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GluRdelta2 expression in the mature cerebellum of hotfoot mice promotes parallel fiber synaptogenesis and axonal competition.

Mandolesi G, Autuori E, Cesa R, Premoselli F, Cesare P, Strata P - PLoS ONE (2009)

Bottom Line: In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input.Moreover, ectopic expression of GluRdelta2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts.Collectively, our observations show that GluRdelta2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain.

View Article: PubMed Central - PubMed

Affiliation: EBRI-Santa Lucia Foundation (IRCCS), Rome, Italy. g.mandolesi@hsantalucia.it

ABSTRACT
Glutamate receptor delta 2 (GluRdelta2) is selectively expressed in the cerebellum, exclusively in the spines of the Purkinje cells (PCs) that are in contact with parallel fibers (PFs). Although its structure is similar to ionotropic glutamate receptors, it has no channel function and its ligand is unknown. The GluRdelta2- mice, such as knockout and hotfoot have profoundly altered cerebellar circuitry, which causes ataxia and impaired motor learning. Notably, GluRdelta2 in PC-PF synapses regulates their maturation and strengthening and induces long term depression (LTD). In addition, GluRdelta2 participates in the highly territorial competition between the two excitatory inputs to the PC; the climbing fiber (CF), which innervates the proximal dendritic compartment, and the PF, which is connected to spiny distal branchlets. Recently, studies have suggested that GluRdelta2 acts as an adhesion molecule in PF synaptogenesis. Here, we provide in vivo and in vitro evidence that supports this hypothesis. Through lentiviral rescue in hotfoot mice, we noted a recovery of PC-PF contacts in the distal dendritic domain. In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input. Moreover, ectopic expression of GluRdelta2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts. Collectively, our observations show that GluRdelta2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain.

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In vivo injection of L7-GFP and L7-GluRδ2 viruses in the mature cerebellum of ho-4J mice (δ2/GFP-ho mice).(A–D). Immunostaining of PCs labeled with anti-calbindin (green) and anti-GluRδ2 (red) antibodies in wild-type mice (A–B) and ho-4j mice (C–D). In the ho-4j mice the GluRδ2 truncated protein is retained in the PC soma. (E–F–G) Immunostaining of a cerebellar sagittal section from δ2/GFP-ho mice 4 weeks after in vivo injection. The infected PCs express GFP (green, E) and GluRδ2 (red, F). VGluT1 antibody (blue, G), used as an endogenous marker, labels the mossy fibers and the PF terminals in the granular (gl) and molecular layers (ml), respectively. (H–K) High magnification images of PCs expressing GFP (green) and GluRδ2 (red) in the distal dendrites of δ2/GFP-ho mice. Two populations of GFP-positive PCs are shown: PCs expressing GluRδ2 (red) (H–I) and the PCs with undetectable levels of GluRδ2 (δ2/GFP-ho CTR group) (J–K). The arrowheads indicate ectopic GluRδ2 in a different cell. Scale bars: A–D = 20 µm, E–G = 200 µm, H–N = 2 µm.
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pone-0005243-g002: In vivo injection of L7-GFP and L7-GluRδ2 viruses in the mature cerebellum of ho-4J mice (δ2/GFP-ho mice).(A–D). Immunostaining of PCs labeled with anti-calbindin (green) and anti-GluRδ2 (red) antibodies in wild-type mice (A–B) and ho-4j mice (C–D). In the ho-4j mice the GluRδ2 truncated protein is retained in the PC soma. (E–F–G) Immunostaining of a cerebellar sagittal section from δ2/GFP-ho mice 4 weeks after in vivo injection. The infected PCs express GFP (green, E) and GluRδ2 (red, F). VGluT1 antibody (blue, G), used as an endogenous marker, labels the mossy fibers and the PF terminals in the granular (gl) and molecular layers (ml), respectively. (H–K) High magnification images of PCs expressing GFP (green) and GluRδ2 (red) in the distal dendrites of δ2/GFP-ho mice. Two populations of GFP-positive PCs are shown: PCs expressing GluRδ2 (red) (H–I) and the PCs with undetectable levels of GluRδ2 (δ2/GFP-ho CTR group) (J–K). The arrowheads indicate ectopic GluRδ2 in a different cell. Scale bars: A–D = 20 µm, E–G = 200 µm, H–N = 2 µm.

Mentions: We extended our study in vivo to determine whether the free spines that are abundant in mature GluRδ2- PCs become innervated on expression of GluRδ2. We used the DBA ho-4j strain of the hotfoot mouse, which carries a 170-amino acid deletion of the N-terminal region of GluRδ2 [9], [23]. Because this region is essential for GluRδ2 localization to the spine membrane, its truncated form is retained inside the PC soma [24] (Fig. 2A–D).


