P2Y1receptor switches to neurons from glia in juvenile versus neonatal rat cerebellar cortex
© Amadio et al; licensee BioMed Central Ltd. 2007
Received: 12 April 2007
Accepted: 28 June 2007
Published: 28 June 2007
In the CNS, several P2 receptors for extracellular nucleotides are identified on neurons and glial cells to participate to neuron-neuron, glia-glia and glia-neuron communication.
In this work, we describe the cellular and subcellular presence of metabotropic P2Y1 receptor in rat cerebellum at two distinct developmental ages, by means of immunofluorescence-confocal and electron microscopy as well as western blotting and direct membrane separation techniques. At postnatal day 21, we find that P2Y1 receptor in addition to Purkinje neurons, is abundant on neuronal specializations identified as noradrenergic by anatomical, morphological and biochemical features. P2Y1 receptor immunoreactivity colocalizes with dopamine β-hydroxylase, tyrosine hydroxylase, neurofilament light chain, synaptophysin and flotillin, but not with glial fibrillary acidic protein for astrocytes. P2Y1 receptor is found enriched in membrane microdomains such as lipid rafts, in cerebellar synaptic vesicles, and is moreover visualized on synaptic varicosities by electron microscopy analysis. When examined at postnatal day 7, P2Y1 receptor immunoreactivity is instead predominantly expressed only on Bergmann and astroglial cells, as shown by colocalization with glial fibrillary acidic protein rather then neuronal markers. At this age, we moreover identify that P2Y1 receptor-positive Bergmann fibers wrap up doublecortin-positive granule cells stretching along them, while migrating through the cerebellar layers.
Membrane components including purinergic receptors are already known to mediate cellular contact and aggregation in platelets. Our results suggesting a potential role for P2Y1 protein in cell junction/communication and development, are totally innovative for the CNS.
Extracellular nucleotides partake to excitatory neurotransmission and neuromodulation in the CNS and are capable of intervening in a broad array of physiopathological functions acting on different P2 purinergic receptors [1–3]. Because of their heterogeneous faculty of responding to several diverse nucleotides, interest is growing on discovering the exact localization of the various P2 receptor subunits in selected organs, tissues and cellular phenotypes [4, 5]. To date, seven different ionotropic P2X (P2X1–7)  and eight distinct metabotropic P2Y (P2Y1,2,4,6,11,12,13,14)  receptors were cloned from mammalian species. In contrast to P2X ion channels, P2Y proteins hold the general feature of G protein-coupled receptors with seven hydrophobic transmembrane domains. Since activation of P2Y subtypes leads to second messenger cascades, their response is slower than that mediated by P2X subunits [8–10]. Among P2Y receptors, an elevated expression particularly of the P2Y1 subtype was detected in both human  and rat brain . In human tissue, expression of P2Y1 protein is reported exclusively on neuronal cells in cerebral and cerebellar cortex, in hippocampus, caudate-putamen nuclei, globus pallidus, subthalamic nucleus and midbrain . Moreover, P2Y1 protein appears associated to neurofibrillary tangles and neuritic plaques in postmortem brain of Alzheimer's disease patients . On the contrary, P2Y1 receptor in rat is detected not only on grey matter, particularly cerebellar, cortical and hippocampal neurons, but also on white matter of corpus callosum and optic nerve .
Many studies have already highlighted a general role for purinergic signaling in brain development  and in neuron and/or glia function and communication [15–17]. For instance, activation particularly of P2Y1 receptor is suggested to regulate oligodendrocyte progenitor functions , whereas reduced levels of P2Y1 protein apparently affect proliferation and migration, but not differentiation of neural progenitor cells during early CNS development . With the present work, we further study P2Y1 receptor and compare protein distribution in juvenile versus neonatal rat cerebellum. We show that during postnatal growth, the phenotypic appearance of P2Y1 protein undergoes a drastic switch from glial to neuronal localization, therefore suggesting this receptor as a novel marker of cerebellar development.
P2Y1receptor is present on tyrosine hydroxylase- and dopamine β-hydroxylase-positive neurons in juvenile rat cerebellum
On the other hand, P2Y1 receptor does not colocalize with glial fibrillary acidic protein (GFAP), therefore excluding its concurrent expression on astrocytes (Fig. 8B, lower-right inset).
Ultrastructural analysis indicates that P2Y1receptor is present on both axon terminals and dendrites
P2Y1receptor localizes in lipid rafts and synaptosomes
Since fluorescence microscopy also demonstrated a high degree of co-localization between P2Y1 receptor and synaptophysin (Fig. 4), fractions were also prepared directly from cerebellar synaptosomes, in order to confirm the synaptic localization of lipid rafts-resident P2Y1 receptor. Our results again demonstrate that only the P2Y1 receptor oligomer is present in synaptosomal lipid rafts-enriched fractions (Fig 7C), together with the lipid rafts markers flotillin-2 and GM1ganglioside (fractions 4–7). Neither monomer nor oligomer receptor is instead detected in the high density synaptosomal fractions (fractions 10–12) containing transferrin receptor, a protein known to be excluded from lipid rafts microdomains.
