Ferguson SC and McFarlane S (2002), GABA and development of the Xenopus optic proje...

XB-ART-6763

J Neurobiol 2002 Jun 15;514:272-84. doi: 10.1002/neu.10061.

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GABA and development of the Xenopus optic projection.

Ferguson SC , McFarlane S .

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In the developing visual system of Xenopus laevis retinal ganglion cell (RGC) axons extend through the brain towards their major target in the midbrain, the optic tectum. Enroute, the axons are guided along their pathway by cues in the environment. In vitro, neurotransmitters have been shown to act chemotropically to influence the trajectory of extending axons and regulate the outgrowth of developing neurites, suggesting that they may act to guide or modulate the growth of axons in vivo. Previous work by Roberts and colleagues (1987) showed that populations of cells within the developing Xenopus diencephalon and mid-brain express the neurotransmitter gamma amino butyric acid (GABA). Here we show that Xenopus RGC axons in the midoptic tract grow alongside the GABAergic cells and cross their GABA immunopositive nerve processes. Moreover, RGC axons and growth cones express GABA-A and GABA-B receptors, and GABA and the GABA-B receptor agonist baclofen both stimulate RGC neurite outgrowth in culture. Finally, the GABA-B receptor antagonist CGP54626 applied to the developing optic projection in vivo causes a dose-dependent shortening of the optic projection. These data indicate that GABA may act in vivo to stimulate the outgrowth of Xenopus RGC axons along the optic tract.

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Species referenced: Xenopus laevis
Genes referenced: gabbr1 hspa9 igf2bp3 isl1 tec
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	Figure 1 Developmental expression of GABA in the optic tract coincides spatially and temporally with the developing optic projection. Whole-mount double immunohistochemistry for GABA (green) and the HRP-labeled optic projection (red) at stages 35/36 (A) and 37/38 (B). (A1–B1). Lateral view of double-immunolabeled wholemount Xenopus brains double-immunolabeled for GABA and HRP. (A2–B2). GABA immunocytochemistry. GABA positive cells are found in the mid-optic tract, tectum as well as in the hindbrain. (A3–B3): HRP-labeled optic projection. (C–D). Double immunocytochemistry for GABA (green) and HRP-labeled RGC axons (red) in a transverse cryostat section through the brain and eyes of a stage 35/36 embryo. RGC axons grow alongside GABAergic cells in the mid-diencephalon. Amacrine cells in the inner nuclear layer (INL) of the retina are also immunopositive for GABA. (D) is a 1-m confocal section of RGC axons growing in the mid-diencephalon. RGC axons grow either in close proximity to GABAergic cells in the diencephalon (arrow), or alongside GABA-positive processes in the neuropil (arrowhead). Di, diencephalon; Np, neuropil; Tec, tectum; Tel, Telencephalon; Hb, hindbrain; A, anterior; V, ventral; Mot, mid optic tract. Scale bar in (B3) is 100 m for (A–B) Scale bar in (C) is 30 m, and bar in (D) is 25 m.
	Figure 2 The GABA-A and GABA-B receptor antibodies recognize Xenopus proteins of the appropriate size. Western blots of stage 35/36–37/38 Xenopus head tissue showing GABA-A (A) and GABA-B (B) receptor protein expression. Molecular weights based on standards run at the same time as the sample tissue are indicated. (A) A prominent protein band with an approximate molecular weight of 58 kDa was observed, which corresponds to the weight of known GABA-A receptor -subunits. (B) A single band with an approximate molecular weight of 100 kDa was observed using a polyclonal antibody (Santa Cruz) against the GABA-B receptor.
	Figure 3 GABA receptors are expressed by developing Xenopus RGCs. GABA-B receptor expression as revealed by immunocytochemistry with an antibody (Santa Cruz) against the GABA-B receptor. (A–B) Transverse sections through a stage 35/36 Xenopus eye and brain. GABA-B receptor immunoreactivity was found in the diencephalic neuropil (A), the optic nerve [see arrowheads in (B)], and the retina. (C) No retinal labeling was seen in a similar section when the primary antibody was preincubated with a blocking peptide (Santa Cruz). (D) GABA-B receptor immunolabeling is seen in the stage 37/38 embryonic Xenopus retina shown at higher power. Immunoreactivity was observed in the inner nuclear layer (INL), retinal ganglion cell layer (RGCL), and the inner (IPL) and outer (OPL) plexiform layers. The optic nerve head (onh) was also immunopositive (arrow). Di, diencephalon; PE, pigment epithelium; L, lens; Np, neuropil; D, dorsal; V, ventral. Scale bar in (D) is 200 m for (A)–(C), and 100 m for (D).
	Figure 4 GABA receptors are expressed by developing Xenopus RGC growth cones. Stage 24 eye buds were cultured as explants for 40–48 h and processed for GABA receptor immunocytochemistry. (A) The GABA-A receptor antibody revealed relatively uniform labeling of RGC axons and growth cones. (B) Intense immunolabeling for GABAB receptors was found in the axons and body of RGC growth cones, with lower level labeling in the filopodia and lamellopodia. (C–D) GABA-B receptor immunoreactivity in a growth cone (C) could be eliminated by incubation with a blocking peptide for the GABA-B receptor antibody (D). Scale bar in (A) is 20 m for (A) and (B), and 25 m for (C) and (D).

	Figure 6 CGP54626 applied to the exposed brain preparation inhibits the outgrowth of RGC axons in vivo: the GABA-B receptor antagonists phaclofen (200–500 M) and CGP54626 (100– 500 M) were applied for 20 h to the developing optic projection starting at stage 33/34 when the RGC axons are first entering the contralateral brain. Shown are lateral views of the HRP-labeled optic projection in wholemount stage 40 brains (A–B) exposed to either control solution (A) or 500 M phaclofen (B). No obvious effect on the optic projection was observed. (C) Optic projection exposed to a control solution containing the same DMSO concentration as the 500 M CGP54626 exposed brain in (F). (D–F) Optic projections exposed to either 100 M CGP54626 (D), 250 M CGP54626 (E), or 500 M CGP54626 (F). Scale bar in (F) is 150 m. Tec, tectum; Tel, telencephalon; ot, optic tract.
	Figure 7 (A–F) Immunolabeling with various neuroepithelial cell markers indicates that the neuroepithelium was not obviously affected by treatment with 250 and 500 M CGP54626. (A–B) Immunocytochemistry with an antibody against islet-1 shows ventrally located cells in both control (0.5% DMSO)- (A) and 500 M CGP54626- (B) treated brains. (C–D) Zn-12, a general neural marker, exhibits comparable labeling in control (1% DMSO)- (C) and 250 M CGP54626- (D) treated brains. (E) Unexposed (UnEx) and exposed (Ex) sides of a 250 M CGP54626-treated brain show comparable immunolabeling with an antibody against -tubulin. (F) GABA immunoreactivity is unaffected by treatment with 500 M CGP54626. Bar in F is 100 m. (G) Mean normalized optic tract length in control and GABA-B receptor antagonist treated brains. For each CGP54626 concentration, control brains were exposed to equivalent levels of the DMSO carrier. CGP54626 caused a dose-dependent shortening of the optic projection, whereas phaclofen had no obvious effect on the optic projection. p .05, *p .01 (unpaired, two-tailed Student t test). Numbers in brackets are the number of embryos from three to five separate experiments.