Activation and habituation of extracellular signal-regulated kinase phosphorylation in zebra finch auditory forebrain during song presentation

Abstract

The sound of tape-recorded birdsong triggers a set of behavioral and physiological responses in zebra finches, including transcriptional activation of the zenk gene in the auditory forebrain. Song repetition leads to the stimulus-specific habituation of these responses. To gain insight into the mechanisms that couple auditory experience to gene regulation, we monitored the phosphorylation of the zebra finch extracellular signal-regulated kinase (ERK) protein by immunoblotting. Initial presentations of novel song (but not tones or noise) resulted in a rapid increase in ERK phosphorylation, followed by a return to basal levels within 5 min. This response was localized to the auditory forebrain where the zenk gene is activated. Sustained repetition of one song caused a selective habituation of the ERK response: a different song triggered another cycle of ERK phosphorylation without altering the habituated response to the first. To test directly for a role of ERK in experience-dependent zenk gene regulation, we infused an inhibitor of mitogen-activated and extracellular-regulated protein kinase kinase (MEK-1; the enzyme responsible for ERK activation) unilaterally into one auditory lobule just before song stimulation. The song-induced increase in zenk mRNA was blocked on the side of the injection, but not on the contralateral (uninfused) side. These results show that ERK phosphorylation is necessary for the initiation of the zenk gene response to novel song and identify ERK as a plausible site of signal integration underlying the selective habituation of genomic responses to a repeated song.

Figure 1.

The songbird (canary) zenk promoter. The diagram shows the location of consensus binding sites for several transcription factors of interest relative to the start site (+1); similar binding sites are conserved in mammalian orthologs of the gene (Changelian et al., 1989).

Figure 3.

ERK phosphorylation is induced by song playback in zebra finch. A, Representative immunoblots of zebra finch AL extracts probed with pERK-specific and cyclophilin antibodies; the cyclophilin immunoreactivity was used to correct for possible loading differences in different lanes. AL was collected from birds immediately after exposure to two repetitions (lasting 75 sec), 30× (∼30 min), 60× (∼60 min), and 90× (∼90 min) of song playback. B, Group data of corrected pERK signal in AL from birds exposed to 2× (n = 5), 30× (n = 4), 60× (n = 3), and 90× (n = 3) song repetitions, shown as fold increase relative to average intensities of corrected pERK in controls exposed to silence (n = 4). Error bars represent SEM. The gray fill identifies the only group statistically significant from the silence controls (p < 0.05, one-way ANOVA and post hoc Dunnett's t test). Note that the pERK signal was increased immediately after just two repetitions of the song stimulus.

Figure 4.

Stimulus specificity of ERK phosphorylation. A, Representative immunoblots of zebra finch AL extracts probed with pERK-specific and cyclophilin antibodies as in previous figures. Birds were exposed to two repetitions of a stimulus made from three different songs (triple song, described in Materials and Methods) or eight repetitions of one song (single song) or eight repetitions of either ascending tones or white noise over a period of 72-75 sec and then were killed immediately. B, Group data of corrected pERK signal in AL from birds exposed to triple song (n = 7), single song (n = 7), tone (n = 10), and noise (n = 9) shown as fold increase relative to average intensities of corrected pERK in silence control (n = 12). Gray fill indicates the two groups with statistically significant differences from the silence controls (p < 0.05, one-way ANOVA and Dunnett's t test in comparison to the silence group). Error bars represent SEM.

Figure 5.

Anatomical localization of pERK increase. A, Representative immunoblots of extracts from AL, anterior forebrain (AF), cerebellum (Ce), hippocampus (Hp), and optic tectum (OT) of birds exposed to silence or two repetitions of triple song, probed with pERK and ERK antibodies. B, Mean intensity of ERK signal in brain regions as in A, normalized to intensity in AL from silence controls; birds were exposed to silence or song (n = 3 for each group). C, Mean intensity of pERK signal in brain regions as in A, normalized to intensity in AL from silence controls for birds exposed to silence or song (n = 3 for each group). Note that song stimulation induces a large increase in pERK only in AL. Error bars represent SEM.

Figure 6.

Transient induction of ERK phosphorylation. A, Representative immunoblots of zebra finch AL extracts probed with pERK-specific and cyclophilin antibodies. Birds were exposed either to silence or to eight repetitions of a single song over 72 sec (A8) and then were killed for collection either immediately or after 5 min (A8-5′), 15 min (A8-15′), 30 min (A8-30′), or 60 min (A8-60′) in silence. B, Group data from birds under conditions A8 (n = 7), A8-5′ (n = 6), A8-15′ (n = 6), A8-30′ (n = 4), and A8-60′ (n = 3) shown as fold increase relative to average intensities of corrected pERK in silence control (n = 8). Gray fill indicates the group with a statistically significant difference from the silence controls (p < 0.05, one-way ANOVA and Dunnett's t test in comparison to the silence group). Error bars represent SEM.

