Direct interaction of GluRdelta2 with Shank scaffold proteins in cerebellar Purkinje cells

Glutamate receptor (GluR) delta2 selectively expressed in cerebellar Purkinje cells plays a central role in cerebellar long-term depression (LTD), motor learning, and formation of parallel fiber synapses. By yeast two-hybrid screening, we identified members of the Shank family of scaffold proteins as major GluRdelta2-interacting molecules. GluRdelta2 bound directly to the PDZ domain of Shank proteins through an internal motif in the carboxyl-terminal putative cytoplasmic domain. Shank1 and Shank2 proteins as well as GluRdelta2 proteins were localized in the dendritic spines of cultured Purkinje cells. Anti-GluRdelta2 antibodies immunoprecipitated Shank1, Shank2, Homer, and metabotropic GluR1alpha proteins from the synaptosomal membrane fractions of cerebella. Furthermore, Shank2 interacted with GRIP1 in the cerebellum. These results suggest that through Shank1 and Shank2, GluRdelta2 interacts with the metabotropic GluR1alpha, the AMPA-type GluR, and the inositol 1,4,5-trisphosphate receptor (IP3R) that are essential for cerebellar LTD.     

The postsynaptic scaffold proteins ProSAP1/Shank2 and Homer1 are associated with glutamate receptor complexes at rat retinal synapses

The postsynaptic density (PSD) at glutamatergic synapses is a macromolecular complex of various molecules that organize the different glutamate receptors spatially and link them to their appropriate downstream signaling pathways and to the cytoskeleton. Recently, a new family of multidomain proteins called Shanks or ProSAPs (proline-rich synapse-associated proteins) has been identified. They are suggested to be central adaptor proteins of the PSD of glutamatergic synapses, bridging different types of glutamate receptor complexes. With immunocytochemistry and light and electron microscopy, we examined the cellular, synaptic, and postnatal developmental expression of ProSAP1/Shank2 at the synapses of rat retina. With double-labeling experiments and confocal microscopy, we analyzed the association of ProSAP1/Shank2 with proteins specific for glutamatergic, glycinergic, and gamma-aminobutyric acid (GABA)ergic synapses and with proteins known to be involved in the structural and functional organization of PSDs containing N-methyl-D-aspartate receptors [95-kDa postsynaptic density protein (PSD-95)], group I metabotropic glutamate receptors (Homer1), and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors [glutamate receptor-interacting protein (GRIP)]. ProSAP1/Shank2 was present postsynaptically at the glutamatergic ribbon synapses of photoreceptor and bipolar cells, and it was absent from glycinergic and GABAergic amacrine cell synapses. The double-labeling experiments revealed a high rate of colocalization of ProSAP1/Shank2 with Homer1 and PSD-95, and little colocalization with GRIP. These data suggest that ProSAP1/Shank2 acts as an organizer at PSDs of different glutamatergic retinal synapses.     

Distinct spatiotemporal expressions of five NMDA receptor channel subunit mRNAs in the cerebellum

The distribution of five NMDA receptor channel subunit mRNAs was examined in the mouse cerebellum from embryonic day 13 through postnatal day 56, by in situ hybridization with subunit-specific oligonucleotide probes. At postnatal days 21 and 56, each cerebellar neuron displayed differential expressions of the ∈ subunit mRNAs. The granule cells showed hybridizing signals for the ∈1 and ∈3 subunit mRNAs, the molecular layer neurons for the ∈4 subunit mRNA, and the cerebellar nucleus neurons for the ∈1 and ∈4 subunit mRNAs, whereas the Purkinje cells did not express any ∈ subunit mRNAs. At early postmitotic stages of development, the ∈2 subunit mRNA appeared in each cerebellar neuron, including the Purkinje cells, and the ∈4 subunit mRNA appeared in neurons of the molecular layer and the cerebellar nuclei. The expression patterns in the cerebellum altered drastically during the first 2 postnatal weeks: the ∈1 and ∈3 subunit mRNAs appeared in the granule cells and the cerebellar nucleus neurons, whereas the ∈2 subunit mRNA disappeared from each neuron and the signal levels of the ∈4 subunit mRNA decreased remarkably. In contrast to the differential expressions of the four ∈ subunit mRNAs, intense signals for the ζ 1 subunit mRNA were observed in each cerebellar neuron from early postmitotic stages through the mature stage. These findings suggest that anatomical organization of the ∈ subunits is heterogeneous in the cerebellum both spatially and temporally, which would give rise to functional diversity of the NMDA receptor channel. © 1994 Wiley-Liss, Inc.     

Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol-12-myristate-13-acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.     

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs, for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly. © 1994 Wiley-Liss, Inc.     

Lack of NMDA receptor subunit exchange alters Purkinje cell dendritic morphology in cerebellar slice cultures

Early postnatal developmental changes in N-methyl-d-aspartate (NMDA) receptor (NR) subunits regulate cerebellar granule cell maturation and potentially Purkinje cell development. We therefore investigated Purkinje cell morphology in slice cultures from mice with genetic subunit exchange from NR2C to NR2B (NR2C-2B). NR2C-2B Purkinje cells after 12 days in vitro showed a significantly impaired dendritic arbour complexity with reduced branching density as compared to wild-type cells, a phenotype that was reversed by NMDA treatment. These data support the concept that in cerebellar slice cultures, Purkinje cell dendritic outgrowth is regulated by granule cell inputs.     

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.     

In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain

Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha-Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta-subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co-localization of translational machinery and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.     

