BLA inactivation during fear memory consolidation affects learning-induced LTP in cerebellum when performed 5 min, but not 6 h, after training.

<p><b>A</b>. Representative traces of PF-PC EPSC from conditioned animals and from those that received anisomycin 5 min or 6 h after acquisition. <b>B</b>. Input-output data from conditioned subjects (circle), and those injected with anisomycin 5 min (square) and 6 h (triangle) after training. <b>C</b>. Paired-pulse facilitation in the three groups. Data are expressed as mean ± SEM.</p

Effects of pretraining BLA inactivation on spontaneous and conditioned fear behavior.

<p><b>A.</b> Experimental design. The arrow indicates pretraining muscimol (M) injection. <b>B.</b> Photomicrograph (magnification 4X) showing the position of the needle track in animals that received muscimol into BLA. Scale bars, 300 µm. <b>C.</b> Spontaneous activities showed by control (filled columns) and muscimol-injected (empty columns) animals before shock presentation. <b>D.</b> Long-term memory retention evaluated 24 h after conditioning by measuring freezing 2 min before (gray columns) and during CS presentation (filled columns). All values are mean ± SEM.</p

Pretraining BLA inactivation prevents learning-induced LTP in cerebellum.

<p><b>A.</b> Electrophysiological recordings were performed on lobules V and VI (gray area) of cerebellar vermis. <b>B</b>. PF-PC EPSCs were recorded (R) at the PC soma by stimulating (S) PF in the molecular layer. <b>C.</b> Input-output data from naïve (square), conditioned (circle) and muscimol-injected (triangle) animals. <b>D.</b> Representative traces of EPSCs obtained by paired PF stimuli with 100 ms interval in naïve, conditioned and muscimol-injected subjects. <b>E.</b> Paired-pulse facilitation is similar in the three groups. All values are mean ± SEM.</p

BLA reversible blockade and fear memory consolidation.

<p><b>A.</b> BLA role in fear memory consolidation was studied by injecting into this site a) muscimol (M) 5 min after training; b) muscimol (M) 5, 90 and 180 min; c) anisomycin (A) 5 min; d) anisomycin (A) 6 h after training. <b>B.</b> Histological control of the location of anisomycin injection into BLA (magnification 10X). Scale bars, 200 µm. <b>C.</b> Memory retention tested in conditioned (C) subjects and in those that received one (M) or three (3M) injection of muscimol, anisomycin (A) 5 min or 6 h (A 6 h) after conditioning.</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 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 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

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 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