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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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GluRδ2 promotes formation of PF contacts in the PC distal domain of δ2/GFP-ho mice.(A–D) Immunostaining of PF innervations on PC distal dendrites of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). GFP spines bearing GluRδ2 (red, A) are contacted by PF terminals labeled by VGluT1 antibody (blue) (B). (E–F) Histograms show the mean density of spines emerging from the distal dendritic domain and the percentage of spines contacted by the PFs in this compartment. (E) The mean spine density does not change between the experimental groups (p = 0.096). (F) The mean percentage of spines overlapping with VGluT1 is increased in δ2/GFP-ho mice relative to control ho groups (GFP-ho and δ2/GFP-ho CTR), while there is no significant difference between δ2/GFP-ho mice and the GFP-wt group. In the presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of PF contacts reaches that of wild-type mice. *** p<0.001. Error bars indicate SE. Scale bars:  = 2 µm.
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pone-0005243-g003: GluRδ2 promotes formation of PF contacts in the PC distal domain of δ2/GFP-ho mice.(A–D) Immunostaining of PF innervations on PC distal dendrites of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). GFP spines bearing GluRδ2 (red, A) are contacted by PF terminals labeled by VGluT1 antibody (blue) (B). (E–F) Histograms show the mean density of spines emerging from the distal dendritic domain and the percentage of spines contacted by the PFs in this compartment. (E) The mean spine density does not change between the experimental groups (p = 0.096). (F) The mean percentage of spines overlapping with VGluT1 is increased in δ2/GFP-ho mice relative to control ho groups (GFP-ho and δ2/GFP-ho CTR), while there is no significant difference between δ2/GFP-ho mice and the GFP-wt group. In the presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of PF contacts reaches that of wild-type mice. *** p<0.001. Error bars indicate SE. Scale bars:  = 2 µm.

Mentions: To detect morphological changes in the cerebellar cortex, we performed immunofluorescence experiments and confocal microscopy four weeks after injection. We first observed that in δ2/GFP-ho mice, GluRδ2 was expressed in several neurons and restricted to the dendritic spines of the PC distal dendritic domain (Fig. 3A). Then, we investigated whether GluRδ2 induced these distal spines to establish contacts with the VGluT1-labeled PF terminals (Fig. 3A–D). Therefore, in each experimental group, we counted the GFP-positive spines (δ2/GFP-ho n = 1135, GFP-ho n = 1295, GFP-wt n = 925) in samples of distal dendritic segments whose diameters were less than 2 µm. In GFP-wt and GFP-ho mice, the mean spine density, expressed as the number of spines per square micrometer (µm2) of dendritic surface, was 4.95 (±0.062 SE) and 4.11 (±0.18 SE), respectively. In δ2/GFP-ho mice the value was 3.88 (±0.13 SE, δ2/GFP-ho) for the PCs expressing the L7-driven GluRδ2 and 4.57 (±0.25 SE, δ2/GFP-ho CTR) for the PCs only GFP-positive. The difference between the groups was not significant (one-way ANOVA, p = 0.096) (Fig. 3E). These results indicate that in the distal dendritic domain, the expression of GluRδ2 does not effect any increase in spine density.


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)

GluRδ2 promotes formation of PF contacts in the PC distal domain of δ2/GFP-ho mice.(A–D) Immunostaining of PF innervations on PC distal dendrites of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). GFP spines bearing GluRδ2 (red, A) are contacted by PF terminals labeled by VGluT1 antibody (blue) (B). (E–F) Histograms show the mean density of spines emerging from the distal dendritic domain and the percentage of spines contacted by the PFs in this compartment. (E) The mean spine density does not change between the experimental groups (p = 0.096). (F) The mean percentage of spines overlapping with VGluT1 is increased in δ2/GFP-ho mice relative to control ho groups (GFP-ho and δ2/GFP-ho CTR), while there is no significant difference between δ2/GFP-ho mice and the GFP-wt group. In the presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of PF contacts reaches that of wild-type mice. *** p<0.001. Error bars indicate SE. Scale bars:  = 2 µm.
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pone-0005243-g003: GluRδ2 promotes formation of PF contacts in the PC distal domain of δ2/GFP-ho mice.(A–D) Immunostaining of PF innervations on PC distal dendrites of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). GFP spines bearing GluRδ2 (red, A) are contacted by PF terminals labeled by VGluT1 antibody (blue) (B). (E–F) Histograms show the mean density of spines emerging from the distal dendritic domain and the percentage of spines contacted by the PFs in this compartment. (E) The mean spine density does not change between the experimental groups (p = 0.096). (F) The mean percentage of spines overlapping with VGluT1 is increased in δ2/GFP-ho mice relative to control ho groups (GFP-ho and δ2/GFP-ho CTR), while there is no significant difference between δ2/GFP-ho mice and the GFP-wt group. In the presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of PF contacts reaches that of wild-type mice. *** p<0.001. Error bars indicate SE. Scale bars:  = 2 µm.
Mentions: To detect morphological changes in the cerebellar cortex, we performed immunofluorescence experiments and confocal microscopy four weeks after injection. We first observed that in δ2/GFP-ho mice, GluRδ2 was expressed in several neurons and restricted to the dendritic spines of the PC distal dendritic domain (Fig. 3A). Then, we investigated whether GluRδ2 induced these distal spines to establish contacts with the VGluT1-labeled PF terminals (Fig. 3A–D). Therefore, in each experimental group, we counted the GFP-positive spines (δ2/GFP-ho n = 1135, GFP-ho n = 1295, GFP-wt n = 925) in samples of distal dendritic segments whose diameters were less than 2 µm. In GFP-wt and GFP-ho mice, the mean spine density, expressed as the number of spines per square micrometer (µm2) of dendritic surface, was 4.95 (±0.062 SE) and 4.11 (±0.18 SE), respectively. In δ2/GFP-ho mice the value was 3.88 (±0.13 SE, δ2/GFP-ho) for the PCs expressing the L7-driven GluRδ2 and 4.57 (±0.25 SE, δ2/GFP-ho CTR) for the PCs only GFP-positive. The difference between the groups was not significant (one-way ANOVA, p = 0.096) (Fig. 3E). These results indicate that in the distal dendritic domain, the expression of GluRδ2 does not effect any increase in spine density.

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