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Minimal Experience Required for Immediate-Early Gene Induction in Zebra Finch Neostriatum
Neurobiology of Learning and Memory 74, 179–184 (2000) doi:10.1006/nlme.2000.3968, available online at http://www.idealibrary.com on
RAPID COMMUNICATION Minimal Experience Required for Immediate-Early Gene Induction in Zebra Finch Neostriatum Amy A. Kruse, Roy Stripling, and David F. Clayton Neuroscience Program, Beckman Institute and Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801
We show that a single presentation of a zebra finch song, 2 s in duration, will induce an “immediate-early gene” response in the caudomedial neostriatum of zebra finches (Poephila guttata). Repetition of this stimulus 10 times is sufficient to induce a maximal increase in RNA and protein, detected 30 and 90 min later respectively. Thus very brief stimuli can set in motion a slow genomic process in the brain which takes hours to resolve. Immediate-early gene function is often considered in the context of a “feedback” model (i.e., to consolidate memories of the inducing event). However, based on the long lag observed here between initiation and full expression of the molecular response, we suggest an alternative, ethologically based, “feed-forward” model in which exposure to a novel or significant context triggers an increase in the efficiency of memory capture processes for subsequent experiences. q 2000 Academic Press Key Words: songbird; zebra finch; immediate-early gene; zenk; zif-268; egr-1; consolidation; caudal neostriatum.
Behavioral and perceptual activities can induce transient increases in gene expression in various brain regions. This phenomenon is often referred to as the immediate-early gene (IEG) response and is widely assumed to contribute to the consolidation of memories of the inducing event (reviewed in Tischmeyer & Grimm, 1999). However, its significance for normal brain function is not yet clear. Although a few studies have demonstrated IEG responses in freely behaving animals following natural stimuli (Jarvis, Schwabl, Ribeiro, & Mello, 1997; Jin & Clayton, 1997), most investigations have used intense or highly artificial laboratory-based paradigms, often of long duration, to induce the response (e.g., Work supported by an NSF Graduate Fellowship (A.K.) and NIH Grant R01 MH52086. We thank Dr. Telsa Mittelmeier and Karen Pilcher for assistance with immunoblotting and immunohistochemistry and Bridget Carragher and Steve Rogers (Beckman Imaging Technology Group) for assistance with quantitative microscopy. Correspondence and reprint requests to David F. Clayton, B107 Chemical and Life Sciences Laboratories, 601 S. Goodwin Ave., Urbana, IL 61801. Fax: (503) 213-6611. E-mail: [email protected]. 179
1074-7427/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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Tischmeyer & Grimm, 1999). Prior research into the IEG response thus leaves mostly unaddressed a fundamental question: What is the minimal amount of behavioral experience necessary to induce the IEG response? A clearer definition of the temporal relationship between circumstances that lead to IEG induction and the time course of the IEG response itself would help in developing a physiological model of IEG function during ongoing brain activity. We therefore sought to establish the minimal temporal stimulus necessary and sufficient for zenk gene induction in a natural model of the phenomenon, adult zebra finches responding to tape-recorded song playbacks (reviewed in Clayton, 1997, 2000a). Adult zebra finches (n 5 18, Magnolia Bird Farms) were housed for at least a month in the Beckman Animal Care facility prior to use in experiments. All procedures involving animals were conducted with the guidance and formal approval of the Illinois Laboratory Animal Care Advisory Committee. Prior to each experiment, birds were individually isolated for 24 h in an anechoic chamber (birds that produced any spontaneous song during the final hour were not used in experiments). Tape-recorded stimuli (70 dB) were then played over a loudspeaker in the chamber, in one of five different stimulation paradigms. Birds (n 5 4) hearing the “13” stimulus were played one presentation of a single tape-recorded zebra finch song, ,2 s in duration. The “triple song” group (n 5 2) heard three different songs presented without separation for a single 15-s period. The “103” (n 5 4) and positive control (n 5 4) groups heard a single song repeated once every 10 s for a total of 10 or 180 presentations, respectively. Silent controls (n 5 4) were not presented with a song stimulus. Birds were sacrificed 30 min after the onset of song playback to allow time for induced mRNA to accumulate following the time course for gene expression established in previous studies (Mello & Clayton, 1994). zenk mRNA was detected in fixed brain slices using