The phenotypic expression of P2Y1receptor switches during cerebellar development
We next investigated the expression of P2Y1 receptor at postnatal day 7 (P7), when the maturation of synapses is just taking place between granule neuron dendrites and fiber terminals , and the internal granular layer (igl) rapidly expands beneath the monocellular sheet of pc. With the only exception of calbindin-positive pc (Fig. 8A), we find that P2Y1 receptor localization is completely different from that observed at P21 (Fig. 8A, inset). At P7, P2Y1 receptor immunoreactivity is indeed absent from noradrenergic fibers of white matter, but predominantly shown: a) on fibers irradiating the thin ml (above the pl) and the external granule (or germinal) layers (egl) (Fig. 8B); b) on cell bodies aligned at the interface between the pc and ml (Fig. 8B, upper-left inset); c) on isolated cells located in the igl (Fig. 8B). P2Y1 receptor immunostaining is thus totally absent from NFL-positive (Fig. 8C) and TH- or DβH-positive fibers (data not shown), which already span into the gl at this age of development. By means of distinctive morphology of cerebellar astrocytes and positive colocalization between P2Y1 receptor and GFAP (Fig. 8B), at P7 we do identify P2Y1 receptor on both Bergmann cell bodies/fibers and astroglial cells scattered among granule neurons. This is confirmed for postnatal day one (data not shown), but is the opposite of what was shown at P21, when P2Y1 receptor is totally absent from GFAP-positive structures (Fig. 8B, lower-right inset) and present only on neuronal fibers (Figs. 2, 3, 4). Conversely, the enrichment of P2Y1 receptor in lipid rafts, but not synaptosomal fractions, is confirmed also at P7 (data not shown), therefore appearing as a conserved feature of this receptor.
The main goal of our study was to characterize the cellular and subcellular distribution and features of the purinergic metabotropic P2Y1 receptor in the cerebellum, and to gain insights on its potential function during development of the cerebellar circuitry. To this purpose, we analyzed P2Y1 receptor expression at P21, when the major structural changes already took place in the developing cerebellum, and compared it with expression at P7, when migration of granule neurons from the molecular to the granular layer is still occurring [23, 26, 27], and when the neuronal bodies in the locus coeruleus of origin for the dorsal bundle of noradrenergic neurons innervating the cerebellum are spreading out their projections to the granular, Purkinje and molecular layers . In juvenile rat cerebellum we find that, in addition to Purkinje cell bodies and ramifications , P2Y1 receptor is abundant on neuronal specializations identified as noradrenergic by anatomical (the fibers travel in linear, sparse and different thickness profiles through the gl, and branch into radially and longitudinally oriented chains), ultrastructural (abundance of synaptic varicosities and winding, beaded appearance) and biochemical features (immunoreactivity with noradrenergic-specific DβH absent from Purkinje neurons in rat) . Moreover, we observe that P2Y1 receptor is preferentially localized on synaptic varicosities of the noradrenergic fibers and synaptic glomeruli, which can be recognized by the presence of synaptophysin. The ultrastructural analysis confirms the localization of P2Y1 receptor on varicosities of axon terminals, moreover showing features indicative of a particular modality of transmitter release. As generally reported for noradrenergic fibers , the absence of conventional synaptic junctions led us to suppose that in the cerebellum also extracellular nucleotides might exert their modulatory action through paracrine release or simple diffusion in the extracellular space. This modality has the advantage to extend the potential effects of extracellular nucleotides, and neurotransmitters in general, to structures equipped with adequate receptors (extra-synaptic receptors) , but distant from the source of release (volume transmission) . The enrichment in these P2Y1-positive varicosities of both synaptophysin and the lipid rafts marker flotillin-2 (together with the biochemical observation that P2Y1 receptor is indeed a lipid rafts-resident protein in total cerebellar tissue and cerebellar synaptosomes) furthermore suggests that the role of P2Y1 receptor is likely mediated by lipid microdomains. In this regard, it is already well-known that lipid rafts display a key part in the targeting and functional organization of proteins at both synapses and spines .
Since a further specification of the central noradrenergic system consists in the frequency of co-transmission phenomena, the presence in the cerebellum of P2Y1 receptor on noradrenergic neurons in addition to Purkinje cells might signify that extracellular nucleotides such as the natural agonists ATP and ADP could contribute in this brain region to the physiological role of noradrenaline . In this regard, ATP is already well known to be co-released with noradrenaline in various PNS and CNS neurons [35, 36]. Thus, if co-release and synergism occur at cerebellar synapses as well, nucleotide-dependent increase of cerebellar noradrenergic signaling could be achieved. Homologous recombination techniques have shown that complete removal of either the enzymes responsible for noradrenaline metabolism or the vesicular monoamine transporter has deleterious consequences for foetal survival . Moreover, blockade of postsynaptic noradrenergic receptors decreases the rate of learning in several cerebellar-dependent motor tasks . The possibility of integrating and complementing noradrenergic with purinergic mechanisms to increase the strength of synaptic connections through activation of both noradrenergic and P2Y1 receptors might therefore constitute also a novel powerful approach against neuronal degeneration and/or malfunctioning in the cerebellum.