Figure 7.

pERK is habituated by repeated playback. A, Representative immunoblots of zebra finch AL extracts probed with pERK-specific and cyclophilin antibodies as in previous figures. AL was collected from birds exposed to eight (A8) or 908 × (A908) repetitions of a single song or to 900 repetitions of one song, followed by eight repetitions of a different song (A900B8). B, Group data of corrected pERK signal in AL from birds under conditions A8 (n = 8), A908 (n = 6), and A900B8 (n = 6) shown as fold increase relative to average intensities of corrected pERK in silence controls (n = 8). Gray fill indicates statistically significant difference from silence controls (p < 0.05, one-way ANOVA and Dunnett's t test). Note that extended song repetition (A908) led to suppression of pERK, but brief exposure to a novel song then reactivated pERK phosphorylation (A900B8). Error bars represent SEM.

Figure 8.

pERK habituation is maintained after periods of silence. A, Representative immunoblots of zebra finch AL extracts probed with pERK-specific and cyclophilin antibodies. The nomenclature indicates the sequence of exposures to song or silence before death, as follows: A₁₀ (killed immediately after 10 repetitions of song A), A₂₀₀60′A₁₀ (200 repetitions of A, followed by 60 min of silence, and then 10 more repetitions of A), A₂₀₀5′A₁₀ (200 repetitions of song A, followed by 5 min of silence, and then 10 more repetitions of A), A₂₀₀5′B₁₀ (200 repetitions of song A, followed by 5 min of silence, and then 10 more repetitions of a novel song, B). B, Mean corrected data for pERK in AL from birds under conditions A₁₀ (n = 6), A₂₀₀60′A₁₀ (n = 4), A₂₀₀5′A₁₀ (n = 5), and A₂₀₀5′B₁₀ (n = 4), shown as fold increase relative to average intensities of corrected pERK in silence controls (n = 6). Gray fill indicates statistically significant difference from silence controls (p < 0.05, one-way ANOVA and Dunnett's t test). Note that habituation to song A persisted after intervening periods of silence (A₂₀₀60′A₁₀, A₂₀₀5′A₁₀), but exposure to a new song reactivated ERK (A₂₀₀5′B₁₀). Error bars represent SEM.

Figure 9.

Song-specific pERK habituation is maintained after intervening exposure to a different song. Three different unfamiliar zebra finch songs (designated A, B, C) or silence (-) was used in the experiment, each presented in the single-song format (2 sec of song every 10 sec). The nomenclature indicates the sequence of exposures as follows: A₁₀, 10 repetitions of song A, followed by immediate death; ABC₂₀₀A-B, 10 repetitions each of songs A, B, and C, followed by 200 repetitions to habituate song C, then 10 presentations of A, then 5 min of silence, and then a final exposure to 10 repetitions of song B; ABC₂₀₀A-C, same as ABC₂₀₀A-B except that the final exposure was to the habituated stimulus, song C. A, Representative immunoblots of zebra finch AL extracts probed with pERK-specific and cyclophilin antibodies. B, Mean corrected pERK signal intensity relative to silence control group; n = 3 each group. Gray fill indicates statistically significant difference from silence controls (p < 0.05, one-way ANOVA and Dunnett's t test). Note that after intervening exposure to song A the response to song C remained selectively habituated. Error bars represent SEM.

Figure 10.

Inhibition of ERK phosphorylation inhibits zenk induction by song playback. A, Representative immunoblots showing suppression of song-induced pERK levels by unilateral AL-directed infusion of MEK-1 inhibitor U0126. At 1 hr after infusion the birds were exposed to eight repetitions of single song playback and assayed for pERK and cyclophilin immunoreactivity in AL as in Figure 6 (group A8). D-i, AL ipsilateral to injection of DMSO vehicle; D-c, AL contralateral to injection of DMSO vehicle; U-i, AL ipsilateral to injection of U0126; U-c, AL contralateral to injection of U0126. B, Representative in situ hybridization images of zenk expression in zebra finch AL. Ba, Silence control; Bb, 30 min after novel song stimulation; Bc, 30 min after novel song stimulation, ipsilateral side of U0126-injected brain; Bd, 30 min after novel song stimulation, contralateral side of DMSO-injected brain. C, Mean zenk-positive cell counts in AL from birds exposed to silence (n = 4), 30 min of song playback (song, n = 4), ipsilateral DMSO injection followed by 30 min of song playback (DMSO, n = 4), and ipsilateral U0126-injection followed by 30 min of song playback (U0126, n = 4). Gray fill indicates statistically significant difference from silence controls (p < 0.05, one-way ANOVA and Dunnett's t test). Note that zenk levels are increased in birds hearing song and in DMSO-injected controls, but not in U0126-injected birds, when compared with silence controls. Error bars represent SEM.

Figure 2.

Commercial ERK and pERK antibodies recognize a zebra finch protein. Shown are representative immunoblots of HEK-293 (HEK) and zebra finch AL extracts (ZF) probed with commercial ERK (left) and pERK (right) antibodies, with size standards as indicated. HEK-293 extracts contain two species recognized by the antibodies, as expected (42 and 44 kDa); however, only one protein band in zebra finch AL is recognized by these antibodies.