Song-Induced ZENK Gene Expression in Auditory Pathways of Songbird Brain and Its Relation to the Song Control System
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The Journal of Neuroscience, November 1994, 14(11): 6652-6666 Song-induced ZENK Gene Expression in Auditory Pathways of Songbird Brain and Its Relation to the Song Control System Claudio V. Mello’ and David F. Clayton2 ‘Laboratory of Animal Behavior, The Rockefeller University, New York, New York 10021 and *Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801 The ZENK gene encodes a zinc-finger-containing transcrip- Marler and Peters, 1977; Welty and Baptista, 1988; Godard, tional regulator and can be rapidly activated in songbird brain 1991). Considerableprogress has been achieved in defining the by presentation of birdsong (Mello et al., 1992). Here we map neural substrate of normal singing behavior by using a combi- the areas of the songbird forebrain that show this genomic nation of brain lesions,electrophysiological recordings and an- response to birdsong, using in situ hybridization. After 30 atomical tract-tracing techniques. A seriesof distinct intercon- min of song presentation ZENK mRNA levels reach a peak nected brain nuclei is necessaryfor normal song production in the caudomedial telencephalon, in areas adjacent to or (Nottebohm et al., 1976, 1982); these include a neostriatal nu- closely related with primary auditory structures. These areas cleus,HVC (high vocal center), an archistriatal nucleusto which include subfields of field L (Ll and L3), the caudomedial HVC projects (RA or robustus archistriatalis), and RA’s pro- neostriatum (NCM), the caudomedial hyperstriatum ventrale jections onto the motoneurons of the XII cranial nerve that (CMHV) anterior to field L, the caudal paleostriatum, and two innervate the syrinx, the vocal organ of songbirds.An alternative field L targets, HVC shelf and RA cup. In contrast, ZENK pathway leading from HVC to RA includes a seriesof paleo- induction is absent in some areas that show a response to striatal, thalamic, and neostriatal nuclei, and seemsto be es- song by other measures and where ZENK induction might sential for songlearning, but not necessarilyfor songproduction have been expected. These include the direct thalamo-re- in adults (Bottjer et al., 1984; Sohrabji et al., 1990; Scharff and cipient field L subfield L2, and the nuclei of the circuit in- Nottebohm, 1991). Various of these “song control nuclei” un- volved in the acquisition and production of learned song. dergo anatomical changesassociated with the development of These results demonstrate that ZENK induction following song production in juveniles (Konishi and Akutagawa, 1985; song presentation occurs only in a subset of areas physi- Nordeen and Nordeen, 1988) and with subsequentseasonal ologically activated by song, and draw attention to areas changesin singing behavior in speciessuch as the canary (Not- previously unsuspected as related to processing of complex tebohm, 198 1, 1989; Nottebohm et al., 1986; Alvarez-Buylla auditory stimuli. Based on what is known about ZENK func- et al., 1990). tion in mammalian systems (Christy et al., 1989; Cole et al., Despite considerableprogress in understandingsong produc- 1989; Wisden et al., 1990), we speculate that areas revealed tion, relatively little is known about the neural circuitry re- by ZENK induction might correspond to sites where critical sponsible for song perception and discrimination, or for the neuronal modifications occur in response to birdsong pre- formation of memoriesrelated to the songsthe bird hears.An- sentation, possibly leading to the formation of song-related atomical studieshave revealed that the ascendingauditory path- memories. ways in birds share many homologies with mammals and in- [Key words: song control circuit, birdsong, immediate-ear- clude the midbrain nucleus MLd (homolog of the mammalian ly gene, ZENK, field L, NCM] inferior colliculus) and the thalamic nucleus Ov (homolog of the medial geniculate; seeKarten, 1967, 1968). Projectionsfrom Ov then reach the telencephalonthrough the primary auditory Songbirds have emerged, in the last 3 decades,as a powerful projection area, field L (Karten, 1968; Kelley and Nottebohm, system to study the mechanismsby which experience leads to 1979). However, the anatomy and function of higher auditory the modification of behaviors. Birds must hear and remember processingstations in the avian telencephalon is poorly under- songsfrom other individuals in order to perform various tasks stood. Recent studies of field L cytoarchitectonic organization essentialfor their survival and reproduction, such as songleam- in songbirds, for example, reveal that it is a complex structure ing, territorial defense,and courtship behavior (Kroodsma, 1976; consisting of various subdivisions whose