Spatial and temporal distribution patterns of Na-K-2Cl cotransporter in adult and developing mouse retinas

Abstract

The Na-K-2Cl cotransporter (NKCC) is a Cl(-) uptake transporter that is responsible for maintaining a Cl(-) equilibrium potential positive to the resting potential in neurons. If NKCC is active, GABA and glycine can depolarize neurons. In view of the abundance of GABAergic and glycinergic synapses in retina, we undertook a series of studies using immunocytochemical techniques to determine the distribution of NKCC in retinas of both developing and adult mice. We found NKCC antibody (T4) labeling present in retinas from wild-type mice, but not in NKCC1-deficient mice, suggesting that the NKCC1 subtype is a major Cl(-) uptake transporter in mouse retina. Strong labeling of NKCC1 was present in horizontal cells and rod-bipolar dendrites in adult mice. Interestingly, we also found that a diffuse labeling pattern was present in photoreceptor terminals. However, NKCC1 was barely detectable in the inner retina of adult mice. Using an antibody against K-Cl cotransporter 2 (KCC2), we found that KCC2, a transporter that extrudes Cl(-), was primarily expressed in the inner retina. The expression of NKCC1 in developing mouse retinas was studied from postnatal day (P) 1 to P21, NKCC1 labeling first appeared in the dendrites of horizontal and rod-bipolar cells as early as P7, followed by photoreceptor terminals between P10-P14; with expression gradually increasing concomitantly with the growth of synaptic terminals and dendrites throughout retinal development. In the inner retina, NKCC1 labeling was initially observed in the inner plexiform layer at P1, but labeling diminished after P5. The developmental increase in NKCC expression only occurred in the outer retina. Our results suggest that the distal synapses and synaptogenesis in mouse retinas undergo a unique process with a high intracellular Cl(-) presence due to NKCC1 expression.

Fig. 1

Immunoblotting and immunocytolabeling for NKCC1 in wide-type and NKCC1-deficient mouse retinas. (A) Western Blot assays to detect NKCC1 proteins in homogenates of (1) mouse brain tissues, (2) wild-type mouse retinas, and (3) NKCC1-deficient mouse retinas. The monoclonal antibody T4 recognizes a single protein band at MW 155 ±8 kDa (the predicted MW for NKCC1) in wide-type mouse brain and retinas, but not in a NKCC1-deficient mouse retina homogenate. (B) A confocal single optical section shows that the T4 antibody labels NKCC1 in the outer retina of wild-type mouse. (B′) Enlarged inset from (B) shows a detailed pattern of NKCC1 labeling in the outer retina. Two sub-layers are differentiated in the OPL: a faint, diffuse layer in the pre-synaptic area, and a strong, clear staining layer in the postsynaptic area. (C and C′) The labeling is completely eliminated in NKCC1-deficient mouse retina, shown in both a vertical section and an enlarged inset in the OPL. (BV: blood vessel.)

Fig. 2

Colocalization of NKCC1 with photoreceptor terminal markers in adult mouse retinas. (A1 and A2) Single-labeling patterns for NKCC1 (green) and PSD95 (red, a photoreceptor terminal marker) in the outer retina of a vertical section double-labeled for both antibodies. (A3) The merged image indicates that a faint, diffuse staining of NKCC1 is colocalized with PSD95 in photoreceptor terminals. The colocalizations can be clearly viewed in higher amplified insets of (A1), (A2), and (A3), focusing at photoreceptor terminals. (B1 and B2) Single-channel optical sections of KCC2 (red) and SV2 (green) in the outer retina. The superimposed image shows that KCC2 staining does not overlap with SV2 in photoreceptor terminals. (ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; BV: blood vessel.)

Fig. 3

Localizations of NKCC1 and KCC2 in adult mouse retinal HCs. HCs were labeled with calbindin. (A) Double-labeling for calbindin and NKCC1 shows NKCC1 binding colocalized with calbindin in the processes and somatic membranes of HCs. (B) KCC2 immunolabeling does not overlap with calbindin-positive HCs. (A1–A3 and B1–B3) Higher magnification views of the insets in (A) and (B) in single-channel optical sections and superimpositions.

