: Nissl-stained section of the similar region in an adjacent section. The immunoreactivity was found in the molecular ŽMo. and Purkinje cell ŽPu. layers and white matter ŽWM.. In the white matter, Purkinje dendrites Žarrowheads. extending from the Purkinje cell layer were clearly detected Žarrowheads.. H: cerebellar cortex in the hemisphere at higher magnification of circle H in panel C. Inset: Nissl-stained adjacent sections of the similar folium. Purkinje dendrites Žarrowheads. and cell bodies were labeled in the molecular ŽMo. and granul cell layer ŽGr., where incomplete granule cell layer was discernible under the molecular layer ŽMo.. Scale bars: A–C: 500 mm; D–H: 30 mm. Fig. 3. Ž see page 236 . Light microscopic localization of the GluR d 2 in the reeler cerebellum. A,B: sagittal sections at low magnification of the vermis ŽA. and hemisphere ŽB.. The GluR d 2-immunoreactivity was detected widely in the cerebellum except for white matter ŽWM. and cerebellar nuclei ŽNu.. C: pial surface composed with the molecular ŽMo. and granule cell ŽGr. layers at higher magnification of circle C in the panel A. The molecular layer ŽMo. was occupied by fine granular immunoreaction product. In the granule cell layer ŽGr., Purkinje dendrites Žarrowheads. were clearly labeled with the antibody. D: white matter and superficial zone of the central cerebellar mass ŽCM. at higher magnification of circle D in panel A in the vermis. Purkinje dendrites Žarrowheads. extending into the white matter were clearly discernible. E: deep zone of the central cerebellar mass near the fourth ventricle at higher magnification of circle E in panel A. Purkinje cell bodies and dendrites Žarrowheads. were labeled. F: border zone between the white matter ŽWM. and central cerebellar mass ŽCM. in the hemisphere at higher magnification of circle F in panel B. In the white matter ŽWM., GluR d 2-immunopositive Purkinje dendrites Žarrowheads. were extending from the central mass. G: deep zone of the central cerebellar mass ŽCM. near the cerebellar nucleus ŽNu. at higher magnification of circle G in panel B. Purkinje dendrites Žarrowheads. extending randomly between Purkinje cell bodies were labeled. Scale bars: A,B: 500 mm; C–F: 30 mm.
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and spines were densely labeled between myelinated fibers ŽFig. 4F.. Immunoreactivity within dendritic shafts was observed not only near the plasma membrane but also in the cytoplasm, whereas organelles such as mitochondria
and endoplasmic reticulum were negative Žthick arrows in Fig. 4F.. Labeled spines often formed synapses with terminals containing numerous synaptic vesicles in the subcortical regions ŽFig. 4E,F..
Fig. 3.
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Fig. 4.
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3.3.2. Reeler cerebellum In the molecular layer beneath the pia mater, immunoreactivity of the GluR d 2 was found in dendritic spines, especially near the synaptic junction site, but was absent within the dendritic shafts ŽFig. 5A,B.. Immunopositive spines often formed synapses with parallel fiber varicosities between parallel fiber bundles ŽFig. 5A,B.. In the granule cell layer, immunoreactivity was also detected in dendritic spines of Purkinje cells remaining there ŽFig. 5C,D.. In the superficial zone near the molecular layer, some of the presynaptic elements of labeled spines might be parallel fiber varicosities, since parallel fiber-like structures were observed ŽFig. 5C.. In contrast, in the deep zone near the CCM, GluR d 2-immunopositive spines formed synapses with axon terminals other than parallel fiber varicosities, as parallel fibers were not easily detected ŽFig. 5D.. Immunoreactivity was absent in the dendritic shafts near the molecular layer ŽFig. 5C., but was present near the plasma membrane within the dendritic shafts in the deep portion of the granule cell layer ŽFig. 5D.. By light microscopy, dense immunoreactivity of the GluR d 2 was localized in Purkinje dendrites in both white matter and CCM ŽFig. 3D–G.. In the white matter, GluR d 2-immunoreaction product was detected in both dendritic shafts and spines of Purkinje cells between myelinated fibers ŽFig. 5E.. Some labeled spines formed synapses with large axon terminals containing numerous synaptic vesicles. In the dendritic shafts, dense immunoreaction product was localized along the plasma membrane and in the cytoplasm ŽFig. 5E.. In the CCM, GluR d 2-immunoreactivity was found in both spines and shafts of Purkinje dendrites as observed in the white matter ŽFig. 5F..