The presence of P2Y1 receptor in postnatal rat cerebellum was then analyzed before the formation of noradrenergic synaptic connections takes place. Surprisingly, we have shown here that P2Y1 undergoes a drastic switch during development, with a phenotypic expression resembling that of Bergmann glia at P7, rather then noradrenergic neurons at P21. In this regard, it is well established that a number of neuronal and glial receptor systems and/or diffusible factors act to induce and maintain Bergmann glia process extension at an early stage of postnatal development [39–41], when Bergmann cells specialize in supporting the migration of granule neurons and migration of granule cells seems to be largely dependent on their interaction with glial processes [42, 43]. In particular, the ErbB4 tyrosine kinase receptor present on Bergmann glia appears to have a distinct role in this process , interacting with neoregulin expressed on migrating granule cells [45, 46]. On the other hand, in vitro studies on the migration of granule neurons demonstrated that the glycoprotein astrotactin provides a neuronal receptor system for migration along glial processes . Thus, P2Y1 receptor expression only on Bergmann glia at an early stage of development might sustain a role for purinergic receptors in signaling events needed for interaction and migration of neurons. This is strongly supported by the result that ADP, preferential ligand for P2Y1receptor, induces Ca2+ mobilization in Bergman glia . The factors produced by granule neurons to induce the glial scaffold might thus comprise the purinergic ligands ATP and ADP directly targeting P2Y1 receptors. To support this hypothesis, we have both shown here that P2Y1 receptor is localized at the interface between Bergmann glia and DCX-positive migrating granule cells, and previously demonstrated that, at least in vitro, granule neurons can release ATP, which is easily degraded to ADP in the extracellular environment . It is finally well known that, once migration across the glia scaffold is completed and cells change their repertoire of adhesive molecules and switch into a static asset: a) the Bergmann glia loses the apposition with granule neurons; b) the granule cells are locked in position by the formation of new specific axon-target interactions . In parallel, we have demonstrated here that in juvenile rat cerebellum: a) the Bergmann glia lose P2Y1 receptors; b) P2Y1 receptors appear on Purkinje and noradrenergic neurons forming new specific axon-target interactions.
Since membrane components of the cell surface are largely known to mediate the close apposition between two cells during all phases of development, our results suggest a novel role for P2Y1 receptor in the CNS, specifically in cell junction/communication. This already occurs on platelets, where P2Y1 receptor is well known to mediate cell contact and aggregation [50–52]. Considering the glia-neuron switch of P2Y1 receptor a novel biological mechanism of development, aim of further studies will be to investigate the potential impact of this receptor on Bergman glia-guided migration of granule neurons; on alteration/reorganization of noradrenergic fibers in the cerebellar cortex in response to the degeneration of their major target, granule and Purkinje neurons; and finally to investigate if the presence of purinergic ligands in the environment surrounding noradrenergic fibers can influence their anatomical integrity and development.
Wistar rats (Harlan, Udine, Italy) of different ages were deeply anesthetized by i.p. injections of sodium pentobarbital (60 mg/kg), and transcardially perfused with saline (0.9% NaCl) followed by 4% paraformaldehyde, in phosphate buffer (PB, 0.1 M pH 7.4). Each brain was immediately removed, post-fixed in the same fixative for 2 hs, and then transferred to 30% sucrose in PB at 4°C, until it sank. The experimental protocol used in this study was approved by the Italian Ministry of Health and was in agreement with the guidelines of the European Communities Council Directive of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and their suffering.
Sagital sections (40 μm thick) were cut on a freezing microtome and were processed for double immunofluorescence studies. Non-specific binding sites were blocked with 10% normal donkey serum in 0.3% Triton X-100, in phosphate buffered saline (PBS) for 30 min at room temperature. The sections were incubated in a mixture of primary antisera for 24 hs in 0.3% Triton X-100 in PBS. Rabbit anti-P2Y1 (1:500, Alomone, Jerusalem-Israel) was used in combination with either mouse anti-Calbindin-D-28K (1:200, Sigma, Mi-Italy), mouse anti-Tyrosine Hydroxylase (TH, 1:500, Sigma), mouse anti-Dopamine β-Hydroxylase (DβH, 1:500, Chemicon International, Inc. Temecula, CA-USA), mouse anti-Glial Fibrillary Acidic Protein (GFAP) (1:400, Sigma), mouse anti-Synaptophysin (1:100, Sigma), goat anti-Doublecortin (DCX, 1:200, Santa Cruz, Mi-Italy), or goat anti-NFL (Neurofilament-L protein, 1:100, Santa Cruz). The secondary antibodies used for double labeling were Cy3-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunoresearch, West Baltimore Pike, PA, USA, red immunofluorescence), Cy2-conjugated donkey anti-mouse IgG (1:100, Jackson Immunoresearch, green immunofluorescence) or Cy2-conjugated donkey anti-goat IgG (1:100, Jackson Immunoresearch, green immunofluorescence). The sections were washed in PBS three times for five min each, and then incubated for 3 hs in a solution containing a mixture of the secondary antibodies in 1% normal donkey serum in PBS. After rinsing, the sections were mounted on slide glasses, allowed to air dry and coverslipped with gel/mount™ anti-fading medium (Biomeda, Foster City, CA-USA).
