Distinct roles of calcineurin-nuclear factor of activated T-cells and protein kinase A-cAMP response element-binding protein signaling in presynaptic differentiation

Abstract

Synaptic vesicle accumulation and morphological changes are characteristic features of axon terminal differentiation during synaptogenesis. To investigate the regulatory mechanism that orchestrates synaptic molecules to form mature presynaptic terminals, we visualized a single axon terminal of zebrafish olfactory sensory neurons in vivo and examined the effects of the neuron-specific gene manipulations on the axon terminal differentiation. Synaptic vesicles visualized with vesicle-associated membrane protein 2 (VAMP2)-enhanced green fluorescent protein (EGFP) fusion protein gradually accumulated in axon terminals, whereas the axon terminals visualized with GAP43 fused with EGFP remodeled from complex shapes with filopodia to simple shapes without filopodia from 50 h postfertilization (hpf) to 84 hpf. Expression of dominant-negative protein kinase A (PKA) or cAMP response element-binding protein (CREB) suppressed the VAMP2-EGFP punctum formation in axon terminals during synaptogenesis. Consistently, constitutively active PKA or CREB stimulated VAMP2-EGFP puncta formation. On the other hand, cyclosporine A treatment or suppression of nuclear factor of activated T cells (NFAT) activation prevented the axon terminal remodeling from complex to simple shapes during synaptogenesis. Consistently, expression of constitutively active calcineurin accelerated the axon terminal remodeling. These results suggest that calcineurin-NFAT signaling regulates axon terminal remodeling, and PKA-CREB signaling controls synaptic vesicle accumulation.

Figure 1.

Development of connections between olfactory sensory neurons and target neurons in the dorsal olfactory bulb in living zebrafish. A, C, E, G, Frontal views of the right olfactory organ of a BODIPY-labeled zebrafish embryo carrying the omp promoter-driven VAMP2-ECFP transgene at 36 hpf (A), 50 hpf (C), 60 hpf (E), and 84 hpf (G). Each panel is a single optical section. VAMP2-ECFP (green) and BODIPY505/515 (red) signals were imaged with a helium-cadmium (HeCd) and an argon (Ar) laser, respectively. Arrows point to BODIPY-labeled areas merging with VAMP2-ECFP signals in olfactory neuron axon terminals. B, D, F, H, Frontal views of the right olfactory organ of a zebrafish embryo carrying both the omp promoter-driven VAMP2-ECFP and tbr1 promoter-driven EYFP transgenes at 36 hpf (B), 50 hpf (D), 60 hpf (F), and 84 hpf (H). These composite images were generated from 15-20 z-series optical sections. VAMP2-ECFP (green) and EYFP (red) signals were imaged with a HeCd and an Ar laser, respectively. Arrowheads indicate EYFP-positive round cells in the dorsal olfactory bulb. Asterisks indicate the nasal pit, and dashed lines indicate the boundary between olfactory placode and olfactory bulb. OB, Olfactory bulb; OP, olfactory placode. CZ, DZ, LZ, MZ, and VZ indicate the central, dorsal, lateral, medial, and ventral zones of glomeruli, respectively. Scale bar, 20 μm.

Figure 2.