digoxygen-labeled antisense RNA probes (Jin & Clayton, 1997), and the density of positively labeled cells was determined using an automated cell counting system (Stripling et al., submitted). Figure 1 shows representative hybridizations from each of the five conditions. Cells positive for zenk mRNA accumulation appear as darkly stained points at this magnification. The fields shown represent sections in the parasagittal plane, taken at a medial level containing the region of greatest zenk responsiveness to song (Mello & Clayton, 1994). The level of staining is similar inside and outside the NCM/CMHV region in birds in the silent control group (Fig. 1C), whereas birds in the positive control group (Fig. 1B) contained a much greater density of labeled cells within NCM/CMHV compared to surrounding brain. A high level of labeling similar to the positive control is seen in sections from birds who heard only 10 presentations of song (Fig. 1F), and increased labeling relative to the surrounding brain is evident in birds in the 13 and triple song groups (Figs. 1D and 1E, respectively). Figure 2 presents quantification of these results, expressed as the density of labeled cells in NCM/CMHV. Relative to nonstimulated controls, zenk expression increased about eightfold in birds hearing 10 song repetitions (P , .01). This increase was equivalent to that produced by 30 min of repeated song stimulation (Fig. 2). The triple song stimulus resulted in a sixfold increase relative to silence, slightly but significantly less than the response to 10 songs or 30 min of song (P , .05 and P , .01, respectively). Presentation of even a single song resulted in an increase in cell labeling that was significant relative to nonstimulated controls (P , .02). These results indicate that a detectable increase in
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FIG. 1. Representative zenk in situ hybridizations for experimental conditions. (A) Line drawing of the relevant anatomical regions in the zebra finch forebrain. Abbreviations correspond to hippocampus (HP), caudomedial neostriatum (NCM), and caudomedial hyperstriatum ventrale (CMHV). Size bar 5 1.0 mm, magnification for all images 5 203. (B) In situ hybridization from the 30-min song presentation condition. Staining is absent in the hippocampus, but is evident throughout NCM and CMHV. (C) The silent control condition yields minimal staining in all areas of the forebrain. (D) In the 13 condition staining is light throughout the hippocampus, but is apparent in CMHV and dorsal NCM. (E) The triple song condition shows increased labeling throughout the NCM/CMHV region. (F) The 103 condition exhibits dense labeling throughout all of NCM/CMHV.
zenk mRNA results from a single song presentation and maximal activation is achieved by as little as 10 repetitions of a single song stimulus over a 2-min period. To determine whether the zenk protein was also increased following such brief stimulation, tissue was collected from the 103 (n 5 2), negative control (n 5 2), and positive control (n 5 2) conditions. The birds were presented with song and then sat in silence until 90 min had lapsed from the beginning of song presentation. Whole-cell extracts were prepared from the NCM tissue of individual birds by dounce homogenization [in 20 mM HEPES (pH 7.9), 100 mM NaCl, 0.5 mM EDTA, 0.7% (v/v) NP-40, 0.5 mM DTT, 10% (w/v) glycerol, plus a cocktail of protease inhibitors]. These extracts were analyzed by immunoblotting with an antiserum used in previous songbird studies (Mello & Ribeiro, 1998). As seen in Fig. 3, an inducible band is evident at ,64 kDa in all three conditions. The silent controls have very low levels of signal as compared to the 103 and 30-min conditions. When this antiserum was used to probe perfusion-fixed sections of song-stimulated zebra finch brains (data not shown), the anatomical pattern of immunocytochemical labeling after the 103 stimulus closely followed the pattern of zenk mRNA labeling and was specific to NCM. Thus a very brief perceptual experience, hearing 2 s of birdsong, is sufficient to induce the immediate-early gene zenk in the zebra finch NCM. Furthermore, a maximal level of zenk RNA was achieved by as little as 20 s of song over a 2-min period. These increases
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FIG. 2. Changes in zenk labeling with increasing song exposure. Positively labeled cells were identified using criteria described elsewhere (Stripling et al., submitted). Bars filled with distinct patterns are statistically different (Kruskal–Wallis test). A single presentation was sufficient to induce zenk over silent controls (P , .02). The triple song stimulus also produced significant induction (P , .01), yet not to levels as high as the 30-min positive control (P , .01). In contrast, 10 presentations of a single song induced zenk to levels not significantly different from the positive control (P . .20).