After double immunofluorescence, the sections were mounted on slide glasses, and allowed to air dry. A rectangle was then drawn around the sections with a PAP pen. To allow the use of a second mouse antibody in the same immunolabeling protocol, the unlabeled monoclonal anti-NeuN (Neuronal Nuclei, mouse IgG1 isotype or anti-Calbindin-D-28K (mouse IgG1 isotype)) or Flotillin-2 (mouse IgG1 isotype) were labeled with Zenon technology (Molecular Probes, Oregon, USA). Briefly, mouse anti-NeuN (1:100, Chemicon International), mouse anti-Flotillin-2 (1:100, BD Biosciences, San José, CA) and mouse anti-Calbindin-D-28K (1:200, Sigma) were separately incubated with Zenon Alexa Fluor 647 mouse IgG1 labeling reagent (molar ratio 6:1), which contains a fluorophore-labeled (Ex/Em 650/668) anti-mouse Fab fragments. The labeled Fab fragments bind to the Fc portion of the monoclonal antibodies and excess Fab fragments are neutralized by the addition of a nonspecific IgG (Zenon blocking reagent-mouse IgG). The addition of non-specific IgG prevents cross-labeling of the Fab fragment, in experiments where multiple primary antibodies of the same type are present. After rehydration in PBS, the sections were incubated with the staining solution in PBS containing 0.5% Triton X-100 (PBT) in a humidified chamber for 2 hs at room temperature. The sections were washed twice in PBT and for 5 min in PBS at room temperature. Sections were then fixed in 4% paraformaldehyde in PB for 15 min at room temperature, to avoid the dissociation of the Zenon Fab fragment from the primary antibody, washed three times with PBS, allowed to air dry and coverslipped with gel/mount anti-fading medium.
Double or triple label immunofluorescence was analyzed by means of a confocal laser scanning microscope (CLSM) (LSM 510, Zeiss, Arese Mi-Italy) equipped with argon laser emitting at 488 nm, helium/neon laser emitting at 543 nm, and helium/neon laser emitting at 633 nm. Specificity of the antibodies was positively proved by performing confocal analysis in the absence of the primary antibodies, but in the presence of either anti-rabbit or anti-mouse secondary antibodies. Specificity was further confirmed for the P2Y1 antiserum by performing immunoreactions in the simultaneus presence of the P2Y1 neutralizing immunogenic peptide.
Electron microscopy sample preparation
Rats (n = 4) were anaesthetised with chloral hydrate (400 mg/kg i.p.), perfused through the ascending aorta with a solution of NaCl 0.9% for 5 min, then followed by 3% paraformaldehyde with 0.4% glutaraldehyde in PB for 30 min. Cerebella were dissected and sagittal sections were cut at 40 μm, and washed several times in PBS. Sections were treated with sodium borohydrate (Sigma) 0.1% in PBS. Immunoreactivity for P2Y1 receptor was detected by means of the avidin-biotin peroxidase method. Briefly, sections from the cerebellum were pre-blocked in a solution containing 10% goat serum in PB for 30 min at room temperature. Then, sections were incubated in a solution containing primary antibody against P2Y1 receptor (rabbit anti-P2Y1diluted 1:200) in PB for 24 hs at 4°C. After several washes in PBS, sections were incubated with biotinylated secondary antibody (goat anti-rabbit diluted 1:100 Vectastain Elite, Vector Laboratories, Peterborough, UK) in PB for 3 hs at room temperature. They were then incubated in avidin-biotin peroxidase complex (diluted 1:100 in PB; Vectastain Elite, Vector Labs.) for 1 h. After washing, immunolabeling was revealed by incubation of the sections in 0.05% 3,3'-diaminobenzidine solution (DAB-Sigma) diluted in Tris-HCl buffer, in the presence of 0.01% H2O2. The reaction was stopped by several washes in Tris-HCl buffer followed by PBS. Sections were post-fixed in osmium tetroxide (1% in PB) for 10 min, dehydrated in ascending series of dilution of ethanol (with the presence of 1% uranyl acetate in 70% ethanol) followed by propylene oxide (Aldrich, MI, Italy) and then embedded overnight in resin (Durcupan ACM-Fluka, Gillingham, Dorset, UK), mounted on glass slides and then cured at 60°C for 48 hs. The areas of interest were examined in the light microscope, cut from the sections and 60 nm ultra-thin sections were obtained with an ultramicrotome (Reichert-Jung Ultracut E, Leica, Nussloch, Germany), and collected on 400-mesh copper grids, counterstained with lead citrate and examined using a Zeiss EM900 electron microscope. Controls were performed omitting the primary antibody from the procedure.