specific connections and functions are not yet well defined (Fortune and Margoliash, 1992a). Metabolic studies and electrophysiological recordings received Nov. 16, 1993; revised Apr. 19, 1994; accepted Apr. 27, 1994. have demonstrated that units in the avian field L have a ton- We gratefully acknowledge the unfailing support and continual inspiration pro- vided by Fernando Nottebohm over many years; without his generous and in- otopic organization and show low selectivity to complex audi- quisitive spirit, this marriage of molecular biology and neuroethology never would tory stimuli (D. Bonke et al., 1979; Mtiller and Leppelsack, have proceeded. We also thank Fernando Nottebohm for providing birds from 1985; Mtiller and Scheich, 1985); auditory responseswith a the Rockefeller University Field Research Center, with partial support of a grant from the National Institutes of Health (DCO0182). Specific grant support for the higher selectivity for complex stimuli have been described in experiments presented here was provided to D.F.C. by the Whitehall Foundation areasadjacent to and receiving an input from field L, for instance and the NIH (NS25742). the caudomedial HV (Mtiller and Leppelsack, 1985). Correspondence should be addressed to Claudio V. Mello, Laboratory ofAnimal Behavior, The Rockefeller University, 1230 York Avenue, New York, NY, 1002 1. Initial studies on field L connectivity in songbirdsrevealed Copyright 0 1994 Society for Neuroscience 0270-6474/94/146652-l 5$05,00/O projections to the shelf and cup areasimmediately adjacent to The Journal of Neuroscience, November 1994, f4(11) 6653 nuclei HVC and RA, respectively (Kelley and Nottebohm, 1979) suggesting a pathway for auditory information to reach the nu- clei that control song production. Because these nuclei are an- atomically well defined and have an obvious function in song behavior, several studies have been directed at testing the hy- pothesis that these song control centers also have a role in song perception and discrimination. From the work of a number of individuals, it is now apparent that selective electrophysiological responses can be evoked by the sound of specific songs at various levels throughout the song control pathway, all the way to the syringeal musculature (Katz and Gurney, 1981; Margoliash, 1983, 1986; Williams and Nottebohm, 1985; Doupe and Kon- Figure 1. Northernblot analysisof the ZENK gene. Shown is an x-ray ishi, 1991; Margoliash and Fortune, 1992; Vicario and Yohai, film autoradiogramof a blot containingsize-fractionated RNA isolated 1993). The identification of these auditory responses confirms from the forebrainof canaries(A andB) or zebrafinches (C), hybridized that the song control pathway receives auditory input and may to a 32P-labeledsingle-stranded antisense riboprobe for the ZENK gene. be tuned to specific song patterns. However, it does not yet A contains30 wug,of total RNA, and B and C contain 3 ueof polyA+ RNA, final washconditions are 0.1 x SSPE,0.1% SDS’it 65°C(hy- establish a functional role for these responses, nor does it es- bridization protocolas described in Claytonet al., 1988). tablish whether the response specificity arises within the song control pathway or in other auditory processing centers that may ultimately impinge upon the song control pathway. striatum (NCM), ZENK gene activation seemsto be specific to More suggestive evidence that the song control nuclei have a song stimulation, in that conspecific songselicit a greater re- function in song perception and behavioral modification has sponsethan heterospecific songs, and pure auditory tones do been provided by Brenowitz (1991) who showed that lesions not causeit (Mello et al., 1992). This responseappears to be directed at HVC will disrupt the selectivity of the behavioral specifically linked to perceptual processesas opposedto motor response of females to conspecific song. However, these “HVC” activity, as it occurs independent of song production by the lesions were not limited to HVC proper but also included sur- birds that hear the song.Although the functional significanceof rounding neo- and hyperstriatum, areas that could also be in- the ZENK responseis yet unknown, it neverthelessprovides a volved in auditory processing and perception (B. A. Bonke et way to map sites in the brain that undergo a physiological re- al., 1979; Kelley and Nottebohm, 1979; Saini and Leppelsack, sponseto a song stimulus. Since the areaswhere we detect this 1981; Fortune and Margoliash, 1992; Mello et al., 1992). Fur- responsediffer from the areas one might have expected based thermore, behavioral responses by females to conspecific song on previous studies, the present methodology may highlight can be elicited in species in which females lack a complete song heretofore unappreciated aspectsof the organization of the song control