in mRNA were accompanied by increases in zenk protein levels in NCM detected 90 min after stimulus onset. Previous reports have documented the avian response to birdsong using a much longer stimulus, typically several hundred seconds of song over a 30-min period (Mello, Vicario, & Clayton, 1992; Mello & Clayton, 1994; Jarvis et al., 1997; Mello & Ribeiro, 1998). These results provide evidence that the IEG response is engaged during normal dayto-day brain function and not simply reserved for extremes of metabolic function or stressful circumstances. Other experiments also indicate that induction of zenk and other IEGs is a common, presumably daily, occurrence in freely behaving animals. Free-ranging song sparrows in the wild show a zenk response in NCM to tape-recorded birdsongs presented via loudspeaker on their home territories (Jarvis et al., 1997), and birds singing spontaneously at dawn show zenk activation in telencephalic nuclei of the vocal control system (Jin & Clayton, 1997). Cells positive for zenk and/or c-fos can be detected throughout the rodent brain even in animals under “baseline” control conditions (Cirelli, Pompeiano, & Tononi, 1996; Campeau et al., 1997; Rosen et al., 1998). The functional significance of the genomic response to experience is not well understood—roles in memory consolidation and/or cellular homeostasis have been widely considered (Tischmeyer & Grimm, 1999). The results here reveal a dramatic temporal dissociation between the initiation of the process (within a few seconds) and its fulfillment (hours before induced protein levels have returned to baseline). As reviewed in detail elsewhere (Clayton, 2000b), this temporal dissociation suggests another hypothesis: that increases in IEG proteins may serve to alter the system’s responsiveness to subsequent
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FIG. 3. ZENK protein levels in individual birds exposed to different stimulation conditions. (A) Western blot showing two silent controls (S), two controls hearing 30 min of one song (308), and two subjects exposed to 10 presentations of song (103). Blots were probed with an affinity-purified rabbit polyclonal antibody (Santa Cruz no. sc-189, 1:1000). Three ZENK-specific bands are evident, with the 64-kDa band (arrow) showing inducibility under these conditions. (B) MAP kinase levels from the same blot (stripped and reprobed with ap44/42 MAPK, 1:2000, New England Biolabs) indicating evenness of loading for the individual lanes. Numbers across the bottom represent relative zenk signal intensity compared to the mean for the silent controls (after normalization to MAPK signal for each lane).
events. According to this hypothesis, experiences that occur during the peak of an IEG wave might be consolidated even more effectively than memories of the inducing experience itself. REFERENCES Campeau, S., Falls, W. A., Cullinan, W. E., Helmreich, D. L., Davis, M., & Watson, S. J. (1997). Elicitation and reduction of fear—Behavioural and neuroendocrine indices and brain induction of the immediate-early gene c-fos. Neuroscience, 78, 1087–1104. Cirelli, C., Pompeiano, M., & Tononi, G. (1996). Neuronal gene expression in the waking state: A role for the locus coeruleus. Science, 274, 1211–1215. Clayton, D. F. (1997). Role of gene regulation in song circuit development and song learning. Journal of Neurobiology, 33, 549–571. Clayton, D. F. (2000a). Neural basis of avian song learning and perception. In J. Bolhuis (Ed.), Brain, perception, memory: Advances in cognitive neuroscience. New York: Oxford Univ. Press. Clayton, D. F. (2000b). The genomic action potential. Neurobiology of Learning and Memory, 74, 185–216. Jarvis, E. D., Schwabl, H., Ribeiro, S., & Mello, C. V. (1997). Brain gene regulation by territorial singing behavior in freely ranging songbirds. NeuroReport, 8, 2073–2077. Jin, H., & Clayton, D. F. (1997). Localized changes in immediate-early gene regulation during sensory and motor learning in zebra finches. Neuron, 19, 1049–1059.
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Mello, C. V., & Clayton, D. F. (1994). Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system. Journal of Neuroscience, 14, 6652–6666. Mello, C. V., & Ribeiro, S. (1998). Zenk protein regulation by song in the brain of songbirds. Journal of Comparative Neurology, 393, 426–438. Mello, C. V., Vicario, D. S., & Clayton, D. F. (1992). Song presentation induces gene expression in the songbird forebrain. Proceedings of the National Academy of Sciences U.S.A., 89, 6818–6822. Rosen, J. B., Fanselow, M. S., Young, S. L., Sitcoske, M., & Maren, S. (1998). Immediate-early gene expression in the amygdala following footshock stress and contextual fear conditioning. Brain Research, 796, 132–42. Tischmeyer W., & Grimm, R. (1999). Activation of immediate early genes and memory formation. Cellular and Molecular Life Sciences, 55, 564–574.
