They’re Playing Our Song
Authors: Gregory F Ball, Timothy Q Gentner
Journal: Neuron (1998)

Neuroethology, a hybrid discipline straddling the behavioral and neural sciences, grew out of the field of ethology, the biological study of animal behavior. Neuroethology has benefited from the application of new technical and conceptual breakthroughs derived from other subdisciplines of neuroscience; the use of these new approaches has, in turn, produced principles of general significance to the field of neuroscience. Recent studies of immediate-early gene (IEG) induction in relation to birdsong perception are an excellent illustration of this cross-fertilization process. One of the important legacies of ethology to behavioral neuroscience is the experimental documentation of the selectivity of perception. Based on experiments with many taxa, it has become clear that animals attend to only the particular stimulus attributes needed to make adaptive behavioral responses. In a variety of sensory systems, one finds neurons with tuning characteristics directly related to the behavioral relevance of a signal (see 10Konishi M Neural Comput. 1991; 3: 1-18Crossref Google Scholar, for a general review of these phenomena). For example, barn owls (Tyto alba) possess specialized sensory systems to detect and process the interaural time and intensity differences in acoustic signals for prey localization. Several species of electric fish (genus Eigenmannia) possess highly specialized neural circuits to process the phase and amplitude information in conspecific electric signals. Likewise, it is becoming clear that songbirds also possess specialized neural circuitry for the production and perception of birdsong. This minireview will focus on studies of IEG expression in relation to the elucidation of neural systems underlying birdsong perception. Birdsongs are species-typical vocal signals that are generally more elaborate than calls and are used in the context of courtship and mating. The term birdsong is often limited to such vocalizations produced by species in the songbird order (order passeriformes). Unlike most vocalizations produced by nonhuman animals, these songs are learned based on auditory experiences early in ontogeny. Decades of work by ethologists have established that songs are powerful releasers of behavioral responses in conspecifics and that specific acoustic attributes of song can elicit aggressive challenges from males and sexual responses from females (Reviewed by 1Ball G.F Hulse S.H Am. Psychol. 1998; 53: 37-58Crossref PubMed Scopus (71) Google Scholar). Detailed studies of two commonly studied songbird species, zebra finches (Taeniopygia guttata) and canaries (Serinus canaria) have revealed a vocal control circuit underlying these behavioral responses. Many nuclei in this circuit are unique to songbird species and thus represent part of the neural specialization that has evolved in association with the ability to learn, produce, and perceive these complex species-typical vocalizations. A key nucleus in this circuit is HVc (sometimes referred to as the "high vocal center"). This nucleus projects to another forebrain nucleus RA (the robust nucleus of the archistriatum) to form part of a motor pathway that lesion studies demonstrate is required for song production (Figure 1). Electrophysiological studies in the zebra finch brain provide evidence that these motor pathways are hierarchically organized: populations of neurons in HVc appear to code for song syllables (large units within songs), while RA neurons code for song notes (smaller units within song syllables; 13Margoliash D Neuron. 1997; 19: 963-966Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Still other brain areas are involved in song perception: auditory information is critical for song learning, needed for the maintenance of song, and obviously essential for complex song perception. As one would expect, auditory pathways in the songbird brain are interconnected with the song control circuit (Figure 1) and also show evidence of hierarchical organization. For example, the sensitivity to local temporal structures in the perception of conspecific song increases substantially between the primary auditory projection region in the telencephalon (field L) and the song control nucleus HVc (see Figure 1) where neurons are tuned exclusively or preferentially to a bird's own song (13Margoliash D Neuron. 1997; 19: 963-966Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Based on the work of David Clayton and colleagues, starting in the 1980s, a number of genes expressed as messenger RNA in the songbird forebrain were cloned (reviewed by 4Clayton D.F J. Neurobiol. 1997; 33: 549-571Crossref PubMed Scopus (75) Google Scholar). Although no genes were found that were specific to the song control system, several recognized classes of IEG transcription factors were identified in songbirds (4Clayton D.F J. Neurobiol. 