Effects of constitutively active calcineurin and constitutively active PKA on the development of VAMP2-EGFP puncta in axon terminals of olfactory sensory neurons. A, Omp promoter-driven expression vectors for VAMP2-EGFP (top), for caCN and VAMP2-EGFP (middle), and for PKA^* and VAMP2-EGFP (bottom) in olfactory sensory neurons. Black boxes, The omp promoter; cross-hatched boxes, the 3′ downstream sequence of the omp gene; hatched boxes, SV40 polyadenylation signal sequence; lines, pBluescript II SK+. B, A single axon terminal of an olfactory sensory neuron in a zebrafish embryo injected with omp promoter-driven VAMP2-EGFP and Bassoon-EYFP vectors at 50 hpf. VAMP2-ECFP (left; green) and Bassoon-EYFP (middle; red) signals were merged on the right. Arrowheads point to axonal shafts. Scale bar, 5 μm. C, Confocal fluorescent images of immunostaining with anti-GFP (left; green) and anti-syntaxin (middle; red) antibodies in the olfactory bulb from an omp promoter-driven VAMP2-ECFP transgenic fish embryo at 60 hpf. Merged image is on the right. Scale bar, 10 μm. D-F, Representative VAMP2-EGFP signals in axon terminals of olfactory sensory neurons in Pomp-VG-injected embryos at 36 hpf (D), 60 hpf (E), and 84 hpf (F). Scale bar, 5 μm. G-I, The threshold images of VAMP2-EGFP signals in D-F for evaluation of VAMP2-EGFP puncta. J-O, Representative VAMP2-EGFP signals in axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-VG-caCN (J-L) or Pomp-VG-PKA^* (M-O) at 36 hpf (J, M), 60 hpf (K, N), and 84 hpf (L, O). Arrowheads point to axonal shafts. P, The area of VAMP2-EGFP puncta in axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-VG (open bars), Pomp-VG-caCN (black bars), and Pomp-VG-PKA^* (gray bars) expression vectors at 36, 60, and 84 hpf. Pomp-VG, n = 37, 26, and 30 at 36, 60, and 84 hpf, respectively, 21-23 embryos; Pomp-VG-caCN, n = 40, 34, and 34 at 36, 60, and 84 hpf, respectively, 24-26 embryos; Pomp-VG-PKA^*, n = 25, 37, and 39 at 36, 60, and 84 hpf, respectively, 20-22 embryos. All values represent mean ± SEM. ^**p < 0.01.

Figure 3.

Effects of dominant-negative PKA on the VAMP2-EGFP puncta formation in axon terminals of olfactory sensory neurons. A, Omp promoter-driven double-cassette expression vector for VAMP2-EGFP and dominant-negative PKA. Black boxes, The omp promoter; cross-hatched box, the 3′ downstream sequence of the omp gene; hatched box, SV40 polyadenylation signal sequence; line, pBluescript II SK+. B-G, Representative VAMP2-EGFP signals in axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-VG (B-D) or Pomp-VG-dnPKA (E-G) at 36 hpf (B, E), 60 hpf (C, F), and 84 hpf (D, G). Arrowheads point to axonal shafts. Scale bar, 5 μm. H, The area of VAMP2-EGFP puncta in axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-VG (open bars) and Pomp-VG-dnPKA (filled bars) expression vectors at 36, 60, and 84 hpf. Pomp-VG, n = 31, 33, and 34 at 36, 60, and 84 hpf, respectively, 25-31 embryos; Pomp-VG-dnPKA, n = 35, 35, and 35 at 36, 60, and 84 hpf, respectively, 25-30 embryos. All values represent mean ± SEM. ^*p < 0.05; ^**p < 0.01.

Figure 4.

Effects of dominant-negative CREB and constitutively active CREB on the development of VAMP2-EGFP puncta in axon terminals of olfactory sensory neurons. A, Omp promoter-driven expression vectors for A-CREB and VAMP2-EGFP (top) and for VP16-CREB and VAMP2-EGFP (bottom) in olfactory sensory neurons. Black boxes, The omp promoter; cross-hatched boxes, the 3′ downstream sequence of the omp gene; hatched boxes, SV40 polyadenylation signal sequence; lines, pBluescript II SK+. B-J, Representative VAMP2-EGFP signals in axon terminals of olfactory sensory neurons in embryos injected with Pomp-VG (B-D), Pomp-VG-ACREB (E-G), or Pomp-VG-VP16CREB (H-J) at 36 hpf (B, E, H), 60 hpf (C, F, I), and 84 hpf (D, G, J). Arrowheads point to axonal shafts. Scale bar, 5 μm. K, The area of VAMP2-EGFP puncta in axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-VG (open bars), Pomp-VG-ACREB (black bars), and Pomp-VG-VP16CREB (gray bars) expression vectors at 36, 60, and 84 hpf. Pomp-VG, n = 32, 35, and 37 at 36, 60, and 84 hpf, respectively, 22-26 embryos; Pomp-VG-ACREB, n = 35, 41, and 36 at 36, 60, and 84 hpf, respectively, 18-19 embryos; Pomp-VG-VP16CREB, n = 33,33, and 34 at 36,60, and 84 hpf, respectively,22-27 embryos. All values represent mean ± SEM. ^**p < 0.01; ^***p < 0.001.

