We investigated the participation of genomic regulatory events in the response of the songbird brain to a natural auditory stimulus of known physiological and behavioral relevance, birdsong. Using in situ hybridization, we detected a rapid increase in forebrain mRNA levels of an immediate-early gene encoding a transcriptional regulator (ZENK; also known as zif-268, egr-1, NGFI-A, or following presentation of tape-recorded songs to canaries (Sefinus canaria) and zebra finches (Taeniopygia guttata). ZENK induction is most marked in a forebrain region believed to participate in auditory processing and is greatest when birds hear the song of their own species. A signifkantly lower level of induction occurs when birds hear the song of a different species and no induction ls seen after exposure to tone bursts. Cellular analysis indicates that the level ofinduction reflects the proportion of neurons recruited to express the gene. These results suggest a role for genomic responses in neural processes linked to song pattern recognition, discrimination, or the formation of auditory asocations.Songbirds hear the song of other individuals of their species and respond by modifying their own vocal and social behavior (1)(2)(3)(4)(5) Study of brain areas related to normal singing behavior has revealed a specialized brain circuit that includes a welldefined series of sexually dimorphic nuclei essential for song production (6)(7)(8). This circuit ultimately controls the output of motoneurons of the XII nerve, which then project to muscle fibers of the syrinx, the vocal organ of songbirds. Compared to areas involved in the motor control of song, however, brain areas related to perceptual aspects such as analysis and storage of complex auditory patterns are still poorly defined. A primary auditory area of the avian forebrain, field L, has been described based on the projection from the thalamic auditory relay, nucleus ovoidalis (9, 10). In songbirds, conspecific song is known to be an effective stimulus for neurons in field L and is the preferred stimulus in parts of the song control circuit (11-15). However, the detailed connectivity of auditory areas and the pathway(s) that conveys information from primary auditory areas to song-selective brain areas, such as parts of the song control circuit (13-15), remains to be determined. Similarly, the molecular and cellular consequences of exposure to song are still largely unknown as is the mechanism(s) by which these responses may ultimately translate into physiological and behavioral changes.We have begun to investigate these issues by focusing on genes that respond rapidly to various signals associated with neural activity and growth (16)(17)(18)(19)(20) , here referred to by the acronym "ZENK". METHODSSong Exposure. Twenty-four adult male songbirds of two different species, zebra finches (Taeniopygia guttata) and canaries (Serinus canaria) in the spring, were obtained from Canary Bird Farms (Englishtown, NJ) and from closed colonies maintained at the Rockefeller University ...

Earlier work showed that playbacks of con- Songbirds use their songs and calls to communicate with members of their own species. Male songbirds typically learn these vocalizations from adult conspecifics during a sensitive period in development. These learned songs and calls can differ markedly between individuals, though they also share species-typical features (1-3). Much is known about the brain pathways birds use for the production of learned vocalizations (4, 5), but our understanding of circuits that discriminate and store these signals is just beginning to develop (6, 7). One forebrain area that may play a role in perceptual aspects of vocal communication is the caudo-medial neostriatum (NCM), where playback of conspecific song is more effective than heterospecific song or non-song sounds in inducing the expression of the immediate early gene ZENK (also known as zif-268, egr-1, NGFI-A, or Krox-24) (8, 9). The ZENK response in NCM disappears when the same song is played back repeatedly, but a full ZENK response is elicited again if a novel song is presented (10). ZENK is one of a group of immediate early genes that encode transcriptional regulators thought to mediate the long-term effects of depolarization on neural activity and thus to play a role in memory formation (11)(12)(13)(14).These observations suggest that NCM is part of a system that processes and memorizes species-specific sounds.In the present study we recorded the electrophysiological responses of NCM neurons to conspecific and heterospecific song in the presence or absence of protein or RNA synthesis inhibitors. We describe a long-lasting decrease in responsive-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ness of NCM neurons to repetitions of the same song stimulus and suggest that this may be a good system in which to study the cellular and molecular basis of perceptual learning.* METHODS Experimental Animals and Surgical Procedures. Forty adult male zebra finches (Taeniopygia guttata) obtained from our breeding colony or from a local supplier were used. Under anesthesia, birds were surgically prepared for recording. Twenty-four to 48 h later, the awake-animals were restrained for recordings and microinjections. Recording sessions typically lasted 4-5 h, conforming to an approved animal use protocol. At the end of each experiment, small electrolytic lesions were made for use in locating the recording sites. The birds were then killed and the brains were processed for histology. For all procedural details, see ref. 15.Auditory Stimulation. The songs of 50 male zebra finches and 5 canaries from our archives were digitized at 20 kHz (Signal, Engineering Design, Belmont, MA) to provide a set of conspecific and heterospecific songs that the birds had never heard before. The zebra finch songs were 1.2-2.0 s long and contained introductory notes and one or two motifs. The canary...

Auditory responses in the caudomedial neostriatum (NCM) of the zebra finch (Taeniopygia guttata) forebrain habituate to repeated presentations of a novel conspecific song. This habituation is long lasting and specific to individual stimuli. We here test the acoustic and ethological basis of this stimulus-specific habituation by recording extracellular multiunit activity in the NCM of awake male and female zebra finches presented with a variety of conspecific and heterospecific vocalizations, white noise, and tones. Initial responses to conspecific song and calls and to human speech were higher than responses to the other stimuli. Immediate habituation rates were high for all novel stimuli except tones, which habituated at a lower rate. Habituation to conspecific calls and songs outlasted habituation to other stimuli. The extent of immediate habituation induced by a particular novel song was not diminished when other conspeeific songs were presented in alternation. In addition, the persistence of habituation was not diminished by exposure to other songs before testing, nor was it influenced by gender or laterality. Our results suggest that the NCM is specialized for remembering the calls and songs of many individual conspecifics.Vocal communication using songs and calls is an important part of social and reproductive behavior in many songbird species (1). Songbirds learn their songs (and in some cases calls) from conspecific tutors, but include minor variations; as a result, these vocalizations have an acoustic form that is specific to each individual. We have previously shown that playbacks of novel conspecific songs elicit vigorous auditory responses in neurons in the caudomedial neostriatum (NCM) of the zebra finch (Taeniopygia guttata) brain. These responses habituate with repeated presentations of the same song, and the habituation persists for up to 48 h (2). We have now tested the specificity of these responses by using a larger variety of stimuli, presented in various temporal combinations to both male and female zebra finches. The results show that the initial activity elicited by noise, tones, and various conspecific or heterospecific vocalizations does not predict whether or not repeated exposure to any of these signals will produce longterm habituation. In addition, long-term habituation remains specific for individual songs and calls even when the bird is exposed to large numbers of similar stimuli that could potentially interfere with this form of memory.

In both humans and songbirds, infants learn vocalizations by imitating the sounds of adult tutors with whom they interact during an early sensitive period. Vocal learning occurs in few animal taxa; similarities in the imitation process between humans and songbirds make the songbird a unique system in which vocal learning mechanisms can be studied at the neurobiological level. One theory of vocal learning proposes that early auditory experience generates auditory memories that subsequently guide vocal imitation. We now present a combination of behavioral and neurophysiological results, obtained in a songbird, that support this theory. We show that neurons in a forebrain auditory area of adult male zebra finches are selectively tuned to the song of a tutor heard early in development. Furthermore, the strength of this selectivity shows a striking correlation with the fidelity of vocal imitation, suggesting that this auditory memory may have served as the model for song learning.development ͉ electrophysiology ͉ memory ͉ caudal medial nidopallium ͉ zebra finch