1997; 33: 549-571Crossref PubMed Scopus (75) Google Scholar). One IEG in particular, named ZENK (15Mello C.V Vicario D.S Clayton D.F Proc. Natl. Acad. Sci. USA. 1992; 89: 6818-6822Crossref PubMed Scopus (517) Google Scholar) because it is homologous to genes cloned in other species (ZENK is an acronym for zif-268, egr-1, NGFI-A, and krox-24), has been found to be regulated specifically in response to conspecific song in the songbird brain. The initial observation was that the ZENK mRNA and protein is expressed at high levels relatively rapidly (i.e., in less than 1 hr) in the auditory telencephalon in response to conspecific song (15Mello C.V Vicario D.S Clayton D.F Proc. Natl. Acad. Sci. USA. 1992; 89: 6818-6822Crossref PubMed Scopus (517) Google Scholar, 14Mello C.V Ribeiro S J. Comp. Neurol. 1998; 393: 426-438Crossref PubMed Scopus (200) Google Scholar). Expression is especially high in areas of the auditory telencephalon such as NCM and cHV, regions not previously identified as specifically involved in song perception. ZENK expression is also high in other areas such as the HVc "shelf" and the RA "cup," areas that were previously thought to receive auditory inputs specific to song (see Figure 1). Further studies showed that the ZENK response is specifically tuned to novel conspecific song. ZENK expression is basal in response to simple tones, but it is twice as high in response to conspecific as opposed to heterospecific song (15Mello C.V Vicario D.S Clayton D.F Proc. Natl. Acad. Sci. USA. 1992; 89: 6818-6822Crossref PubMed Scopus (517) Google Scholar). Furthermore, repeated presentations of the same conspecific song lead to a diminution of the ZENK response (i.e., habituation), while the response to a different conspecific song is still observed (16Mello C.V Nottebohm F Clayton D.F J. Neurosci. 1995; 15: 6919-6925PubMed Google Scholar). The ZENK response to song is dependent on early experience; zebra finches raised in social isolation do not exhibit this response (8Jin H Clayton D.F Neuron. 1997; 19: 1049-1059Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). It is also important to note that the ZENK response is not limited to the laboratory. The playback of song to free-living, wild song sparrows (Melospiza melodia) results in the induction of the ZENK mRNA with a magnitude and distribution in the brain similar to that observed in laboratory-housed zebra finches and canaries (7Jarvis E.D Schwabl H Ribeiro S Mello C.V Neuroreport. 1997; 8: 2073-2077Crossref PubMed Scopus (51) Google Scholar). IEG expression is known to be affected by changes in neuronal activity, although the exact relationship between IEG expression and neural activity is not well understood. Many neurons in the song circuit (i.e., areas essential for the learning and production of birdsong) exhibit increased electrophysiological activity in response to song (13Margoliash D Neuron. 1997; 19: 963-966Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). It might therefore be expected that ZENK expression would localize to the song circuit. Surprisingly, however, none of the studies of song perception identified ZENK expression in brain regions considered to be part of the song system per se. Therefore, the ZENK response, although clearly influenced by neuronal activation, does not simply map onto brain areas that exhibit enhanced physiological responses to song. However, it should be noted that IEG expression is high in areas within the song control circuit during song production. Two IEGs, fos as well as ZENK, have been found to be induced at high levels in HVc and RA, two nuclei essential for motor production, while male zebra finches are producing song (6Jarvis E.D Nottebohm F Proc. Natl. Acad. Sci. USA. 1997; 94: 4097-4102Crossref PubMed Scopus (316) Google Scholar, 8Jin H Clayton D.F Neuron. 1997; 19: 1049-1059Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Kimpo R.R Doupe A.J Neuron. 1997; 18: 315-325Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). This high induction is specifically tied to motor production, given that it even occurs when deaf birds sing. Thus, specific IEG induction can be observed in songbird forebrain in association with both the perception and the production of song. One important contribution of the ZENK induction studies outlined above has been their inspiration for several anatomical, neurophysiological, and lesion studies that have, in turn, furthered our knowledge about the organization and function of the neural circuits mediating birdsong perception. Many of the connections between the auditory system and the song system illustrated in Figure 1 were only discovered after studies of ZENK induction made it clear that the connectivity of NCM and cHV is important for our understanding of the neural basis of song perception (e.g., 20Vates G.E Broome B.M Mello C.V Nottebohm F J. Comp. Neurol. 1996; 366: 613-642Crossref PubMed Scopus (438) Google Scholar). Likewise, recent electrophysiological studies of NCM have placed further emphasis on the importance of this region for the processing of conspecific signals (2Chew S.J Mello C Nottebohm F Jarvis E Vicario D.S Proc. Natl. Acad. Sci. USA. 1995; 92: 3406-3410Crossref PubMed Scopus (217) Google Scholar, 3Chew S.J Vicario D.S Nottebohm F Proc. Natl. Acad. Sci. USA. 1996; 93: 1950-1955Crossref PubMed Scopus (199) Google Scholar, 19Stripling R Volman S.F Clayton D.F J. Neurosci. 