Article Figures & Data
Figures
- Fig. 1.
Schematic sagittal drawing of the avian song system. The thin lines represent the motor control pathway that is essential for the song production. The thick lines show the anterior forebrain pathway necessary for song acquisition. The white arrows show the pathway from the auditory area to the song system. Field L is the primary auditory area in the bird brain. NCM, in which the immediate early gene ZENK was induced by song, is one indirect source of auditory inputs to the song system. HVC, Higher vocal center;RA, robust nucleus of the archistriatum;nXIIts, tracheosyringeal part of the hypoglossal nucleus; Uva, nucleus uvaeformis of the thalamus;NIf, nucleus interface of the neostriatum;X, area X of the parolfactory lobe; DLM, medial part of the dorsolateral nucleus of the thalamus;LMAN, lateral magnocellular nucleus of the anterior neostriatum; NCM, caudomedial neostriatum.
- Fig. 2.
Immunohistochemical localization of CREB-IR structures in a sagittal section through the adult zebra finch brain.HV, Hyperstriatum ventrale; PA, paleostriatum augmentatum; LPO, lobus parolfactorius.B–D, Higher magnifications of the three main song control nuclei, HVC (B),X (C), and RA(D). A number of CREB-IR cell nuclei with intense staining were found in the HVC and area X (B, C) but not LMAN (A). In the RA, there were CREB-IR cell nuclei with a light staining intensity (D). Dorsal is toward the top and anterior is to the right in all photos. Scale bars: A, 500 μm;B–D, 100 μm.
- Fig. 3.
Combined neuron labeling in the HVC with a retrograde tracer, FRe. A, Ipsilateral RA, injected with FRe. Retrogradely labeled RA-projecting neurons within HVC (pink label; white arrowheadspoint to tracer-labeled neurons) do not overlap with CREB-IR cell nuclei (black label; arrows point to CREB-IR nuclei without cytoplasmic retrograde tracer).B, Double-labeled HVC neurons were projected in area X and were visible when FRe was injected into area X (arrows point to typical neurons). Scale bar, 20 μm.
- Fig. 4.
Primary structure of zebra finch CREB.A, The zebra finch CREB sequence was aligned with mammalian CREB proteins (human, rat, mouse, and bovine δ CREB). Amino acids that are identical in all five sequences are displayed with the figure and ground reversed. The GenBank accession numbers for the human, rat, mouse, and bovine sequences are X60003, X60002, s20955, andAF006042, respectively. B, The domains of CREB are schematically represented. The small numbers refer to amino acid residues at the boundaries of each domain. Q1, Q2, Glutamine-rich regions; KID, kinase-inducible domain; DBD/LZ, DNA-binding/leucine zipper domain. The percentages in each domain represent the percentage identity of CREB amino acid sequences between zebra finch and rat. The epitope for antiserum 5322 is shown below, and the putative protein kinase A (PKA) phosphoacceptor site is enclosed by arectangle.
- Fig. 5.
Immunoblots of zebra finch CREB. A, Immunoblots of zebra finch homogenates (whole brain, lane 1; HVc region, lane 2) and recombinant CREB proteins (rat, lane 3; zebra finch,lane 4) detected with the CREB-1 monoclonal antibody. This antibody does not discriminate between phosphorylated and unphosphorylated CREB. B, In vitrophosphorylation of recombinant zebra finch CREB with the catalytic subunit of protein kinase A (PKA). Zebra finch recombinant CREB (zCREB) protein was incubated with (+) or without (−) the catalytic subunit of PKA in the reaction mixtures containing [γ-32P]ATP and resolved by SDS-PAGE. Phosphorylation of recombinant rat CREB (rCREB) is shown as a positive control. C, Immunoblots of unphosphorylated (ATP−) or phosphorylated (ATP+) CREB. Recombinant CREB proteins (lanes 1–4) and crude homogenates of brain tissues (lanes 5–8) were incubated with or without ATP in the presence of PKA. Only the phosphorylated form of zCREB in the brain tissue (lane 8) and recombinant zCREB (lane 4) were recognized with antibody 5322. A crude rat homogenate (lane 6) and recombinant CREB (lane 2) are shown as positive controls. The positions (in kilodaltons) of the molecular size markers are shown to the left of the gels.
- Fig. 6.
Song-induced CREB phosphorylation in the HVC. Shown are CREB-IR (A, B) and PCREB-IR (C–F) cell nuclei in the HVC after hearing white noise (A, C, E) or a zebra finch conspecific song (B, D, F) for 30 min. The song induced phosphorylation of CREB in the HVC, whereas white noise did not. CREB immunoreactivity was not affected after either stimuli.E and F are higher magnifications of the HVCs shown in C and D, respectively. The sections were cut in the sagittal plane. Dorsal is toward the top and anterior is to the right in all photos. Scale bars: B, 100 μm; F, 20 μm.
- Fig. 7.
Time-dependency of song-induced phosphorylation of CREB in the HVC. Values represent the number of phosphorylated CREB-IR cells/mm2 in HVC: n = 4 for 0 min, n = 5 for 30 min, and n = 1 for the others. Values for 0 and 30 min are the mean ± SD. The difference between 0 and 30 min is statistically significant (p < 0.001).
- Fig. 8.
Quantitative analysis of CREB-IR and PCREB-IR nuclei induced in area X by hearing a song. C, Control;S, zebra finch conspecific song for 30 min. Values are the mean ± SD.
- Fig. 9.
Comparative analysis of CREB-IR and PCREB-IR nuclei induced by hearing three auditory stimuli: white noise (NOIS), a canary song (CAN), and a zebra finch conspecific song (CON). Values are the mean ± SD. The differences between the conspecific song group and the other groups are statistically significant (***p < 0.001).
- Fig. 10.
No induction of CREB phosphorylation in HVC by undirected or directed singing. The number of PCREB-IR cells/mm2 in the HVC was plotted as a scatter diagram against the number of song bouts during 30 min. ○, Undirected singing (solo); ●, directed singing.