Electron microscopy data analysis
The ultrastructural analysis was performed exclusively on the most superficial portions of the tissue in contact with the embedding plastic, in order to minimize artificial differences in labeling attributed to potential differences in the penetration reagents. Regions used for this analysis were chosen on the basis of P2Y1 receptor immunoreactivity and the morphological integrity of the tissue. The labelled profiles were examined in thirty-two ultra-thin sections from three separate rats, in four sections each taken from the vermis, the cerebellar hemispheres and the cerebellar nuclei. Electron micrographs of immunoreactive structures were taken at magnifications of 7000–30000 X, then printed and used as the sampling region of each block. The classification of neuronal elements was made according to the description of Peters et al., . Neuronal somata were identified by the nucleus, Golgi apparatus, and rough endoplasmic reticulum; un-myelinated axons were distinguished from dendrites by their larger diameter and/or the abundance of uniformly distributed microtubules and synaptic inputs from axon terminals. Neuronal profiles were classified as un-myelinated axons if they were 0.1–0.25 μm in cross sectional diameter and contained microtubules and/or small vescicles. Axon terminals were defined as elements 0.25 μm or larger in diameter containing numerous small synaptic vescicles. Synapses formed by axon terminals were defined as asymmetric, when their post-synaptic density was thicker that the pre-synaptic one, and as symmetric, when both membranes showed equal electron density. Two structures were considered adjacent, when the two plasma membranes were parallel and not separated by glial processes, but no membrane specialization was visible.
Preparation of low density membrane fractions
Detergent-insoluble glycolipid fractions were prepared following an established method , with minor modifications . Briefly, cerebella were removed from 21 days old Wistar rats, cut with a McIlwain tissue chopper (400 μm) in two orthogonal directions and resuspended in 10 volumes (w/v) of ice-cold lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 20 μM Leupeptin) by vortexing. After 30 min, the lysate was centrifuged at 2000 × g for 10 min, to remove nuclei and large debris. The resulting supernatant (500 μl, 2–2.5 mg) was mixed with an equal volume of 85% sucrose in TBS (10 mM Tris-HCl pH 7.5, 150 mM NaCl), and placed at the bottom of the centrifuge tube. A volume of 700 μl of 35% sucrose in TBS and 400 μl of 5% sucrose were layered on the top of the lysate. The gradient was centrifuged for 14 hs at 200.000 × g in a TLS 55 rotor (Beckman Instruments, Porterville, CA). Four fractions of 150 μl and five fractions of 300 μl were collected from the top of the tube. The entire procedure was performed at 4°C. Equal volumes, containing a range of approximately 0.5–100 μg of total protein depending on the fraction, were loaded on SDS-PAGE electrophoresis.
Synaptosome Triton X-100 solubilization and sucrose floatation gradients
Synaptosomes were obtained from rat cerebella by means of differential centrifugation, as previously described . Briefly, post-nuclear supernatants were centrifuged at 9200 × g for 15 min to yield a pellet corresponding to partially purified synaptosomes. These pellets (6 mg of proteins) were resuspended in 0.75 ml of buffer A (150 mM NaCl, 2 mM EGTA, 50 mM Tris-HCl, pH 7.5, protease inhibitors) containing 1% (w/v) Triton X-100. After 30 min on ice, each sample was adjusted to 1.2 M sucrose, placed in a centrifuge tube and overlaid with a linear gradient ranging from 30 to 5% sucrose (all prepared in buffer A). The gradients were centrifuged at 190000 × g for 19 hs using a rotor SW 41 Ti (Beckman Instruments). Fifteen fractions (0.8 ml each), and the pellets resuspended in 0.8 ml of buffer A were collected and analysed by means of SDS-PAGE and western blotting. The sucrose concentration in each fraction was determined by refractometry.
Western blot analysis
Equal amount of sucrose gradient samples was separated by electrophoresis on 10%–12% SDS-PAGE and transferred to nitrocellulose membranes Hybond-C extra (Amersham Biosciences, Cologno Monzese, Italy). The filters were pre-wetted in 5% non-fat milk in TBS-T (10 mM Tris pH 8, 150 mM NaCl, 0.1%Tween 20), hybridized overnight with rabbit anti-P2Y1 (1:400) and with mouse anti-Flotillin-2 (1:1000), followed by horseradish peroxidase-coupled secondary antibody, and analysed by ECL chemiluminescence (Amersham Biosciences), using Kodak Image Station (KDS IS440CF).