1997; 17: 3883-3893PubMed Google Scholar). Neurons throughout NCM show a robust initial response to the presentation of conspecific vocalizations that quickly falls off upon subsequent presentations of the same song. Thereafter, although repeated presentation of a single song continues to elicit significant responses from single cells (relative to spontaneous firing rates), the initial modulation in the firing rate of these cells disappears and is observed again only upon presentation of a novel song. This link between the electrophysiological response properties of neurons in NCM and stimulus-specific variation among songs has led to the hypothesis that NCM is an important site for individual vocal recognition (3Chew S.J Vicario D.S Nottebohm F Proc. Natl. Acad. Sci. USA. 1996; 93: 1950-1955Crossref PubMed Scopus (199) Google Scholar). Another area exhibiting high ZENK expression in response to conspecific song is cHV (see Figure 1). Recent studies of female zebra finches provide a possible behavioral correlate to this expression by suggesting that an intact cHV is required for the discrimination of conspecific from heterospecific song (12MacDougall-Shackleton S.A Hulse S.H Ball G.F Neuroreport. 1998; in pressGoogle Scholar). Following electrolytic lesions to HVc, females maintained preferences (as measured by their sexual display rate) for conspecific as opposed to heterospecific song. However, following lesions of cHV, females performed courtship displays at a high rate in response to both conspecific and heterospecific song. Thus, an intact cHV, a nucleus involved in the processing of auditory information, but not HVc, appears necessary for female zebra finches to exhibit normal song preferences. Studies of IEG expression in the forebrain of songbirds and the subsequent investigations inspired by them have greatly increased our knowledge of the structure and function of the song perception pathways, particularly with regard to species recognition. However, a wealth of field data demonstrates that birdsong functions primarily as a communication mechanism within species, and so it follows that songbird perceptual systems have adapted to attend to variation among conspecific songs. Thus, while it is an important first step to establish that the brains of songbirds respond differentially to conspecific and heterospecific songs, our understanding of the selectivity of perception exhibited by birds and other species is contingent upon our ability to measure changes in brain and behavior that are correlated with relevant variation among conspecific vocalizations. Current neuroethological studies of song perception focus upon at least two critical questions. First, what are the physical parameters of song that elicit or allow for predictable changes in functionally relevant (adaptive) behavior? Second, how is the information provided by variation among these functional parameters represented in the nervous system? Two recent advances demonstrate the progress that contemporary neuroethologists have made in addressing these questions. 5Gentner T.Q Hulse S.H Anim. Behav. 1998; in pressGoogle Scholar have demonstrated how behaviorally relevant variation among the syllabic structure of conspecific songs can be closely examined in the context of individual vocal recognition, and work on ZENK expression in canaries by 18Ribeiro S Cecchi G.A Magnasco M.O Mello C.V Neuron. 1998; 21 (this issue,): 359-371Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar reported in this issue of Neuron demonstrates a novel method for quantitating the neuronal representations of song syllables. Individual vocal recognition, or the ability to associate specific songs with specific singers, is an important behavioral adaptation for the manipulation and maintenance of territorial boundaries and female mate selection (see 11Kroodsma D.E Miller E.H Ecology and Evolution of Acoustic Communication in Birds. Cornell University Press, Ithaca, NY1996Google Scholar). Although nearly every species of songbird studied to date is capable of recognizing individual conspecifics by their song(s), the specific acoustic features of song that birds use for recognition are not yet well understood. Some species rely on relatively simple frequency cues to discriminate among the songs of neighboring conspecifics (and heterospecifics), while others, such as European starlings (Sturnus vulgaris), rely on the perception of more complex acoustic patterns. Male starlings present their songs in long elaborate bouts made up of sequentially patterned shorter syllables, and the syllables in one bout may or may not overlap with the syllables in other bouts from that same bird. Using operant conditioning techniques, 5Gentner T.Q Hulse S.H Anim. Behav. 