Identification and characterization of a novel Delphilin variant with an alternative N-terminus

Delphilin is identified as a Glutamate receptor delta2 (GluRdelta2) subunit interacting protein, consisting of a PDZ domain and formin homology (FH) domains 1 and 2, in addition to a C-terminal coiled-coil structure. Delphilin has been shown to be selectively expressed in cerebellar Purkinje cells where it co-localizes with the GluRdelta2 subunit at the Purkinje cell-parallel fiber synapses. Although Delphilin specifically interacts with the GluRdelta2 C-terminus via its PDZ domain, the physiological role of the interaction is not yet understood. Here, we report that the Delphilin protein exhibits diversity at its N-terminus by variable usage of the first several exons. Interestingly, the two Delphilin mRNAs which correspond to the first one initially identified (now designated as Delphilin alpha) and the second that contains a newly identified first exon (designated as Delphilin beta), show different chronological expression profiles. Delphilin beta mRNA was not decreased throughout the cerebellar development in vivo and in vitro, while in vivo Delphilin alpha mRNA gradually decreases following the first postnatal week. Delphilins alpha and beta also revealed different subcellular distribution with some overlap. Specifically, the cerebellar synaptosomal membrane fraction contained the Delphilin beta protein. Both Delphilin alpha and beta localized at the dendritic spines with GluRdelta2; however, dendritic shafts in cultured Purkinje cells also included Delphilin beta. In MDCK cells upon becoming confluent, Delphilin alpha moved to the cell-cell junction area, whereas Delphilin beta maintained a diffuse distribution pattern throughout the cytoplasm. Taken as a whole, these two different Delphilins seemed to play functionally different roles in developing and matured cerebellar Purkinje cells.     

Immunogold localization of phocein in dendritic spines

Phocein, a widely expressed intracellular protein involved in clathrin- and dynamin-dependent membrane dynamics, has been shown to interact with members of the striatin family of proteins, striatin, SG2NA, and zinedin. Immunogold labeling was performed to assess subcellular localization of phocein in neurons of the rodent cerebellar cortex and hippocampal Ammon's horn. Most of the phocein-bound gold particles were located within dendritic thorns and spines of the cerebellar Purkinje cells and hippocampal pyramidal neurons, as observed previously for striatin in striatal neurons. The postsynaptic profiles containing phocein were engaged in asymmetric synapses with the main types of afferents in the cerebellum and in the hippocampus. In the cerebellum, phocein-bound immunogold particle numbers ranged from 1-20 in approximately 50% of the Purkinje cell spines. In these spines most of the immunogold particles were found in the neuroplasm ( approximately 70%) and on nonsynaptic plasma membrane domains and related structures such as endocytic-like profiles ( approximately 18%). As soon as the first postnatal week, phocein was detected in the Purkinje cell somatic and dendritic thorns making asymmetric synapses with climbing fibers. During the following weeks the protein was located in the dendritic spines, as observed in the adult molecular layer. Finally, double immunogold labeling revealed a distribution of phocein and SG2NA suggesting that the two proteins could interact in the Purkinje cell spines. The early postnatal expression of phocein, a protein involved in membrane dynamics, suggests that it may have functional relevance in dendritic remodeling during development and potentially in spine plasticity during adulthood.     

Transient expression of GABAA receptor alpha2 and alpha3 subunits in differentiating cerebellar neurons

In the adult mammalian brain, synaptic transmission mediated by gamma-amino butyric acid (GABA) plays a role in inhibition of excitatory synaptic transmission. During brain development, GABA is involved in brain morphogenesis. To clarify how GABA exerts its effect on immature neurons, we examined the expression of the GABAA receptor alpha2 and alpha3 subunits, which are abundantly expressed before alpha1 and alpha6 subunits appear, in the developing mouse cerebellum using in situ hybridization. Proliferating neuronal precursors in the ventricular zone and external granular layer expressed neither alpha2 nor alpha3 subunits. Hybridization signals for the alpha2 and alpha3 subunit mRNAs first appeared in the differentiating zone at embryonic day 13 (E13). The alpha2 subunit was detected in the migrating and differentiating granule cells and cerebellar nucleus neurons until postnatal day 14 (P14). Hybridization signals for the alpha3 subunit mRNA, on the other hand, were localized in the developing Purkinje cells and cerebellar nucleus neurons, and disappeared from Purkinje cells by the end of first postnatal week. Taken together, this indicated that the alpha2 and alpha3 subunits were abundantly expressed in distinct types of cerebellar neurons after completing cell proliferation while forming the neural network. These results suggest that GABA might extrasynaptically activate the GABAA receptors containing alpha2 and/or alpha3 subunits on the differentiating neurons before finishing the formation of synapses and networks, and could be involved in neuronal differentiation and maturation in the cerebellum.     

Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.     

Computer-assisted analysis of the developing Purkinje neuron. I. Effects of the age of the animal at the moment of explantation on the subsequent dendritic development in organotypic cultures

Purkinje cell dendritic arborization were studied in intracellularly horseradish peroxidase-stained mature neurons grown during 30-40 days in organotypic cerebellar cultures from kittens of various ages. The effects of the age of the animal at the moment of explantation upon the subsequent dendritic and axonal development were studied in kitten cerebella of 1, 12 and 21 postnatal days old. These effects were investigated by computer-assisted methods. Qualitative data were found to be different in these 3 in vitro models explanted at 3 different ages except for the spine development which remained the same. Quantitative data, obtained from 15 measurable parameters, showed that the degree of dendritic development was higher for the Purkinje cells explanted at the latest postnatal age: the older the animal, the larger the dendritic tree in culture. A discrimination analysis permitted a recognition of the 3 differently aged populations (94% well classified cells) based on 3 selected variables, number of primary dendritic trunks, number of rows and dendritic field areas. These results lend further support to the major role played in the final dendritic arrangement by extrinsic (discriminant factors, their pre-existence in vivo being responsible for the subsequent degree of the in vitro development of the Purkinje cell dendritic arbor.