4. Discussion The GluR channel d 2 subunit was identified by molecular cloning, and is uniquely distributed in the central nervous system w1,28x. The GluR d 2 mRNA is expressed abundantly and specifically in cerebellar Purkinje cells, and its transcript is localized in the dendritic spines of Purkinje cells w32,57x. Our previous study demonstrated that relocalization of the GluR d 2 protein occurred during the Purkinje cell maturation w58x. In the present study, to
elucidate possible cellular mechanisms underlying expression and relocalization of the GluR d 2 in Purkinje cells, we employed weaver and reeler mutant mice whose cerebelli show abnormal cytoarchitecture and neural circuitry around Purkinje cells, and examined expression and distribution of the GluR d 2 in these mutant cerebelli by in situ hybridization and immunohistochemistry. Although GluR d 2-expression occurred in mutant Purkinje cells normally, GluR d 2 protein did not accumulated to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers in the subcortical regions of mutant cerebelli. These results suggested that GluR d 2-expression was independent of abnormalities in the mutant cerebelli, whereas relocalization of the GluR d 2 protein might be strongly related to parallel fiber input. 4.1. Mutations in weaÕer and reeler genes did not directly affect the GluR d 2-expression and distribution The weaver mutant gene was identified to be a single base-pair substitution of one of the potassium channels, GIRK2 w41x, which is highly and specifically expressed in both external and internal granule cell layers in cerebellum w26,38x. During cerebellar ontogeny, mutant GIRK2 might affect the normal process of granule cell differentiation, including proliferation, migration and synapse formation, and give rise to granule cell death w12,15,16,38x. Thus, gene mutation in weaver mice might primarily attack granule cells, causing subsequent granule cell loss and finally less granular cerebellum to be formed. The malpositioning of neurons in reeler brain is caused by a lack of reelin w7,21x or CR-50 antigen w40x. These molecules are expressed temporarily in Cajar-Retzius cells of the cerebral cortex and considered to play critical roles in regulating the final migratory phase and primordial cortical organization w7,10,47x. It has not been clearly demonstrated which cells express reelin and how reelin acts during the cerebellar development, although a previous study showed that reelin mRNA was detectable at embryonic and early postnatal stages w7x. In the cerebellum, however, reelin might play roles in neuronal cell migration and cortical organization, based on the case in cerebral cortex. Therefore, mutations in two mutant mice might not directly affect expression and distribution of
Fig. 4. Ž see page 237 . Ultrastructural localization of the GluR d 2 in the weaver cerebellum. A–D: molecular ŽA–C. and Purkinje cell ŽD. layers. The GluR d 2-immunoreactivity was detected in dendritic spines Žasterisks. including the neck portion Žarrowheads. of Purkinje cells. Some of labeled spines Žarrows. missed synaptic contact in the processes of Bergmann glial cells ŽBG.. Dendritic shafts ŽDN. were negative. E: cluster of Purkinje cells shown in Fig. 2F. Intense immunoreactivity was detected in dendritic spines Žasterisks. including neck portion Žarrowheads.. Within the dendritic shafts ŽDN., weak signal was detected near the plasma membrane Žthick arrows.. F: white matter shown in Fig. 2G. The GluR d 2-immunoreactivity was detected within the dendritic shafts Žthick arrows. and dendritic spines Žasterisks.. Scale bars: 1 mm. Fig. 5. Ultrastructural localization of the GluR d 2 in the reeler cerebellum. A,B: molecular layer. The GluR d 2-immunoreactivity was detected in dendritic spines Žasterisks., whereas dendritic shafts ŽDN. was negative. C,D: granule cell layer. Dendritic spines were immunopositive Žasterisks. in both the superficial zone ŽC. and deep zone ŽD.. In the deep zone near the central cerebellar mass, the immunoreaction product was detected inside of the dendritic shafts, near the plasma membrane Žthick arrows.. E,F: white matter ŽE. and central cerebellar mass ŽF.. Dense immunoreaction product was localized widely in both dendritic shafts ŽDN. and spines Žasterisks.. Scale bars: 1 mm.
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Fig. 5.
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GluR d 2 and altered intracellular localization of the GluR d 2 in the mutant cerebelli might be due to their abnormal cytoarchitecture and changed neural circuits. 4.2. Mutant Purkinje cells expressed the GluR d 2 mRNA normally in an abnormal enÕironment The Purkinje cell is a pivotal neuron in the cerebellar cortex, and expresses various types of glutamate receptor channel subunits, which are localized at the postsynaptic site of Purkinje dendrites and are involved in excitatory synaptic transmission w2,14,39,42,43x. Expression of the AMPA-type GluR subunits is preserved in mutant cerebelli w49x Žsee also Takayama et al., unpublished data.. Although the distribution of their mRNAs in the cerebelli seems abnormal, the abnormality corresponds to the alteration in cytoarchitecture. In contrast, expression of NMDA-type receptor subunits is altered in reeler and staggerer mutant Purkinje cells w36,60x. Thus, it was suggested that there are two groups of GluR channel subunits; one includes subunits whose expression is independent of the abnormal environment around Purkinje cells, and the other consists of subunits whose expression is altered under abnormal environmental conditions. The present study showed that GluR d 2 is expressed in all Purkinje cells and the intense signal is restricted to only Purkinje cells, even in the aberrant environment of the mutant cerebelli. Thus, abnormalities in cytoarchitecture and changed neural circuitry did not affect the GluR d 2-expression. 