The polyclonal P2Y1 antiserum used in this study was raised against a P2Y1 receptor highly purified peptide (identity confirmed by mass spectroghraphy and aminoacid analysis), corresponding to a specific epitope not present in any other known protein: residues 242–258 of rat and human P2Y1 (3rd intracellular loop). The specificity of the P2Y1 receptor signal was assessed by incubating western blots either in the absence of the primary antiserum, or in the presence of the primary antiserum together with the neutralizing P2Y1 immunogenic peptide (μg protein ratio 1:1 between peptide and antiserum). Furthermore, the P2Y1 receptor polyclonal antiserum was proved to efficiently immunoprecipitate the recombinant Myc-P2Y1 human receptor transiently transfected in SH-SY5Y cells.
external granule layer
glial fibrillary acidic protein
internal granule layer
- postnatal day 7:
- postnatal day 21:
The research presented was supported by Cofinanziamento MIUR "Purinoceptors and Neuroprotection", and grant from Ministero della Salute RF05.105V. We thank Dr. Nadia D'Ambrosi for experiments with recombinant Myc-P2Y1 human receptor transiently transfected in SH-SY5Y cells, and Dr. Patrizia Rosa and Elena Saba for the preparation of synaptosomal lipid rafts-enriched fractions.
- Illes P, Ribeiro JA: Neuronal P2 receptors of the central nervous system. Curr Top Med Chem. 2004, 4: 831-838. 10.2174/1568026043451032.View ArticlePubMedGoogle Scholar
- Burnstock G: Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev. 2006, 58: 58-86. 10.1124/pr.58.1.5.View ArticlePubMedGoogle Scholar
- Burnstock G: Historical review: ATP as a neurotransmitter. Trends Pharmacol Sci. 2006, 27: 166-176. 10.1016/j.tips.2006.01.005.View ArticlePubMedGoogle Scholar
- Burnstock G, Knight GE: Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol. 2004, 240: 31-304.View ArticlePubMedGoogle Scholar
- Volonté C, Amadio S, D'Ambrosi N, Colpi M, Burnstock G: P2 receptor web: complexity and fine-tuning. Pharmacol Ther. 112 (1): 264-280. 10.1016/j.pharmthera.2005.04.012.
- Köles L, Furst S, Illes P: Purine ionotropic (P2X) receptors. Curr Pharm Des
- Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, Weisman GA: Update of the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev. 2006, 58 (3): 281-341. 10.1124/pr.58.3.3.PubMed CentralView ArticlePubMedGoogle Scholar
- Ralevic V, Burnstock G: Receptors for purines and pyrimidines. Pharmacol Rev. 1998, 50: 413-492.PubMedGoogle Scholar
- Communi D, Gonzalez NS, Detheux M, Brezillon S, Lannoy V, Parmentier M, Boeynaems JM: Identification of a novel human ADP receptor coupled to G(i). J Biol Chem. 2001, 276: 41479-41485. 10.1074/jbc.M105912200.View ArticlePubMedGoogle Scholar
- Abbracchio MP, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Miras-Portugal MT, King BF, Gachet C, Jacobson KA, Weisman GA, Burnstock G: Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends Pharmacol Sci. 2003, 24: 52-55. 10.1016/S0165-6147(02)00038-X.View ArticlePubMedGoogle Scholar
- Moore D, Chambers J, Waldvogel H, Faull R, Emson P: Regional and cellular distribution of the P2Y(1) purinergic receptor in the human brain: striking neuronal localisation. J Comp Neurol. 2000, 421: 374-384. 10.1002/(SICI)1096-9861(20000605)421:3<374::AID-CNE6>3.0.CO;2-Z.View ArticlePubMedGoogle Scholar
- Moran-Jimenez MJ, Matute C: Immunohistochemical localization of the P2Y(1) purinergic receptor in neurons and glial cells of the central nervous system. Brain Res Mol Brain Res. 2000, 78: 50-58. 10.1016/S0169-328X(00)00067-X.View ArticlePubMedGoogle Scholar
- Moore D, Iritani S, Chambers J, Emson P: Immunohistochemical localization of the P2Y1 purinergic receptor in Alzheimer's disease. Neuroreport. 2000, 11: 3799-3803. 10.1097/00001756-200011270-00041.View ArticlePubMedGoogle Scholar
- Franke H, Illes P: Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacol Therapeut. 2006, 109: 297-324. 10.1016/j.pharmthera.2005.06.002.View ArticleGoogle Scholar
- James G, Butt AM: P2Y and P2X purinoceptor mediated Ca2+ signalling in glial cell pathology in the central nervous system. Eur J Pharmacol. 2002, 447: 247-260. 10.1016/S0014-2999(02)01756-9.View ArticlePubMedGoogle Scholar
- Fields RD, Burnstock G: Purinergic signalling in neuron-glia interactions. Nat Rev Neurosci. 2006, 7: 423-436. 10.1038/nrn1928.PubMed CentralView ArticlePubMedGoogle Scholar
