GluRδ2 Expression in the Mature Cerebellum of Hotfoot Mice Promotes Parallel Fiber Synaptogenesis and Axonal Competition

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-null 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

rSK1 in rat neurons: a controller of membrane rSK2?

In mammalian neurons, small conductance calcium-activated potassium channels (SK channels) are activated by calcium influx and contribute to the afterhyperpolarization (AHP) that follows action potentials. Three types of SK channel, SK1, SK2 and SK3 are recognized and encoded by separate genes that are widely expressed in overlapping distributions in the mammalian brain. Expression of the rat genes, rSK2 and rSK3 generates functional ion channels that traffic to the membrane as homomeric and heteromeric complexes. However, rSK1 is not trafficked to the plasma membrane, appears not to form functional channels, and the role of rSK1 in neurons is not clear. Here, we show that rSK1 co-assembles with rSK2. rSK1 is not trafficked to the membrane but is retained in a cytoplasmic compartment. When rSK2 is present, heteromeric rSK1-rSK2 channels are also retained in the cytosolic compartment, reducing the total SK channel content on the plasma membrane. Thus, rSK1 appears to act as chaperone for rSK2 channels and expression of rSK1 may control the level of functional SK current in rat neurons

GluRδ2 expressed by HEK293 promotes formation and differentiation of GC axonal contacts.

<p>(A–F) Merge of light microphotographs of GCs in coculture with fluorescent 293 cells expressing GFP and GluRδ2 (in red) (A) or GFP alone (E). The corresponding immunofluorescence images are magnified as a single optical section in B–F. The GluRδ2 labeling around the 293 cell perimeter is shown in (C). GluRδ2 expression induces an increase in synaptic contacts, as indicated by the corresponding VGluT1 labeling (B–D). No contacts are visible around the perimeter of the 293-GFP cells; the blue labeling indicates synaptic contacts on a GC cluster (F). (G) EM quantitative analysis of the GC axonal contacts on the 293 cell perimeter. The 293-GluRδ2 cells (black columns) are in contact with a higher number of GC round terminals relative to the control (white columns); in both groups, most of the round terminals contained vesicles. In the 293-GluRδ2 cells, more terminals with vesicles oriented toward the postsynaptic membrane were observed. (H–I) EM images of differentiation of the presynaptic GC terminals induced by 293-GluRδ2 cells. (H) Contact between 293-GFP cell and a round GC terminal containing homogeneously distributed vesicles. (I) A 293-GluRδ2 cell contacted by round GC terminal containing oriented vesicles; the arrow indicates the vesicle cluster. Scale bars: A and E = 20 µm. B-C-D-F = 10 µm. H and I = 0.25 µm. *** p<0.001; ** p<0.01.</p

GluRδ2 induces spinogenesis in the PC proximal dendritic compartment of δ2/GFP-ho mice.

<p>(A–D) Immunostaining of PC proximal dendrites in δ2/GFP-ho (A–C) and GFP-ho mice (D). In δ2/GFP-ho mice, many new spines, expressing the GluRδ2 subunit (red) (B and C), appears in the proximal dendrite relative to GFP-ho mice (D). (E) Histogram shows the mean spine density in the proximal dendritic domain. In the presence of GluRδ2, the number of spines significantly increases relative to control groups (GFP-wt; GFP-ho and δ2/GFP-ho CTR). *** p<0.001. Error bars indicate SE. Scale bars: A–E = 2 µm.</p

GluRδ2 promotes formation of PF contacts in the PC distal domain of δ2/GFP-ho mice.

<p>(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.</p

Difference in the distribution of PF contacts along the ascending domain of Golgi cell expressing GluRδ2.

<p>(A–D) Immunostaining of the ascending dendritic tract of a Golgi cell (green, A) characterized by differential localization of GluRδ2 (arrowheads) (red, B) and relative VGluT1 (blue, C) signals (D, merge). GluRδ2 expression gradually increases in the proximal domain (gl) at the level of the PC layer (pl), reaching high levels in the distal tract (ml). Although the expression of GluRδ2 is less prominent in the proximal domain, the area that is in contact with the PF inputs is significantly increased relative to both the control groups and the negative control. (E) Histogram shows the mean percentage of the GFP area that colocalizes with GluRδ2 in Golgi cell dendrites of GFP/δ2 mice. A significant reduction of GluRδ2 expression in the proximal tract is observed. (F) Histogram shows the mean percentage of the GFP area that colocalizes with VGluT1 in Golgi cells of GFP-wt, GFP-ho, and δ2/GFP-ho mice. The white columns represent the value obtained in the distal dendritic domain; the light gray columns are the value in the proximal dendritic tract; and the dark gray columns are the negative control value of colocalized GFP-VGluT1 in the rosette. The ectopic expression of GluRδ2 induces a significant increase in PF contacts in both layers. * p<0.05; ***p<0.001. Error bars indicate SE. Scale bar A–D: 10 µm.</p

GluRδ2 promotes an increase in PF inputs on the PC proximal dendrite of δ2/GFP-ho mice.

<p>(A–F) Immunostaining of PF innervations (blue) on the PC proximal domain of δ2/GFP-ho mice (A–D) and GFP-ho mice (E–F). (A) In the δ2/GFP-ho group, numerous spines (arrowheads) bearing GluRδ2 (red, B) appear in the proximal domain, and the PF contacts, labeled with VGluT1 antibody (blue, C and D), are more numerous relative to GFP-ho mice (E–F). The overlap between GluRδ2 and the PF synaptic terminals appears as fuchsia (D). (G) Histogram shows the mean percentage of spines overlapping with VGluT1. A significant increase 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 PF input has a competitive advantage. ***p<0.001. Error bars indicate SE. Scale bar: A–F = 2 µm.</p

GluRδ2 increases PF contacts on Golgi cell dendrites of δ2/GFP-ho mice.

<p>(A–C and J–K) Immunostaining of transfected Golgi cells in δ2/GFP-ho mice (A–C) and in control GFP-ho mice (J–K). The cell bodies (A, J) are in the granular layer (gl), and the ascending dendrites also are visible in the molecular layer (ml). In δ2/GFP-ho mice, GluRδ2 is ectopically expressed in the Golgi dendrites (red, B). (D–I and L–N) High magnification of two Golgi cell dendrites in the molecular layer of δ2/GFP-ho and GFP-ho mice, respectively. (D–I) In a Golgi cell expressing GluRδ2 (in red, E–G–H), the dendritic area that is in contact with the PF inputs is higher (blue, F–H–I) (arrowheads) relative to that (M–N) of the Golgi cell (L) in GFP-ho mice. Scale bars: A–E = 20 µm. F–N = 2 µm.</p

Morphological analysis of CF varicosities in hotfoot and wild type mice

<p>Mean values of major axis length (MA), minor axis length (ma), and ratio (MA/ma); one- way ANOVA; * post-hoc Holm-Sidack test, p<0.05.</p

GluRδ2 causes a reduction of CF inputs on the PC proximal dendrite in δ2/GFP-ho mice.

<p>(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.</p