4.3. Parallel fiber input was related to relocalization of the GluR d 2 In weaver cerebellum except white matter, intracellular localization of the GluR d 2 in Purkinje cells was almost identical to that in normal adult mice, and the immunoreactivity was exclusively localized to dendritic spines. In the cortex of weaver cerebellum, the number of granule cells is remarkably reduced w48x. Synapses between Purkinje dendrities and parallel fibers are rare. Dendritic spines without synapses are frequently found in processes of Bergmann glial cells, and some spines form heterologous synapses with mossy fibers and Golgi cells w19,44,45,54x. Therefore, neither reduction of parallel fiber input nor formation of heterologous synapses affect the relocalization of GluR d 2 protein. On the other hand, in the reeler cerebellum, intracellular localization of GluR d 2 showed regional differences. In the cortical surface of the reeler cerebellum, the GluR d 2 protein was localized selectively in Purkinje dendritic spines as is observed in normal adult mice. In the subcortical region, however, the immunoreactivity was not restricted to dendritic spines, but was found widely in Purkinje dendrites. The altered intracellular localization detected in the subcortical region was similar to that at early postnatal stages of normal mice w58x. In this region, gran-
ule cells are absent and dendritic spines form heterologous synapses with terminals other than parallel fibers w3,30,46,55,61x. Since heterologous synapses are also observed in the weaver cortex where the GluR d 2-immunoreactivity was restricted in dendritic spines, they did not influence on the accumulation. Thus, the accumulation of GluR d 2 protein might be disturbed by an absence of synaptic contact between parallel fibers and Purkinje cells. These results suggest that parallel fiber input might be crucial for relocalization of the GluR d 2 protein and formation of synapses with parallel fibers, even whose number is reduced, might confine the receptor protein to the postsynaptic site. 4.4. Particular input was necessary for relocalization of the receptor protein In the subcortical region of the reeler cerebellum, dendritic spines of Purkinje cells directly form synapses with mossy fiber terminals instead of parallel fibers. Although the neurotransmitter of mossy fibers is considered to be the same as that of parallel fibers; e.g., glutamic acid w9x, accumulation of the GluR d 2 protein had not occurred and the GluR d 2-immunoreactivity was found widely in Purkinje dendrites. Thus, mossy fiber input is not effective for the accumulation of GluR d 2 protein and parallel fiber input might be necessary for relocalization of the GluR d 2 protein. It is well known that proteins are sorted, transported and confined to the programmed place, during the brain development w6,25,62x. Accumulation of receptor proteins to the postsynaptic site during synaptogenesis is considered to be a protein targeting phenomenon. In the case of nicotinic acetylcholine receptors, it was clearly demonstrated that motor nerve innervation confines the receptor proteins to the postsynaptic site of motor end plate w50x. In the central as well as peripheral nervous system, synapse formation appears to trigger the accumulation of the channel receptor proteins to the postsynaptic site. We previously demonstrated that the GluR d 2 protein accumulates at the synaptic site during Purkinje cell maturation w58x. The present study of mutant mice suggests that only parallel fiber input might influence the relocalization of the GluR d 2 protein. More generally, particular input may be necessary for relocalization of receptor protein in the central nervous system.
Acknowledgements The authors would like to thank Mr. Hideo Umeda and Miss Miho Kobayashi at Department of Anatomy and Mr. Yoshihiko Ogawa at Central Research Institute for their technical assistance. We are also grateful to Drs. David R. Kornack and Hitoshi Komuro at Yale University School of Medicine, for their valuable suggestions.
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Research report
Altered intracellular localization of the glutamate receptor channel d 2 subunit in weaver and reeler Purkinje cells Chitoshi Takayama a
a, )
, Shin Nakagawa a , Masahiko Watanabe a , Hideo Kurihara a , Masayoshi Mishina b , Yoshiro Inoue a
Department of Anatomy, Hokkaido UniÕersity School of Medicine, Kita-15 Nishi-7, Kita-ku, Sapporo 060, Japan b Department of Pharmacology, Faculty of Medicine, UniÕersity of Tokyo, Tokyo 113, Japan Accepted 17 September 1996