Figure 5.

Effects of constitutively active calcineurin and constitutively active PKA on the axon terminal morphology of olfactory sensory neurons in living zebrafish embryos. A, Omp promoter-driven expression vectors for GAP43-EGFP (top), for caCN and GAP43-EGFP (middle), and for PKA^* and GAP43-EGFP (bottom) in olfactory sensory neurons. Black boxes, The omp promoter; cross-hatched boxes, the 3′ downstream sequence of the omp gene; hatched boxes, SV40 polyadenylation signal sequence; lines, pBluescript II SK+. B-J, Representative images of the axon terminal morphology of olfactory sensory neurons in zebrafish embryos injected with Pomp-GG (B-D), Pomp-GG-caCN (E-G), or Pomp-GG-PKA^* (H-J) vectors at 36 hpf (B, E, H), 60 hpf (C, F, I), and 84 hpf (D, G, J). Scale bar, 5 μm. K-M, The area (K), perimeter (L), and complexity (M) values of axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-GG (open bars), Pomp-GG-caCN (black bars), and Pomp-GG-PKA^* (gray bars) expression vectors at 36, 60, and 84 hpf. Pomp-GG, n = 25, 29, and 28 at 36, 60, and 84 hpf, respectively, 20 embryos; Pomp-GG-caCN, n = 26, 21, and 25 at 36, 60, and 84 hpf, respectively, 17-20 embryos; Pomp-GG-PKA^*, n = 19, 21, and 29 at 36, 60, and 84 hpf, respectively, 14-18 embryos. All values represent mean ± SEM. ^*p < 0.05; ^**p < 0.01.

Figure 6.

Effects of cyclosporine A treatment on the axon terminal morphology of olfactory sensory neurons. A-D, Representative images of the axon terminal morphology of olfactory sensory neurons in mock-treated (A, B) and cyclosporine A-treated (C, D) embryos injected with Pomp-GG vector at 60 hpf (A, C) and 84 hpf (B, D). Scalebar, 5 μm. E-G, The area (E), perimeter (F), and complexity (G) values of axon terminals of olfactory sensory neurons in mock-treated and cyclosporine A-treated (CsA) embryos injected with Pomp-GG vector at 60 and 84 hpf. Mock treatment, n = 43 and 42 for 28 and 26 embryos at 60 and 84 hpf, respectively; cyclosporine A treatment, n = 30 and 30 for 18 and 19 embryos at 60 and 84 hpf, respectively. All values represent mean ± SEM. ^*p < 0.05; ^***p < 0.001.

Figure 7.

Effects of suppression of NFAT on the axon terminal morphology of olfactory sensory neurons. A, Omp promoter-driven expression vectors for GST-CP and GAP43-EGFP (top) and for GST-VIVIT and GAP43-EGFP (bottom) in olfactory sensory neurons. Black boxes, The omp promoter; cross-hatched boxes, the 3′ downstream sequence of the omp gene; hatched boxes, SV40 polyadenylation signal sequence; lines, pBluescript II SK+. B-E, Representative GAP43-EGFP signals in axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-GG-GST-CP (B, C) or Pomp-GG-GST-VIVIT (D, E) at 60 hpf (B, D) and 84 hpf (C, E). Scale bar, 5 μm. F-H, The area (F), perimeter (G), and complexity (H) values of axon terminals of olfactory sensory neurons in zebrafish embryos injected with Pomp-GG-GST-CP (open bars) and Pomp-GG-GST-VIVIT (black bars) expression vectors at 36, 60, and 84 hpf. Pomp-GG-GST-CP, n = 41, 36, and 39 at 36, 60, and 84 hpf, respectively, 23-25 embryos; Pomp-GG-GST-VIVIT, n = 37, 41, and 39 at 36, 60, and 84 hpf, respectively, 24-30 embryos. All values represent mean ± SEM. ^***p < 0.001.