- Amadio S, Tramini G, Martorana A, Viscomi MT, Sancesario G, Bernardi G, Volonté C: Oligodendrocytes express P2Y12 metabotropic receptor in adult rat brain. Neuroscience. 2006, 141: 1171-1180. 10.1016/j.neuroscience.2006.05.058.View ArticlePubMedGoogle Scholar
- Agresti C, Meomartini ME, Amadio S, Ambrosini E, Serafini B, Franchini L, Volonté C, Aloisi F, Visentin S: Metabotropic P2 receptor activation regulates oligodendrocyte progenitor migration and development. Glia. 2005, 50: 132-144. 10.1002/glia.20160.View ArticlePubMedGoogle Scholar
- Scemes E, Duval N, Meda P: Reduced expression of P2Y1 receptors in connexin43-null mice alters calcium signaling and migration of neural progenitor cells. J Neurosci. 2003, 23: 11444-11452.PubMed CentralPubMedGoogle Scholar
- Nelson TE, King JS, Bishop GA: Distribution of tyrosine hydroxylase-immunoreactive afferents to the cerebellum differs between species. J Comp Neurol. 1997, 379: 443-454. 10.1002/(SICI)1096-9861(19970317)379:3<443::AID-CNE9>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- Bickel PE, Scherer PE, Schnitzer JE, Oh P, Lisanti MP, Lodish HF: Flotillin and epidermal surface antigen define a new family of caveolae-associated integral membrane proteins. J Biol Chem. 1997, 272: 13793-13802. 10.1074/jbc.272.21.13793.View ArticlePubMedGoogle Scholar
- Wang L, Karlsson L, Moses S, Hultgardh-Nilsson A, Andersson M, Borna C, Gudbjartsson T, Jern S, Erlinge D: P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol. 2002, 40: 841-853. 10.1097/00005344-200212000-00005.View ArticlePubMedGoogle Scholar
- Altman J, Bayer SA: An overview of the postnatal development of the rat cerebellum. Development of the cerebellar system: in relation to its evolution, structure, and functions. 1997, Boca Raton, FL: CRC, 324-333.Google Scholar
- Rakic P: Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus Rhesus. J Comp Neurol. 1971, 141: 283-312. 10.1002/cne.901410303.View ArticlePubMedGoogle Scholar
- Gleeson JG, Lin PT, Flanagan LA, Walsh CA: Doublecortin is a microtubule associated protein and is expressed widely by migrating neurons. Neuron. 1999, 23: 257-271. 10.1016/S0896-6273(00)80778-3.View ArticlePubMedGoogle Scholar
- Altman J: Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J Comp Neurol. 1972, 145: 465-514. 10.1002/cne.901450403.View ArticlePubMedGoogle Scholar
- Liesi P, Akinshola E, Matsuba K, Lange K, Morest K: Cellular migration in the postnatal rat cerebellar cortex: confocal-infrared microscopy and the rapid Golgi method. J Neurosci Res. 2003, 72: 290-302. 10.1002/jnr.10573.View ArticlePubMedGoogle Scholar
- Verney C, Grzanna R, Farkas E: Distribution of dopamine-beta-hydroxylase-like immunoreactive fibers in the rat cerebellar cortex during ontogeny. Dev Neurosci. 1982, 5: 369-374.View ArticlePubMedGoogle Scholar
- Takada M, Sugimoto T, Hattori T: Tyrosine hydroxylase immunoreactivity in cerebellar Purkinje cells of the rat. Neurosci Lett. 1993, 150 (1): 61-64. 10.1016/0304-3940(93)90108-W.View ArticlePubMedGoogle Scholar
- Beaudet A, Descarries L: The monoamine innervation of rat cerebral cortex: synaptic and non-synaptic axon terminals. Neuroscience. 1978, 3: 851-860. 10.1016/0306-4522(78)90115-X.View ArticlePubMedGoogle Scholar
- Li B, Chen N, Luo T, Otsu Y, Murphy TH, Raymond LA: Differential regulation of synaptic and extra-synaptic NMDA receptors. Nat Neurosci. 2002, 5: 833-834. 10.1038/nn912.View ArticlePubMedGoogle Scholar
- Fuxe K, Agnati LF: Two principal modes of electrochemical communication in the brain: volume versus wiring transmission. Volume transmission in the brain. Novel mechanisms for neuronal transmission. Edited by: Fuxe K, Agnati LF. 1991, New York: Raven Press, 1-9.Google Scholar
- Hering H, Lin CC, Sheng M: Lipid rafts in the maintenance of synapses, dendritic spines, and surface AMPA receptor stability. J Neurosci. 2003, 23: 3262-3271.PubMedGoogle Scholar
- Yao ST, Lawrence AJ: Purinergic modulation of cardiovascular function in the rat locus coeruleus. Br J Pharmacol. 2005, 145: 342-352. 10.1038/sj.bjp.0706179.PubMed CentralView ArticlePubMedGoogle Scholar
- Mortani Barbosa EJ, Ferreira ZS, Markus RP: Purinergic and noradrenergic cotransmission in the rat pineal gland. Eur J Pharmacol. 2000, 401: 59-62. 10.1016/S0014-2999(00)00416-7.View ArticlePubMedGoogle Scholar