Abstract The glutamate receptor ŽGluR. channel d 2 subunit is expressed abundantly and specifically in cerebellar Purkinje cells. Our previous study demonstrated that the GluR d 2 mRNA is expressed as early as embryonic day 15 prior to Purkinje cell synaptogenesis, and its protein product accumulates in dendritic spines during normal Purkinje cell maturation. In this study, we examined expression and distribution of the GluR d 2 in the weaver and reeler mutant cerebelli, which show abnormal cytoarchitecture and neural circuitry. In situ hybridization analysis showed that GluR d 2 mRNA was expressed in entire Purkinje cells in both mutant mice. Immunohistochemical analysis revealed that intracellular localization of GluR d 2 was altered in some region of mutant cerebelli. In the cortical surface where Purkinje cells form synapses with parallel fibers, GluR d 2-immunoreactivity was restricted to dendritic spines of Purkinje cells as observed in normal mice. In contrast, in the subcortical region where granule cells and parallel fibers are absent, the immunoreactivity was found widely in Purkinje dendrites. Thus, the GluR d 2 protein did not accumulate to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers. These results suggest that the expression of both GluR d 2 mRNA and protein is independent of abnormalities in the mutant cerebelli, but relocalization of the GluR d 2 protein might depend on the formation of synapses between Purkinje cells and parallel fibers. Keywords: Glutamate receptor channel d 2 subunit; Purkinje cell; Granule cell; Parallel fiber; Weaver mouse; Reeler mouse; In situ hybridization; Immunohistochemistry
1. Introduction The glutamate receptor ŽGluR. channel mediates fast excitatory synaptic transmission, and is involved in various types of synaptic plasticity and development of the central nervous system w5,27,33,34x. In the past decade, more than 17 types of GluR channel subunits were identified by molecular cloning w4,22,35,37,51x. Among them, the GluR d 2 subunit has not been assigned to any of the three major classes of functional GluR channels, i.e., a-amino-3-hydroxy-5-methyl-4-isoxazole propionate ŽAMPA., kainate and N-methyl-D-aspartate ŽNMDA. receptor channels, since no GluR channel activity was detected in Xenopus oocytes or mammalian cells after the expression of the GluR d 2 subunit w1,28x. However, the GluR d 2 has been considered to be related to specific functions of Purkinje
) Corresponding author. Fax: q81 Ž11. 706-7863. E-mail: [email protected]
cells, based on its restricted expression in Purkinje cells w1,28,32,57x. Recently, Mishina and his colleagues reported that the subunit is a functional molecule playing roles in motor coordination, formation of parallel fiberPurkinje cell synapses and climbing fiber-Purkinje cell synapses, and long-term depression ŽLTD. of parallel fiber synaptic transmission by analyses of the GluR d 2-knockout mice w20,24x. We previously demonstrated that the GluR d 2 protein is expressed and localized widely in Purkinje dendrites at early postnatal stages, and subsequently accumulates in the synaptic membrane of the dendritic spines during postnatal development of the cerebellum w58x. To elucidate possible cellular mechanisms underlying expression and relocalization of the GluR d 2 protein, two cerebellar mutant mice, reeler and weaver, were employed in this study. First, abnormality of weaver cerebellum is characterized by a serious loss of granule cells w48x. Although granule cells are produced in the external granule cell layer, as occurs in normal mice, the majority of them die there and do not
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achieve their final position in the internal granule cell layer w15,16,19,44,45,48,54x. In the adult weaver cerebellum, the granule cell layer is not present except in a few folia in the hemisphere, and a few granule cells are scattered between and under Purkinje cells w18x. Accordingly, parallel fibers are few in the molecular and Purkinje cell layers, and full synaptic complexes between dendritic spines and parallel fiber varicosities are rare. Dendritic spines without synaptic contact are frequently found in processes of Bergmann glial cells, and some spines form heterologous synapses with terminals of mossy fibers and Golgi cells w19,44,45,53–55x. In contrast, reeler cerebellum is characterized by the malposition of neurons w13,31,52x. In the surface of the cerebellum, molecular and granule cell layers are present, but the cortical structure is poorly developed beneath the pia matter. Many neurons are localized and intermingled in the subcortical region, and a central cerebellar mass ŽCCM. is formed under the white matter w56x. A few Purkinje cells are found between the molecular and granule cell layers, whereas majority of them remain within the CCM w11,17,23x. Granule cells, in contrast, are localized exclusively in the granule cell layer above the white matter, and absent in the CCM w3,30x. In the cortical surface of the cerebellum, neural circuitry around Purkinje cells is preserved, and parallel fibers form synaptic contact with Purkinje dendrites normally. In the CCM, however, synapses between parallel fibers and Purkinje cells are absent, and spines of Purkinje dendrites form heterologous synapses with mossy fibers and axon terminals of Golgi and stellate cells w3,30,46,55,56,61x. In addition, multiple innervation from climbing fibers is detected in the reeler CCM, even at the adult stage w30x. In the present study, to elucidate possible cellular mechanisms underlying expression and relocalization of the GluR d 2, we examined expression of the GluR d 2 mRNA and distribution of its protein product in these two mutant cerebelli by in situ hybridization and immunohistochemistry and determined whether abnormal cytoarchitecture and changed neural circuitry in the mutant mice affect the expression and relocalization.
2. Materials and methods 2.1. Animals The reeler and weaver mice were obtained from Jackson Laboratories ŽBar Harbor, Maine, USA. and maintained in our laboratory. Two-month-old weaver and reeler mice were used in the present study.