GluRdelta2 expression in the mature cerebellum of hotfoot mice promotes parallel fiber synaptogenesis and axonal competition.

Mandolesi G, Autuori E, Cesa R, Premoselli F, Cesare P, Strata P - PLoS ONE (2009)

In vivo injection of L7-GFP and L7-GluRδ2 viruses in the mature cerebellum of ho-4J mice (δ2/GFP-ho mice).(A–D). Immunostaining of PCs labeled with anti-calbindin (green) and anti-GluRδ2 (red) antibodies in wild-type mice (A–B) and ho-4j mice (C–D). In the ho-4j mice the GluRδ2 truncated protein is retained in the PC soma. (E–F–G) Immunostaining of a cerebellar sagittal section from δ2/GFP-ho mice 4 weeks after in vivo injection. The infected PCs express GFP (green, E) and GluRδ2 (red, F). VGluT1 antibody (blue, G), used as an endogenous marker, labels the mossy fibers and the PF terminals in the granular (gl) and molecular layers (ml), respectively. (H–K) High magnification images of PCs expressing GFP (green) and GluRδ2 (red) in the distal dendrites of δ2/GFP-ho mice. Two populations of GFP-positive PCs are shown: PCs expressing GluRδ2 (red) (H–I) and the PCs with undetectable levels of GluRδ2 (δ2/GFP-ho CTR group) (J–K). The arrowheads indicate ectopic GluRδ2 in a different cell. Scale bars: A–D = 20 µm, E–G = 200 µm, H–N = 2 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666267&req=5

pone-0005243-g002: In vivo injection of L7-GFP and L7-GluRδ2 viruses in the mature cerebellum of ho-4J mice (δ2/GFP-ho mice).(A–D). Immunostaining of PCs labeled with anti-calbindin (green) and anti-GluRδ2 (red) antibodies in wild-type mice (A–B) and ho-4j mice (C–D). In the ho-4j mice the GluRδ2 truncated protein is retained in the PC soma. (E–F–G) Immunostaining of a cerebellar sagittal section from δ2/GFP-ho mice 4 weeks after in vivo injection. The infected PCs express GFP (green, E) and GluRδ2 (red, F). VGluT1 antibody (blue, G), used as an endogenous marker, labels the mossy fibers and the PF terminals in the granular (gl) and molecular layers (ml), respectively. (H–K) High magnification images of PCs expressing GFP (green) and GluRδ2 (red) in the distal dendrites of δ2/GFP-ho mice. Two populations of GFP-positive PCs are shown: PCs expressing GluRδ2 (red) (H–I) and the PCs with undetectable levels of GluRδ2 (δ2/GFP-ho CTR group) (J–K). The arrowheads indicate ectopic GluRδ2 in a different cell. Scale bars: A–D = 20 µm, E–G = 200 µm, H–N = 2 µm.
Mentions: We extended our study in vivo to determine whether the free spines that are abundant in mature GluRδ2- PCs become innervated on expression of GluRδ2. We used the DBA ho-4j strain of the hotfoot mouse, which carries a 170-amino acid deletion of the N-terminal region of GluRδ2 [9], [23]. Because this region is essential for GluRδ2 localization to the spine membrane, its truncated form is retained inside the PC soma [24] (Fig. 2A–D).

Bottom Line: In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input.Moreover, ectopic expression of GluRdelta2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts.Collectively, our observations show that GluRdelta2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain.

View Article: PubMed Central - PubMed

Affiliation: EBRI-Santa Lucia Foundation (IRCCS), Rome, Italy. g.mandolesi@hsantalucia.it

ABSTRACT
Glutamate receptor delta 2 (GluRdelta2) is selectively expressed in the cerebellum, exclusively in the spines of the Purkinje cells (PCs) that are in contact with parallel fibers (PFs). Although its structure is similar to ionotropic glutamate receptors, it has no channel function and its ligand is unknown. The GluRdelta2- mice, such as knockout and hotfoot have profoundly altered cerebellar circuitry, which causes ataxia and impaired motor learning. Notably, GluRdelta2 in PC-PF synapses regulates their maturation and strengthening and induces long term depression (LTD). In addition, GluRdelta2 participates in the highly territorial competition between the two excitatory inputs to the PC; the climbing fiber (CF), which innervates the proximal dendritic compartment, and the PF, which is connected to spiny distal branchlets. Recently, studies have suggested that GluRdelta2 acts as an adhesion molecule in PF synaptogenesis. Here, we provide in vivo and in vitro evidence that supports this hypothesis. Through lentiviral rescue in hotfoot mice, we noted a recovery of PC-PF contacts in the distal dendritic domain. In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input. Moreover, ectopic expression of GluRdelta2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts. Collectively, our observations show that GluRdelta2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain.

Show MeSH
Related in: MedlinePlus