- Vonend O, Stegbauer J, Sojka J, Habbel S, Quack I, Robaye B, Boeynaems JM, Rump LC: Noradrenaline and extracellular nucleotide cotransmission involves activation of vasoconstrictive P2X(1,3)- and P2Y6-like receptors in mouse perfused kidney. Br J Pharmacol. 2005, 145: 66-74. 10.1038/sj.bjp.0706151.PubMed CentralView ArticlePubMedGoogle Scholar
- Thomas SA, Matsumoto AM, Palmiter RD: Noradrenaline is essential for mouse fetal development. Nature. 1995, 374: 643-646. 10.1038/374643a0.View ArticlePubMedGoogle Scholar
- Cartford MC, Gould T, Bickford PC: A central role for norepinephrine in the modulation of cerebellar learning tasks. Behav Cogn Neurosci Rev. 2004, 3: 131-138. 10.1177/1534582304270783.View ArticlePubMedGoogle Scholar
- Yamada K, Watanabe M: Cytodifferentiation of Bergmann glia and its relationship with Purkinje cells. Anat Sci Int. 2002, 77: 94-108. 10.1046/j.0022-7722.2002.00021.x.View ArticlePubMedGoogle Scholar
- Oomman S, Strahlendorf H, Finckbone V, Strahlendorf J: Non-lethal active caspase-3 expression in Bergmann glia of postnatal rat cerebellum. Brain Res Dev Brain Res. 2005, 160: 130-145. 10.1016/j.devbrainres.2005.07.010.View ArticlePubMedGoogle Scholar
- Tohgo A, Eiraku M, Miyazaki T, Miura E, Kawaguchi SY, Nishi M, Watanabe M, Hirano T, Kengaku M, Takeshima H: Impaired cerebellar functions in mutant mice lacking DNER. Mol Cell Neurosci. 2006, 31: 326-333. 10.1016/j.mcn.2005.10.003.View ArticlePubMedGoogle Scholar
- Solecki DJ, Model L, Gaetz J, Kapoor TM, Hatten ME: Par6alpha signaling controls glial-guided neuronal migration. Nat Neurosci. 2004, 7: 1195-1203. 10.1038/nn1332.View ArticlePubMedGoogle Scholar
- Weller M, Krautler N, Mantei N, Suter U, Taylor V: Jagged1 ablation results in cerebellar granule cell migration defects and depletion of Bergmann glia. Dev Neurosci. 2006, 28: 70-80. 10.1159/000090754.View ArticlePubMedGoogle Scholar
- Jones FE, Golding JP, Gassmann M: ErbB4 signaling during breast and neural development: novel genetic models reveal unique ErbB4 activities. Cell Cycle. 2003, 2: 555-559.PubMedGoogle Scholar
- Rio C, Rieff HI, Qi P, Khurana TS, Corfas G: Neuregulin and erbB receptors play a critical role in neuronal migration. Neuron. 1997, 19: 39-50. 10.1016/S0896-6273(00)80346-3.View ArticlePubMedGoogle Scholar
- Hatten ME: Central nervous system neuronal migration. Annu Rev Neurosci. 1999, 22: 511-539. 10.1146/annurev.neuro.22.1.511.View ArticlePubMedGoogle Scholar
- Edmondson JC, Liem RK, Kuster JE, Hatten ME: Astrotactin: a novel neuronal cell surface antigen that mediates neuron-astroglial interactions in cerebellar microcultures. J Cell Biol. 1988, 106: 505-517. 10.1083/jcb.106.2.505.View ArticlePubMedGoogle Scholar
- Kirischuk S, Moller T, Voitenko N, Kettenmann H, Verkhratsky A: ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. J Neurosci. 1995, 15: 7861-7871.PubMedGoogle Scholar
- Merlo D, Volonté C: Binding and functions of extracellular ATP in cultured cerebellar granule neurons. Biochem Bioph Res Co. 1996, 225: 907-914. 10.1006/bbrc.1996.1271.View ArticleGoogle Scholar
- Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP: Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem. 1998, 273: 2024-2029. 10.1074/jbc.273.4.2024.View ArticlePubMedGoogle Scholar
- Jantzen HM, Gousset L, Bhaskar V, Vincent D, Tai A, Reynolds EE, Conley PB: Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb Haemost. 1999, 81: 111-117.PubMedGoogle Scholar
- Gachet C: ADP receptors of platelets and their inhibition. Thromb Haemost. 2001, 86: 222-232.PubMedGoogle Scholar
- Peters A, Palay SL, Webster HD: The fine structure of the nervous system. 1991, Oxford University PressGoogle Scholar
- Iwabuchi K, Yamamura S, Prinetti A, Handa K, Hakomori S: GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. J Biol Chem. 1998, 273: 9130-9138. 10.1074/jbc.273.15.9130.View ArticlePubMedGoogle Scholar
- Vacca F, Amadio S, Sancesario G, Bernardi G, Volonté C: P2X3 receptor localizes into lipid rafts in neuronal cells. J Neurosci Res. 2004, 76: 653-661. 10.1002/jnr.20069.View ArticlePubMedGoogle Scholar
- Huttner WB, Schiebler W, Greengard P, De Camilli P: Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol. 1983, 96: 1374-1388. 10.1083/jcb.96.5.1374.View ArticlePubMedGoogle Scholar
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