mounted on glass slides precoated with 4% 3-aminopropyltriethoxysilane in acetone. The oligonucleotide probe ŽD2B. used for the detection of the GluR d 2 mRNA is complementary to the nucleotide residues 2901–2945 of the GluR d 2 cDNA w1x and the specificity of the probe was verified in previous papers w1,24,58x. In addition, 45-mer antisense oligonucleotide probe for the mouse inositol 1,4,5-triphosphate receptor ŽIP3 R. mRNA was used to detect the Purkinje cells in the weaver and reeler cerebelli w23,29x. The probe sequence is complementary to nucleotide residues 1079–1123 of the IP3 R cDNA w8x. In situ hybridization analysis was carried out as described in a previous paper w59x. Briefly, following fixation with 4% paraformaldehyde and prehybridization, hybridization was performed at 428C for 10 h in buffer containing 50% formamide, 0.1 M Tris-HCl ŽpH. 7.5., 4 = SSC Ž1 = SSC; 150 mM NaCl and 15 mM sodium citrate., 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2% sarkosyl, 250 mgrml of salmon sperm DNA, 10% dextran sulfate, 0.1 M dithiothreitol, and 10 4 dpmrml of 35 S-labeled oligonucleotide. The glass slides were washed at room temperature for 40 min in 2 = SSC containing 0.1% sarkosyl and were washed twice at 558C for 40 min in 0.1 = SSC containing 0.1% sarkosyl. The sections were dipped in nuclear track emulsion ŽNTB2, Kodak., and exposed for one month for the IP3 R and two months for the GluR d 2. 2.3. Immunohistochemistry Under deep ether anesthesia, brains were fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB, pH 7.4., removed from skulls, cryoprotected by immersion in 30% sucrose in PB overnight, and cut sagitally at a thickness of 20 mm. Sections were mounted on gelatin-coated glass slides. Immunohistochemistry for GluR d 2 was performed as described in previous papers w57,58x. For the electron microscopic analysis, the cryostat sections stained as above were postfixed in 0.2% OsO4 in PB for 2 h, followed by staining with 2% uranyl acetate aqueous solution and embedded in epoxy resin. After they were detached from glass slides, immunopositive areas were cut into ultrathin sections. These experiments were permitted by the Animal Care and Use committee of Hokkaido University School of Medicine.
3. Results
2.2. In situ hybridization
3.1. Expression of the GluR d 2 mRNA in the weaÕer and reeler cerebelli
Under deep ether anesthesia, brains were removed from skulls and frozen in powdered dry ice. Sagittal sections at a thickness of 20 mm were prepared by a cryostat and
In weaver cerebellum, hybridizing signal representing GluR d 2 mRNA was detected in the entire Purkinje cell layer ŽFig. 1A–C.. The distribution was almost identical to
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Fig. 1. Expression of the GluR d 2 ŽA–C, G–I. and IP3 R ŽD–F, J–L. mRNAs in the weaver ŽA–F. and reeler ŽG–L. cerebelli. In both types of mutant cerebelli, GluR d 2-expression was identical to that of IP3 R detected in serial sections in lateral ŽA,D,G,J., intermediate ŽB,E,H,K. and medial ŽC,F,I,L. regions. Scale bars: 500 mm.
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that of the IP3 R mRNA which is a marker molecule expressed abundantly and selectively in Purkinje cells in the normal cerebellum ŽFig. 1D–F.. In reeler cerebellum, signal of the GluR d 2 mRNA was found widely ŽFig. 1G–I.. Only a few neurons expressed the GluR d 2 beneath the molecular layer, whereas the mRNA was expressed in the majority of neurons in subcortical regions
under the white matter except for the deep cerebellar nuclei ŽFig. 1G–I.. This distribution in the reeler cerebellum, however, was identical to that of IP3 R mRNA detected in serial sections ŽFig. 1J–L.. These results demonstrated that abundant GluR d 2-expression is restricted to Purkinje cells and all Purkinje cells express the GluR d 2 in both mutant cerebelli.
Fig. 2.
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3.2. Light microscopic localization of the GluR d 2 in the mutant cerebelli 3.2.1. WeaÕer cerebellum The GluR d 2-immunoreactivity was found in throughout the molecular and Purkinje cell layers, whereas the white matter and cerebellar nuclei were negative ŽFig. 2A–C.. In the cortex of the vermis, Purkinje dendrites and cell bodies were discernible in the Purkinje cell layer where few granule cells were scattered ŽFig. 2D,E.. In the para-medial cerebellum, the GluR d 2-immunoreactivity was detected in the subcortical region as well as the cortex. Purkinje cells, remaining under the Purkinje cell layer, formed a large cluster with other neurons at the dorso-posterior portion ŽFig. 2B,F.. Within the cluster, moderate immunoreactivity of the GluR d 2 was detected in Purkinje dendrites and cell bodies ŽFig. 2F.. In the white matter, heavily stained Purkinje dendrites extended towards the deep cerebellar nuclei ŽFig. 2G.. In the hemisphere, Purkinje dendrites and cell bodies were stained similarly to those in the vermis, even in a folium where an incomplete granule cell layer was discernible ŽFig. 2H.. 3.2.2. Reeler cerebellum The GluR d 2-immunoreactivity was widely detected in the cerebellum except for cerebellar nuclei ŽFig. 3A,B.. The molecular layer underlying the pial surface was occupied by punctate immunoreaction product, whereas dendrites were not clearly discernible ŽFig. 3C.. In the granule cell layer, dendrites and cell bodies of malpositioned Purkinje cells were labeled with the antibody ŽFig. 3C.. In the white matter of the vermis and hemisphere, densely stained