1998; in pressGoogle Scholar have demonstrated that both male and female starlings can readily learn to discriminate among the songs of several different males, and that these songs are categorized by starlings on the basis of individual identity. This recognition capability is robust and is maintained under a variety of stimulus conditions, even when the subjects are presented with novel song bouts from familiar singers. This ability for starlings to recognize individual conspecifics on the basis of their vocalizations is mediated primarily by the memorization of the specific syllables in an individual singer's repertoire and by the statistical patterning of the sequences in which those syllables are sung (5Gentner T.Q Hulse S.H Anim. Behav. 1998; in pressGoogle Scholar). What is the neural basis for such discrimination? One of the most important recent advances in the study of the neural basis of song perception has been the discovery of neurons tuned to a bird's own song (13Margoliash D Neuron. 1997; 19: 963-966Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Contemporary research in this area has focused on the ontogenetic development and response properties of these neurons. However, comparatively little is known about the neural correlates that underlie the perception of conspecific song in adults. 18Ribeiro S Cecchi G.A Magnasco M.O Mello C.V Neuron. 1998; 21 (this issue,): 359-371Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have made a significant advance in filling this gap in our knowledge by the discovery of an important step in the transformation of an acoustic signal into a functional neural representation. They have employed sophisticated pattern analysis methods to investigate the collective behavior of large populations of neurons that are responsive to canary song syllables. 18Ribeiro S Cecchi G.A Magnasco M.O Mello C.V Neuron. 1998; 21 (this issue,): 359-371Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have demonstrated that the spatial distribution and immunocytochemical labeling intensities of ZENK-expressing neurons in the brains of canaries hearing conspecific song are closely correlated with variation among the syllables in male canary songs. Moreover, in contrast to more "classical" organization schemes well documented for auditory forebrain regions in other species, where neurons are organized tonotopically and respond best to a specific fundamental frequency, the ZENK expression patterns elicited by various narrow band syllables (i.e., whistles) are not replicated by presenting pure tone stimuli that simply match the fundamental frequencies of these conspecific song syllables. Thus, the spatial organization of neuronal populations in NCM observed by 18Ribeiro S Cecchi G.A Magnasco M.O Mello C.V Neuron. 1998; 21 (this issue,): 359-371Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar is not just another example of simple tonotopy, but instead appears to correlate primarily with variation in conspecific song. This is significant in that it provides compelling evidence of a salient organization underlying populations of neurons in the auditory forebrain of birds that corresponds to behaviorally relevant signals. The recent findings in the neuroethology of song perception discussed here point to important ways in which approaching the question of neural representation in songbirds has led to advances in our understanding of both the neural and the behavioral mechanisms of perception. What remains as a significant challenge is the integration of these components, and one promising possibility in this regard is to examine the functional role of ZENK expression in the context of individual vocal recognition. ZENK expression may be directly related to the categorization of classes of songs on the basis of individual identity, or the consolidation of neuronal representations into long-term memory, as suggested by work in other systems (4Clayton D.F J. Neurobiol. 1997; 33: 549-571Crossref PubMed Scopus (75) Google Scholar). By combining operant conditioning techniques—where the behavioral relevance of different songs can be tightly controlled—with ZENK immunocytochemistry, one could dissociate between IEG responses that are due to basic discrimination among different songs and/or those that are due to higher level cognitive processes such as learning. Finally, now that NCM has been identified as a site for population-level neuronal representations of song, it is important that we extend our knowledge about the real-time dynamics of this region using electrophysiology. Recent technical advances that allow for simultaneous recording from large numbers of neurons (reviewed by 17Nicolelis M.A.L Fanselow E.E Ghazanfar A.A Neuron. 1997; 19: 219-221Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), could be used to examine electrophysiological activity throughout NCM and other forebrain regions, in response to behaviorally relevant songs. For all these behavioral studies, the application of methodologies that could specifically block ZENK expression or the action of the ZENK protein would be especially welcome. The already important advances in the study of perceptual processes made by neuroethological studies will no doubt be augmented by the continued coordination and refinement of neural and behavioral methodologies.