RAPID COMMUNICATION Minimal Experience Required for Immediate-Early Gene Induction in Zebra Finch Neostriatum Amy A. Kruse, Roy Stripling, and David F. Clayton Neuroscience Program, Beckman Institute and Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801
We show that a single presentation of a zebra finch song, 2 s in duration, will induce an “immediate-early gene” response in the caudomedial neostriatum of zebra finches (Poephila guttata). Repetition of this stimulus 10 times is sufficient to induce a maximal increase in RNA and protein, detected 30 and 90 min later respectively. Thus very brief stimuli can set in motion a slow genomic process in the brain which takes hours to resolve. Immediate-early gene function is often considered in the context of a “feedback” model (i.e., to consolidate memories of the inducing event). However, based on the long lag observed here between initiation and full expression of the molecular response, we suggest an alternative, ethologically based, “feed-forward” model in which exposure to a novel or significant context triggers an increase in the efficiency of memory capture processes for subsequent experiences. q 2000 Academic Press Key Words: songbird; zebra finch; immediate-early gene; zenk; zif-268; egr-1; consolidation; caudal neostriatum.
Behavioral and perceptual activities can induce transient increases in gene expression in various brain regions. This phenomenon is often referred to as the immediate-early gene (IEG) response and is widely assumed to contribute to the consolidation of memories of the inducing event (reviewed in Tischmeyer & Grimm, 1999). However, its significance for normal brain function is not yet clear. Although a few studies have demonstrated IEG responses in freely behaving animals following natural stimuli (Jarvis, Schwabl, Ribeiro, & Mello, 1997; Jin & Clayton, 1997), most investigations have used intense or highly artificial laboratory-based paradigms, often of long duration, to induce the response (e.g., Work supported by an NSF Graduate Fellowship (A.K.) and NIH Grant R01 MH52086. We thank Dr. Telsa Mittelmeier and Karen Pilcher for assistance with immunoblotting and immunohistochemistry and Bridget Carragher and Steve Rogers (Beckman Imaging Technology Group) for assistance with quantitative microscopy. Correspondence and reprint requests to David F. Clayton, B107 Chemical and Life Sciences Laboratories, 601 S. Goodwin Ave., Urbana, IL 61801. Fax: (503) 213-6611. E-mail: [email protected]. 179
1074-7427/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
180
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Tischmeyer & Grimm, 1999). Prior research into the IEG response thus leaves mostly unaddressed a fundamental question: What is the minimal amount of behavioral experience necessary to induce the IEG response? A clearer definition of the temporal relationship between circumstances that lead to IEG induction and the time course of the IEG response itself would help in developing a physiological model of IEG function during ongoing brain activity. We therefore sought to establish the minimal temporal stimulus necessary and sufficient for zenk gene induction in a natural model of the phenomenon, adult zebra finches responding to tape-recorded song playbacks (reviewed in Clayton, 1997, 2000a). Adult zebra finches (n 5 18, Magnolia Bird Farms) were housed for at least a month in the Beckman Animal Care facility prior to use in experiments. All procedures involving animals were conducted with the guidance and formal approval of the Illinois Laboratory Animal Care Advisory Committee. Prior to each experiment, birds were individually isolated for 24 h in an anechoic chamber (birds that produced any spontaneous song during the final hour were not used in experiments). Tape-recorded stimuli (70 dB) were then played over a loudspeaker in the chamber, in one of five different stimulation paradigms. Birds (n 5 4) hearing the “13” stimulus were played one presentation of a single tape-recorded zebra finch song, ,2 s in duration. The “triple song” group (n 5 2) heard three different songs presented without separation for a single 15-s period. The “103” (n 5 4) and positive control (n 5 4) groups heard a single song repeated once every 10 s for a total of 10 or 180 presentations, respectively. Silent controls (n 5 4) were not presented with a song stimulus. Birds were sacrificed 30 min after the onset of song playback to allow time for induced mRNA to accumulate following the time course for gene expression established in previous studies (Mello & Clayton, 1994). zenk mRNA was detected in fixed brain slices using digoxygen-labeled antisense RNA probes (Jin & Clayton, 1997), and the density of positively labeled cells was determined using an automated cell