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Purkinje dendrites extended from CCM towards the pia matter ŽFig. 3D,F.. In the CCM, GluR d 2-immunoreactivity was also detected in cell bodies and dendrites of malpositioned Purkinje cells localized near the ventricular wall ŽFig. 3E. and cerebellar nuclei ŽFig. 3G.. 3.3. Electron microscopic localization of the GluR d 2 in the mutant cerebelli We employed an electron microscopic analysis to clarify the intracellular localization in the mutant Purkinje dendrites. 3.3.1. WeaÕer cerebellum In the molecular and Purkinje cell layers, immunoreaction product of GluR d 2 was found in dendritic spines of Purkinje cells, but was absent within the dendritic shafts ŽFig. 4A–D.. The GluR d 2-immunopositive spines often formed synapses with axon terminals containing numerous synaptic vesicles, some of which were considered to be parallel fiber varicosities. However, some labeled spines did not formed synapses with any terminals in processes of Bergmann glia Žarrows in Fig. 4B,C.. By light microscopy, the GluR d 2-immunoreactivity was also detected in subcortical regions, such as a cluster formed under the normal Purkinje cell layer ŽFig. 2B circle F and Fig. 2F. and dendrites extending in the white matter ŽFig. 2B circle G, Fig. 2G.. In the cluster, immunoreaction product was detected in both dendritic spines and shafts ŽFig. 4E.. The immunoreactivity in the dendritic shafts was faint and restricted beneath plasma membrane Žthick arrows in Fig. 4E.. In the white matter, both dendritic shafts
Fig. 2. Light microscopic localization of the GluR d 2 in the weaver cerebellum. A–C: sagittal sections of the vermis ŽA., intermittent region ŽB. and hemisphere ŽC. at low magnification. The GluR d 2-immunoreactivity was detected in molecular and Purkinje cell layers, whereas white matter and cerebellar nuclei were negative except for small subcortical regions Žcircle F and G.. D: cerebellar cortex in the vermis at higher magnification of circle D in the panel A. Dense immunoreactivity was found in the molecular layer ŽMo.. In the Purkinje cell layer ŽPu., Purkinje dendrites Žarrowheads. and cell bodies were moderately labeled. E: Nissl-stained section at the similar region to panel D in the serial section. Granule cell layer was not detected, and granule-like cells were scattered between Purkinje cells. F: a cluster of Purkinje cells formed under the normal Purkinje cell layer at higher magnification of circle F in panel B. Inset: Nissl-stained section of the similar region. Purkinje cell bodies and dendrites Žarrowheads. extending randomly were discernible in a cluster consisting of Purkinje cells and other neurons. G: cerebellar cortex at higher magnification of circle G in the panel B. Inset: Nissl-stained section of the similar region in an adjacent section. The immunoreactivity was found in the molecular ŽMo. and Purkinje cell ŽPu. layers and white matter ŽWM.. In the white matter, Purkinje dendrites Žarrowheads. extending from the Purkinje cell layer were clearly detected Žarrowheads.. H: cerebellar cortex in the hemisphere at higher magnification of circle H in panel C. Inset: Nissl-stained adjacent sections of the similar folium. Purkinje dendrites Žarrowheads. and cell bodies were labeled in the molecular ŽMo. and granul cell layer ŽGr., where incomplete granule cell layer was discernible under the molecular layer ŽMo.. Scale bars: A–C: 500 mm; D–H: 30 mm. Fig. 3. Ž see page 236 . Light microscopic localization of the GluR d 2 in the reeler cerebellum. A,B: sagittal sections at low magnification of the vermis ŽA. and hemisphere ŽB.. The GluR d 2-immunoreactivity was detected widely in the cerebellum except for white matter ŽWM. and cerebellar nuclei ŽNu.. C: pial surface composed with the molecular ŽMo. and granule cell ŽGr. layers at higher magnification of circle C in the panel A. The molecular layer ŽMo. was occupied by fine granular immunoreaction product. In the granule cell layer ŽGr., Purkinje dendrites Žarrowheads. were clearly labeled with the antibody. D: white matter and superficial zone of the central cerebellar mass ŽCM. at higher magnification of circle D in panel A in the vermis. Purkinje dendrites Žarrowheads. extending into the white matter were clearly discernible. E: deep zone of the central cerebellar mass near the fourth ventricle at higher magnification of circle E in panel A. Purkinje cell bodies and dendrites Žarrowheads. were labeled. F: border zone between the white matter ŽWM. and central cerebellar mass ŽCM. in the hemisphere at higher magnification of circle F in panel B. In the white matter ŽWM., GluR d 2-immunopositive Purkinje dendrites Žarrowheads. were extending from the central mass. G: deep zone of the central cerebellar mass ŽCM. near the cerebellar nucleus ŽNu. at higher magnification of circle G in panel B. Purkinje dendrites Žarrowheads. extending randomly between Purkinje cell bodies were labeled. Scale bars: A,B: 500 mm; C–F: 30 mm.
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and spines were densely labeled between myelinated fibers ŽFig. 4F.. Immunoreactivity within dendritic shafts was observed not only near the plasma membrane but also in the cytoplasm, whereas organelles such as mitochondria
and endoplasmic reticulum were negative Žthick arrows in Fig. 4F.. Labeled spines often formed synapses with terminals containing numerous synaptic vesicles in the subcortical regions ŽFig. 4E,F..
Fig. 3.
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Fig. 4.