Fig. 4

NKCC1 and KCC2 in the somatodendritic regions of rod-BCs in the adult mouse retina. Rod-BCs were labeled with PKC in retinal vertical sections. (A) Double-labeling for NKCC1 and PKC shows colocalization in rod-BC dendrites. (B1, B2) Enlarged single-optical sections for NKCC1 and PKC staining in the somas and dendrites of rod-BCs. (B3) The superimposition shows colocalization in the dendrites of rod-BCs. (C) A superimposed image of double-labeling for KCC2 and PKC in a retinal vertical section. The KCC2 and PKC binding is colocalized in some rod-BC dendrites, somas, axons (thick arrows) and axon terminals. Some rod-BC axons are PKC-labeled (arrow) and some are KCC2-labeled (arrowhead). (C1 and C2) The enlarged inset of (C), KCC2, and PKC in single-channel optical sections. (C3) The co-localization is identified in the superimposed image.

Fig. 5

Localization of NKCC1 and KCC2 on OFF-cone bipolar dendrites in the adult mouse retina. The OFF-cone dendrites were labeled with HCN4. (A) Double-labeling for NKCC1 and HCN4 in a vertical section. At a higher magnification, NKCC1 and HCN4 do not colocalize in Off-cone dendrites of the outer retina in a single optical section (A1–A3). In contrast, KCC2 immunolabeling is clearly colocalized with HCN4 in the dendrites of OFF-cone BCs [shown in (B), and (B1–B3)].

Fig. 6

Spatial and temporal expression of NKCC1 in mouse retinal development. NKCC1 was localized in developing mouse retina from postnatal day 1 to day 21 (P1 to P21) in single optical sections. (A, B, C and D) From P1 to P7, NKCC1 staining is present in the IPL, but gradually declines during maturation. At P7, NKCC1 labeling first appears in the outer retina. The intensity of the labeling increases dramatically by P10 (E) but NKCC1 labeling is absent in the IPL after P7. (F and G) The outer retinal synaptogenesis is completed when the mouse’s eyes open at P14. From P14 to P21, the intensity of labeling in the outer retinas remains the same, but the pattern becomes more evident (as seen in enlarged insets of (F) and (G)). Scale bar in (A) to (G) represents 25 μm. (NBL: neuroblast layer.)

Fig. 7

Double labeling for NKCC1 and PSD95 in developing mouse retina from P7 to P21. (A) Single optical image from a doubled retinal section shows weak labeling of NKCC1 in the outer retina at P7. (A′) Superimposed images of PSD95 and NKCC1 labeling show the two antibodies labeled the pre- and post-synaptic sites in the OPL. (B an B′) Single image and the superimposed image of NKCC1 and PSD94 in the P10 mouse retina shows weak colabeling in photoreceptor terminals at this age. (C and C′) A faint band of NKCC1 labeling is present in the upper layer of OPL, colocalized with PSD95 in photoreceptor terminals. (D and D′) Photoreceptor terminals are NKCC1-positive at P21 as depicted in a double-labeled retinal section. The insets show higher magnifications of the OPL at different ages.

Fig. 8

Spatial and temporal localization of NKCC1 in HC development. HCs were double-labeled with NKCC1 and calbindin. Since NKCC1 was undetectable from P1 to P5, colocalization of NKCC1 and calbindin was examined at P7 and later. (A) Only faint labeling (yellow spots show over lapping areas) appears on the distal processes of calbindin-positive HCs at P7. (B) The size and intensity of the colocalization increased in the HC processes and NKCC1 labeling appearing on the somas of HCs at P10 (see arrows). (C) NKCC1 labeling extends to the somatic membranes of HCs at P14. (D) By P21, a great amount of NKCC1 labeling overlaps with calbindin on the somas and processes of HCs. (E) Double-labeling for NKCC1 and calbindin in the retina from a P21 mouse.

Fig. 9

The distribution of NKCC1 in the dendrites of rod-BCs in development. (A) The scattering dual-labeling pattern is present on the dendrites of PKC-positive BCs at P7. (B, C, and D) Gradually, the labeling intensified and spread out on the dendrites from P10 to P21. However, no NKCC labeling is on the somas of the PKC-positive BCs.

Fig. 10

(A) Schematics of the outer retinal morphogenesis in forming “monad,” “dyad,” and “triad” complexes in mouse outer retinal development (redrawn from Blanks et al., 1974; Sherry et al., 2003). Cones begin “monad” formation at P4–P5, “dyad” formation at P6, and “triad” formation at P7–P8. Rods form “monads” at P8, “dyads” at P9, and “triads” at P10. (B) Scale showing the temporal labeling of NKCC1 during outer retinal development. (HCs: horizontal cells, BCs: bipolar cells; IPL: inner plexiform layer.)