Figure 2.
γ-Pcdh proteins localize to perisynaptic astrocyte processes.
A
, Schematic diagram of the Pcdh-γ
fcon3
conditional mutant allele, in which a GFP-fused C exon 3 is flanked by loxP sites. The A–C subfamilies of V exons are in shades of blue, and the 3 C exons are in red.
B
, In Cre-ER; Pcdh-γfcon3/+ mice injected with tamoxifen (
B
, right), Cre excision resulted in loss of the ubiquitous γ-Pcdh-GFP immunofluorescence observed in uninjected or Cre-ER-negative mice (
B
, left). GFP-tagged γ-Pcdhs remained detectable, however, in a small number of GFAP-positive gray matter protoplasmic astrocytes (
B
).
C
, A higher-magnification image of a separate double-immunostained cryosection is shown.
D
,
E
, Confocal analysis of immunostained Cre-ER; Pcdh-γfcon3/+ spinal cord sections demonstrated that astrocytic γ-Pcdh puncta (stained for both GFP and γ-Pcdh; closed arrowheads; yellow in merged images) can localize directly adjacent to neuronal γ-Pcdh at synaptic puncta (stained for both bassoon and γ-Pcdh; open arrowheads, magenta in merged images).
F
,
G
, Immunostaining for GFP and presynaptic and postsynaptic markers confirmed that astrocytic γ-Pcdhs were frequently apposed to and/or wrapped around synaptic terminals. Large images are of single planes from z-stacks; perpendicular cross-sections through the entire z-stacks at locations indicated by white lines are shown at right and below.
H
, Tracings of several such perisynaptic contacts for the indicated boxes in
F
and
G
are presented. Scale bars:
B
, 100 μm;
C
, 6 μm;
D
,
E
, 1 μm;
F
,
G
, 2 μm.