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3.3.2. Reeler cerebellum In the molecular layer beneath the pia mater, immunoreactivity of the GluR d 2 was found in dendritic spines, especially near the synaptic junction site, but was absent within the dendritic shafts ŽFig. 5A,B.. Immunopositive spines often formed synapses with parallel fiber varicosities between parallel fiber bundles ŽFig. 5A,B.. In the granule cell layer, immunoreactivity was also detected in dendritic spines of Purkinje cells remaining there ŽFig. 5C,D.. In the superficial zone near the molecular layer, some of the presynaptic elements of labeled spines might be parallel fiber varicosities, since parallel fiber-like structures were observed ŽFig. 5C.. In contrast, in the deep zone near the CCM, GluR d 2-immunopositive spines formed synapses with axon terminals other than parallel fiber varicosities, as parallel fibers were not easily detected ŽFig. 5D.. Immunoreactivity was absent in the dendritic shafts near the molecular layer ŽFig. 5C., but was present near the plasma membrane within the dendritic shafts in the deep portion of the granule cell layer ŽFig. 5D.. By light microscopy, dense immunoreactivity of the GluR d 2 was localized in Purkinje dendrites in both white matter and CCM ŽFig. 3D–G.. In the white matter, GluR d 2-immunoreaction product was detected in both dendritic shafts and spines of Purkinje cells between myelinated fibers ŽFig. 5E.. Some labeled spines formed synapses with large axon terminals containing numerous synaptic vesicles. In the dendritic shafts, dense immunoreaction product was localized along the plasma membrane and in the cytoplasm ŽFig. 5E.. In the CCM, GluR d 2-immunoreactivity was found in both spines and shafts of Purkinje dendrites as observed in the white matter ŽFig. 5F..
4. Discussion The GluR channel d 2 subunit was identified by molecular cloning, and is uniquely distributed in the central nervous system w1,28x. The GluR d 2 mRNA is expressed abundantly and specifically in cerebellar Purkinje cells, and its transcript is localized in the dendritic spines of Purkinje cells w32,57x. Our previous study demonstrated that relocalization of the GluR d 2 protein occurred during the Purkinje cell maturation w58x. In the present study, to
elucidate possible cellular mechanisms underlying expression and relocalization of the GluR d 2 in Purkinje cells, we employed weaver and reeler mutant mice whose cerebelli show abnormal cytoarchitecture and neural circuitry around Purkinje cells, and examined expression and distribution of the GluR d 2 in these mutant cerebelli by in situ hybridization and immunohistochemistry. Although GluR d 2-expression occurred in mutant Purkinje cells normally, GluR d 2 protein did not accumulated to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers in the subcortical regions of mutant cerebelli. These results suggested that GluR d 2-expression was independent of abnormalities in the mutant cerebelli, whereas relocalization of the GluR d 2 protein might be strongly related to parallel fiber input. 4.1. Mutations in weaÕer and reeler genes did not directly affect the GluR d 2-expression and distribution The weaver mutant gene was identified to be a single base-pair substitution of one of the potassium channels, GIRK2 w41x, which is highly and specifically expressed in both external and internal granule cell layers in cerebellum w26,38x. During cerebellar ontogeny, mutant GIRK2 might affect the normal process of granule cell differentiation, including proliferation, migration and synapse formation, and give rise to granule cell death w12,15,16,38x. Thus, gene mutation in weaver mice might primarily attack granule cells, causing subsequent granule cell loss and finally less granular cerebellum to be formed. The malpositioning of neurons in reeler brain is caused by a lack of reelin w7,21x or CR-50 antigen w40x. These molecules are expressed temporarily in Cajar-Retzius cells of the cerebral cortex and considered to play critical roles in regulating the final migratory phase and primordial cortical organization w7,10,47x. It has not been clearly demonstrated which cells express reelin and how reelin acts during the cerebellar development, although a previous study showed that reelin mRNA was detectable at embryonic and early postnatal stages w7x. In the cerebellum, however, reelin might play roles in neuronal cell migration and cortical organization, based on the case in cerebral cortex. Therefore, mutations in two mutant mice might not directly affect expression and distribution of
Fig. 4. Ž see page 237 . Ultrastructural localization of the GluR d 2 in the weaver cerebellum. A–D: molecular ŽA–C. and Purkinje cell ŽD. layers. The GluR d 2-immunoreactivity was detected in dendritic spines Žasterisks. including the neck portion Žarrowheads. of Purkinje cells. Some of labeled spines Žarrows. missed synaptic contact in the processes of Bergmann glial cells ŽBG.. Dendritic shafts ŽDN. were negative. E: cluster of Purkinje cells shown in Fig. 2F. Intense immunoreactivity was detected in dendritic spines Žasterisks. including neck portion Žarrowheads.. Within the dendritic shafts ŽDN., weak signal was detected near the plasma membrane Žthick arrows.. F: white matter shown in Fig. 2G. The GluR d 2-immunoreactivity was detected within the dendritic shafts Žthick arrows. and dendritic spines Žasterisks.. Scale bars: 1 mm. Fig. 5. Ultrastructural localization of the GluR d 2 in the reeler cerebellum. A,B: molecular layer. The GluR d 2-immunoreactivity was detected in dendritic spines Žasterisks., whereas dendritic shafts ŽDN. was negative. C,D: granule cell layer. Dendritic spines were immunopositive Žasterisks. in both the superficial zone ŽC. and deep zone ŽD.. In the deep zone near the central cerebellar mass, the immunoreaction product was detected inside of the dendritic shafts, near the plasma membrane Žthick arrows.. E,F: white matter ŽE. and central cerebellar mass ŽF.. Dense immunoreaction product was localized widely in both dendritic shafts ŽDN. and spines Žasterisks.. Scale bars: 1 mm.