counting system (Stripling et al., submitted). Figure 1 shows representative hybridizations from each of the five conditions. Cells positive for zenk mRNA accumulation appear as darkly stained points at this magnification. The fields shown represent sections in the parasagittal plane, taken at a medial level containing the region of greatest zenk responsiveness to song (Mello & Clayton, 1994). The level of staining is similar inside and outside the NCM/CMHV region in birds in the silent control group (Fig. 1C), whereas birds in the positive control group (Fig. 1B) contained a much greater density of labeled cells within NCM/CMHV compared to surrounding brain. A high level of labeling similar to the positive control is seen in sections from birds who heard only 10 presentations of song (Fig. 1F), and increased labeling relative to the surrounding brain is evident in birds in the 13 and triple song groups (Figs. 1D and 1E, respectively). Figure 2 presents quantification of these results, expressed as the density of labeled cells in NCM/CMHV. Relative to nonstimulated controls, zenk expression increased about eightfold in birds hearing 10 song repetitions (P , .01). This increase was equivalent to that produced by 30 min of repeated song stimulation (Fig. 2). The triple song stimulus resulted in a sixfold increase relative to silence, slightly but significantly less than the response to 10 songs or 30 min of song (P , .05 and P , .01, respectively). Presentation of even a single song resulted in an increase in cell labeling that was significant relative to nonstimulated controls (P , .02). These results indicate that a detectable increase in
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FIG. 1. Representative zenk in situ hybridizations for experimental conditions. (A) Line drawing of the relevant anatomical regions in the zebra finch forebrain. Abbreviations correspond to hippocampus (HP), caudomedial neostriatum (NCM), and caudomedial hyperstriatum ventrale (CMHV). Size bar 5 1.0 mm, magnification for all images 5 203. (B) In situ hybridization from the 30-min song presentation condition. Staining is absent in the hippocampus, but is evident throughout NCM and CMHV. (C) The silent control condition yields minimal staining in all areas of the forebrain. (D) In the 13 condition staining is light throughout the hippocampus, but is apparent in CMHV and dorsal NCM. (E) The triple song condition shows increased labeling throughout the NCM/CMHV region. (F) The 103 condition exhibits dense labeling throughout all of NCM/CMHV.
zenk mRNA results from a single song presentation and maximal activation is achieved by as little as 10 repetitions of a single song stimulus over a 2-min period. To determine whether the zenk protein was also increased following such brief stimulation, tissue was collected from the 103 (n 5 2), negative control (n 5 2), and positive control (n 5 2) conditions. The birds were presented with song and then sat in silence until 90 min had lapsed from the beginning of song presentation. Whole-cell extracts were prepared from the NCM tissue of individual birds by dounce homogenization [in 20 mM HEPES (pH 7.9), 100 mM NaCl, 0.5 mM EDTA, 0.7% (v/v) NP-40, 0.5 mM DTT, 10% (w/v) glycerol, plus a cocktail of protease inhibitors]. These extracts were analyzed by immunoblotting with an antiserum used in previous songbird studies (Mello & Ribeiro, 1998). As seen in Fig. 3, an inducible band is evident at ,64 kDa in all three conditions. The silent controls have very low levels of signal as compared to the 103 and 30-min conditions. When this antiserum was used to probe perfusion-fixed sections of song-stimulated zebra finch brains (data not shown), the anatomical pattern of immunocytochemical labeling after the 103 stimulus closely followed the pattern of zenk mRNA labeling and was specific to NCM. Thus a very brief perceptual experience, hearing 2 s of birdsong, is sufficient to induce the immediate-early gene zenk in the zebra finch NCM. Furthermore, a maximal level of zenk RNA was achieved by as little as 20 s of song over a 2-min period. These increases
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FIG. 2. Changes in zenk labeling with increasing song exposure. Positively labeled cells were identified using criteria described elsewhere (Stripling et al., submitted). Bars filled with distinct patterns are statistically different (Kruskal–Wallis test). A single presentation was sufficient to induce zenk over silent controls (P , .02). The triple song stimulus also produced significant induction (P , .01), yet not to levels as high as the 30-min positive control (P , .01). In contrast, 10 presentations of a single song induced zenk to levels not significantly different from the positive control (P . .20).
