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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 causes a reduction of CF inputs on the PC proximal dendrite in δ2/GFP-ho mice.(A–D) Immunostaining of CF varicosities (blue) on the PC proximal domain of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). (A–B) In δ2/GFP-ho mice, numerous spines bearing GluRδ2 (red, A) appear in the proximal domain. The number of CF varicosities labeled with the VGluT2 antibody (blue, A–B) is reduced relative to GFP-ho mice (C–D). The arrowheads indicate the CF varicosities in the δ2/GFP-ho that are smaller relative to the control. (E) Histogram shows the mean percentage of spines overlapping with VGluT2. A significant reduction is observed in the δ2/GFP-ho mice relative to the GFP-ho and δ2/GFP-ho CTR groups and also to GFP-wt mice. These results show that in presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of CF contacts is strongly reduced. *** p<0.001. Error bars indicate SEM. Scale bars in A–D = 2 µm.
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pone-0005243-g005: GluRδ2 causes a reduction of CF inputs on the PC proximal dendrite in δ2/GFP-ho mice.(A–D) Immunostaining of CF varicosities (blue) on the PC proximal domain of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). (A–B) In δ2/GFP-ho mice, numerous spines bearing GluRδ2 (red, A) appear in the proximal domain. The number of CF varicosities labeled with the VGluT2 antibody (blue, A–B) is reduced relative to GFP-ho mice (C–D). The arrowheads indicate the CF varicosities in the δ2/GFP-ho that are smaller relative to the control. (E) Histogram shows the mean percentage of spines overlapping with VGluT2. A significant reduction is observed in the δ2/GFP-ho mice relative to the GFP-ho and δ2/GFP-ho CTR groups and also to GFP-wt mice. These results show that in presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of CF contacts is strongly reduced. *** p<0.001. Error bars indicate SEM. Scale bars in A–D = 2 µm.

Mentions: We next investigated whether the expression of GluRδ2 also affected the CF input that abuts clusters of spines in the proximal dendritic domain under normal conditions. To verify the distribution of the CF input, we immunostained VGluT2 and observed a marked decrease in innervation following induction of GluRδ2 expression (Fig. 5A–D). By colocalization analysis, we measured the number of VGluT2-positive spines along the dendrite that was connected to the CF terminals. In the GFP-wt control group, the mean percentage of CF-contacted spines was 37.3 (±0.3 SE, n = 1110), which was designated as the control (see methods). In GFP-ho mice, the percentage was 36.0 (±0.4 SE, n = 742) (one-way ANOVA, p<0.001; post hoc Holm-Sidack test, p>0.05) and decreased to 13.8 in the δ2/GFP-ho group (±0.3 SE, n = 1040; one-way ANOVA, p<0.001; post hoc Holm-Sidack test, p<0.05 versus all 3 groups). As shown in Fig. 5E, in PCs that did not express GluRδ2, we observed a significant difference relative to GluRδ2-positive PCs and no difference compared with the control groups (37.8±0.4 SE, n = 697) (one-way ANOVA, p<0.001; post hoc Holm-Sidack test, p>0.05 versus control and p<0.05 versus δ2/GFP-ho). These results suggest that the decrease in CF synapses in δ2/GFP-ho mice is due to a retraction of the CF input, accompanied by atrophy of the CF varicosities.


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 causes a reduction of CF inputs on the PC proximal dendrite in δ2/GFP-ho mice.(A–D) Immunostaining of CF varicosities (blue) on the PC proximal domain of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). (A–B) In δ2/GFP-ho mice, numerous spines bearing GluRδ2 (red, A) appear in the proximal domain. The number of CF varicosities labeled with the VGluT2 antibody (blue, A–B) is reduced relative to GFP-ho mice (C–D). The arrowheads indicate the CF varicosities in the δ2/GFP-ho that are smaller relative to the control. (E) Histogram shows the mean percentage of spines overlapping with VGluT2. A significant reduction is observed in the δ2/GFP-ho mice relative to the GFP-ho and δ2/GFP-ho CTR groups and also to GFP-wt mice. These results show that in presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of CF contacts is strongly reduced. *** p<0.001. Error bars indicate SEM. Scale bars in A–D = 2 µm.
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pone-0005243-g005: GluRδ2 causes a reduction of CF inputs on the PC proximal dendrite in δ2/GFP-ho mice.(A–D) Immunostaining of CF varicosities (blue) on the PC proximal domain of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). (A–B) In δ2/GFP-ho mice, numerous spines bearing GluRδ2 (red, A) appear in the proximal domain. The number of CF varicosities labeled with the VGluT2 antibody (blue, A–B) is reduced relative to GFP-ho mice (C–D). The arrowheads indicate the CF varicosities in the δ2/GFP-ho that are smaller relative to the control. (E) Histogram shows the mean percentage of spines overlapping with VGluT2. A significant reduction is observed in the δ2/GFP-ho mice relative to the GFP-ho and δ2/GFP-ho CTR groups and also to GFP-wt mice. These results show that in presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of CF contacts is strongly reduced. *** p<0.001. Error bars indicate SEM. Scale bars in A–D = 2 µm.
Mentions: We next investigated whether the expression of GluRδ2 also affected the CF input that abuts clusters of spines in the proximal dendritic domain under normal conditions. To verify the distribution of the CF input, we immunostained VGluT2 and observed a marked decrease in innervation following induction of GluRδ2 expression (Fig. 5A–D). By colocalization analysis, we measured the number of VGluT2-positive spines along the dendrite that was connected to the CF terminals. In the GFP-wt control group, the mean percentage of CF-contacted spines was 37.3 (±0.3 SE, n = 1110), which was designated as the control (see methods). In GFP-ho mice, the percentage was 36.0 (±0.4 SE, n = 742) (one-way ANOVA, p<0.001; post hoc Holm-Sidack test, p>0.05) and decreased to 13.8 in the δ2/GFP-ho group (±0.3 SE, n = 1040; one-way ANOVA, p<0.001; post hoc Holm-Sidack test, p<0.05 versus all 3 groups). As shown in Fig. 5E, in PCs that did not express GluRδ2, we observed a significant difference relative to GluRδ2-positive PCs and no difference compared with the control groups (37.8±0.4 SE, n = 697) (one-way ANOVA, p<0.001; post hoc Holm-Sidack test, p>0.05 versus control and p<0.05 versus δ2/GFP-ho). These results suggest that the decrease in CF synapses in δ2/GFP-ho mice is due to a retraction of the CF input, accompanied by atrophy of the CF varicosities.

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