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Fig. 5.
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GluR d 2 and altered intracellular localization of the GluR d 2 in the mutant cerebelli might be due to their abnormal cytoarchitecture and changed neural circuits. 4.2. Mutant Purkinje cells expressed the GluR d 2 mRNA normally in an abnormal enÕironment The Purkinje cell is a pivotal neuron in the cerebellar cortex, and expresses various types of glutamate receptor channel subunits, which are localized at the postsynaptic site of Purkinje dendrites and are involved in excitatory synaptic transmission w2,14,39,42,43x. Expression of the AMPA-type GluR subunits is preserved in mutant cerebelli w49x Žsee also Takayama et al., unpublished data.. Although the distribution of their mRNAs in the cerebelli seems abnormal, the abnormality corresponds to the alteration in cytoarchitecture. In contrast, expression of NMDA-type receptor subunits is altered in reeler and staggerer mutant Purkinje cells w36,60x. Thus, it was suggested that there are two groups of GluR channel subunits; one includes subunits whose expression is independent of the abnormal environment around Purkinje cells, and the other consists of subunits whose expression is altered under abnormal environmental conditions. The present study showed that GluR d 2 is expressed in all Purkinje cells and the intense signal is restricted to only Purkinje cells, even in the aberrant environment of the mutant cerebelli. Thus, abnormalities in cytoarchitecture and changed neural circuitry did not affect the GluR d 2-expression. 4.3. Parallel fiber input was related to relocalization of the GluR d 2 In weaver cerebellum except white matter, intracellular localization of the GluR d 2 in Purkinje cells was almost identical to that in normal adult mice, and the immunoreactivity was exclusively localized to dendritic spines. In the cortex of weaver cerebellum, the number of granule cells is remarkably reduced w48x. Synapses between Purkinje dendrities and parallel fibers are rare. Dendritic spines without synapses are frequently found in processes of Bergmann glial cells, and some spines form heterologous synapses with mossy fibers and Golgi cells w19,44,45,54x. Therefore, neither reduction of parallel fiber input nor formation of heterologous synapses affect the relocalization of GluR d 2 protein. On the other hand, in the reeler cerebellum, intracellular localization of GluR d 2 showed regional differences. In the cortical surface of the reeler cerebellum, the GluR d 2 protein was localized selectively in Purkinje dendritic spines as is observed in normal adult mice. In the subcortical region, however, the immunoreactivity was not restricted to dendritic spines, but was found widely in Purkinje dendrites. The altered intracellular localization detected in the subcortical region was similar to that at early postnatal stages of normal mice w58x. In this region, gran-
ule cells are absent and dendritic spines form heterologous synapses with terminals other than parallel fibers w3,30,46,55,61x. Since heterologous synapses are also observed in the weaver cortex where the GluR d 2-immunoreactivity was restricted in dendritic spines, they did not influence on the accumulation. Thus, the accumulation of GluR d 2 protein might be disturbed by an absence of synaptic contact between parallel fibers and Purkinje cells. These results suggest that parallel fiber input might be crucial for relocalization of the GluR d 2 protein and formation of synapses with parallel fibers, even whose number is reduced, might confine the receptor protein to the postsynaptic site. 4.4. Particular input was necessary for relocalization of the receptor protein In the subcortical region of the reeler cerebellum, dendritic spines of Purkinje cells directly form synapses with mossy fiber terminals instead of parallel fibers. Although the neurotransmitter of mossy fibers is considered to be the same as that of parallel fibers; e.g., glutamic acid w9x, accumulation of the GluR d 2 protein had not occurred and the GluR d 2-immunoreactivity was found widely in Purkinje dendrites. Thus, mossy fiber input is not effective for the accumulation of GluR d 2 protein and parallel fiber input might be necessary for relocalization of the GluR d 2 protein. It is well known that proteins are sorted, transported and confined to the programmed place, during the brain development w6,25,62x. Accumulation of receptor proteins to the postsynaptic site during synaptogenesis is considered to be a protein targeting phenomenon. In the case of nicotinic acetylcholine receptors, it was clearly demonstrated that motor nerve innervation confines the receptor proteins to the postsynaptic site of motor end plate w50x. In the central as well as peripheral nervous system, synapse formation appears to trigger the accumulation of the channel receptor proteins to the postsynaptic site. We previously demonstrated that the GluR d 2 protein accumulates at the synaptic site during Purkinje cell maturation w58x. The present study of mutant mice suggests that only parallel fiber input might influence the relocalization of the GluR d 2 protein. More generally, particular input may be necessary for relocalization of receptor protein in the central nervous system.
Acknowledgements The authors would like to thank Mr. Hideo Umeda and Miss Miho Kobayashi at Department of Anatomy and Mr. Yoshihiko Ogawa at Central Research Institute for their technical assistance. We are also grateful to Drs. David R. Kornack and Hitoshi Komuro at Yale University School of Medicine, for their valuable suggestions.
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