in mRNA were accompanied by increases in zenk protein levels in NCM detected 90 min after stimulus onset. Previous reports have documented the avian response to birdsong using a much longer stimulus, typically several hundred seconds of song over a 30-min period (Mello, Vicario, & Clayton, 1992; Mello & Clayton, 1994; Jarvis et al., 1997; Mello & Ribeiro, 1998). These results provide evidence that the IEG response is engaged during normal dayto-day brain function and not simply reserved for extremes of metabolic function or stressful circumstances. Other experiments also indicate that induction of zenk and other IEGs is a common, presumably daily, occurrence in freely behaving animals. Free-ranging song sparrows in the wild show a zenk response in NCM to tape-recorded birdsongs presented via loudspeaker on their home territories (Jarvis et al., 1997), and birds singing spontaneously at dawn show zenk activation in telencephalic nuclei of the vocal control system (Jin & Clayton, 1997). Cells positive for zenk and/or c-fos can be detected throughout the rodent brain even in animals under “baseline” control conditions (Cirelli, Pompeiano, & Tononi, 1996; Campeau et al., 1997; Rosen et al., 1998). The functional significance of the genomic response to experience is not well understood—roles in memory consolidation and/or cellular homeostasis have been widely considered (Tischmeyer & Grimm, 1999). The results here reveal a dramatic temporal dissociation between the initiation of the process (within a few seconds) and its fulfillment (hours before induced protein levels have returned to baseline). As reviewed in detail elsewhere (Clayton, 2000b), this temporal dissociation suggests another hypothesis: that increases in IEG proteins may serve to alter the system’s responsiveness to subsequent
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FIG. 3. ZENK protein levels in individual birds exposed to different stimulation conditions. (A) Western blot showing two silent controls (S), two controls hearing 30 min of one song (308), and two subjects exposed to 10 presentations of song (103). Blots were probed with an affinity-purified rabbit polyclonal antibody (Santa Cruz no. sc-189, 1:1000). Three ZENK-specific bands are evident, with the 64-kDa band (arrow) showing inducibility under these conditions. (B) MAP kinase levels from the same blot (stripped and reprobed with ap44/42 MAPK, 1:2000, New England Biolabs) indicating evenness of loading for the individual lanes. Numbers across the bottom represent relative zenk signal intensity compared to the mean for the silent controls (after normalization to MAPK signal for each lane).
events. According to this hypothesis, experiences that occur during the peak of an IEG wave might be consolidated even more effectively than memories of the inducing experience itself. REFERENCES Campeau, S., Falls, W. A., Cullinan, W. E., Helmreich, D. L., Davis, M., & Watson, S. J. (1997). Elicitation and reduction of fear—Behavioural and neuroendocrine indices and brain induction of the immediate-early gene c-fos. Neuroscience, 78, 1087–1104. Cirelli, C., Pompeiano, M., & Tononi, G. (1996). Neuronal gene expression in the waking state: A role for the locus coeruleus. Science, 274, 1211–1215. Clayton, D. F. (1997). Role of gene regulation in song circuit development and song learning. Journal of Neurobiology, 33, 549–571. Clayton, D. F. (2000a). Neural basis of avian song learning and perception. In J. Bolhuis (Ed.), Brain, perception, memory: Advances in cognitive neuroscience. New York: Oxford Univ. Press. Clayton, D. F. (2000b). The genomic action potential. Neurobiology of Learning and Memory, 74, 185–216. Jarvis, E. D., Schwabl, H., Ribeiro, S., & Mello, C. V. (1997). Brain gene regulation by territorial singing behavior in freely ranging songbirds. NeuroReport, 8, 2073–2077. Jin, H., & Clayton, D. F. (1997). Localized changes in immediate-early gene regulation during sensory and motor learning in zebra finches. Neuron, 19, 1049–1059.
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Mello, C. V., & Clayton, D. F. (1994). Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system. Journal of Neuroscience, 14, 6652–6666. Mello, C. V., & Ribeiro, S. (1998). Zenk protein regulation by song in the brain of songbirds. Journal of Comparative Neurology, 393, 426–438. Mello, C. V., Vicario, D. S., & Clayton, D. F. (1992). Song presentation induces gene expression in the songbird forebrain. Proceedings of the National Academy of Sciences U.S.A., 89, 6818–6822. Rosen, J. B., Fanselow, M. S., Young, S. L., Sitcoske, M., & Maren, S. (1998). Immediate-early gene expression in the amygdala following footshock stress and contextual fear conditioning. Brain Research, 796, 132–42. Tischmeyer W., & Grimm, R. (1999). Activation of immediate early genes and memory formation. Cellular and Molecular Life Sciences, 55, 564–574.