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All auditory stimuli were presented using custom-designed software (Roy Stripling, LabView programming environ- ment, National Instruments, Inc.) for a Power ...
Development of Song Responses in the Zebra Finch Caudomedial Neostriatum: Role of Genomic and Electrophysiological Activities Roy Stripling,1 Amy A. Kruse,1,2 David F. Clayton1,2 1

Beckman Institute, University of Illinois, Urbana, Illinois 61801

2

Neuroscience Program, University of Illinois, Urbana, Illinois 61801

Received 16 January 2001; accepted 2 April 2001

ABSTRACT:

Zebra finches first form demonstrable memories of specific songs between 25 and 35 days of age—several days after fledging from the nest. What accounts for the late onset of specific song memory formation? Here we investigated physiological development of the caudomedial neostriatum (NCM), part of the avian analogue of auditory cortex and a probable component of the system involved in song perception. Two types of physiological responses were characterized: electrophysiological (single-unit spike rate) and genomic (induction of the immediate early gene zenk, also known as zif-268, egr-1, ngfi-a, krox-24). We found that by day 20, zebra finches already have robust electrophysiological responses in NCM to song stimulation. Spike activity was greater in response to conspecific songs compared to

INTRODUCTION Songbirds are studied as a model for how complex sensory and motor patterns are learned and represented in specific brain circuits (see Brenowitz et al., 1997, and associated reviews). Mechanistic analyses typically focus on song copying in males—the process by which young birds develop their song by listening to a tutor and correcting their own vocal performance. These studies have defined the “song control system,” a set of interconnected brain nuclei found only in oscine songbirds. These nuclei have a Correspondence to: D.F. Clayton ([email protected]). Contract grant sponsor: NIH; contract grant number: RO1 MH52086. Contract grant sponsor: NSF (A.K.). © 2001 John Wiley & Sons, Inc.

heterospecific songs, white noise, or tones, and ⬇10% of the units showed selective responses to forward versus reversed songs. In contrast, at this age the zenk gene is expressed at a constitutively high level and undergoes no further induction in response to song presentation. At day 30, electrophysiological responses remained similar, but the zenk gene began to shift from a constitutive to an inducible pattern of expression. These results are consistent with a general role for NCM in the representation of song auditory patterns, and with a role for zenk gene expression in governing the efficiency of specific song memory storage at different ages. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 163–180, 2001

Keywords: zebra finch; zenk; immediate early gene; electrophysiology; juvenile

clear and primary role in the motor control of song production, as deduced from electrophysiological and anatomical studies, and from the effects of lesion (e.g., Nottebohm et al., 1976; Bottjer et al., 1984; McCasland, 1987; Yu and Margoliash, 1996). The neural systems responsible for sensory representations of songs are less well defined. Although the song control nuclei show some auditory responses, many of these responses are evident only in anesthetized birds (Capsius and Leppelsack, 1996; Dave et al., 1998; Schmidt and Konishi, 1998). Furthermore, the song control nuclei are underdeveloped in female zebra finches, who nonetheless perceive and discriminate among songs (Miller, 1979; Clayton, 1988; Nixdorf-Bergweiler, 1996). Recently, however, a forebrain region distinct from the song nuclei has emerged as a plausible site for 163

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sensory representations of birdsong. This region was initially identified through studies of immediate-early gene (IEG) responses to tape-recorded birdsong, and includes the caudomedial neostriatum (NCM; Mello et al., 1992; Mello and Clayton, 1994). In addition to robust and selective genomic responses to conspecific songs, NCM neurons also increase their electrophysiological spike activity selectively in response to complex auditory stimuli (Leppelsack, 1983; Mu¨ller and Leppelsack, 1985; Chew et al., 1995; Stripling et al., 1997). NCM receives robust input from field L2a, the avian equivalent of primary auditory cortex, but which shows neither electrophysiological preference for song (Mu¨ller and Leppelsack, 1985) nor songinduced gene responses (Mello and Clayton, 1994). NCM’s projections may innervate at least two song control nuclei: HVC [via the underlying shelf (Vates et al., 1996)] and Area X [via the paraHVC region contained within NCM (Foster and Bottjer, 1998)]. In the context of the avian song model, the potential identification of a structure involved in sensory representations of songs raises an immediate question about how its function may change during juvenile development. In zebra finches of both sexes, behavioral studies suggest that long-lasting song-specific memories begin forming between 25 and 35 days of age (Immelmann, 1969; Arnold, 1975; Clayton, 1988; Bo¨hner, 1990). Do NCM’s electrophysiological responses also emerge on this developmental time course, or are they already in place in younger birds? A previous study suggested that the genomic response to song stimulation was absent in zebra finches at d20 (posthatch day 20), but present by d30 (Jin and Clayton, 1997). What is the developmental relationship between spike production, genomic responsiveness, and the formation of stable song memories? To explore these issues, we characterized single-unit electrophysiological responses in NCM to song in d20 and d30 zebra finches, and compared these to the patterns of genomic activation at the same ages.

METHODS Use of Unanesthetized, Restrained Birds for Electrophysiology To date, all gene induction studies in NCM have been performed using awake, unanesthetized zebra finches. Previous electrophysiological studies attempting to correlate recorded NCM neuronal activity with these genomic responses made use of unanesthetized, restrained animals. To justify the comparison of our results to this body of work, we carried out experiments on juvenile zebra finches that were unanesthetized and restrained as well. We saw no evidence that any aspect of the procedures provoked exces-

sive distress or discomfort and the experiments were carried out under a protocol approved by the University of Illinois Laboratory Animal Care Advisory Committee.

Animal and Surgical Procedures for Electrophysiology Juvenile zebra finches [age ⫽ 20 days, n ⫽ 14 (seven male and seven female); and age ⫽ 30 days, n ⫽ 13 (seven male and six female)] were bred and raised in colonies at the University of Illinois Beckman Institute animal care facility. Two days before each electrophysiological experiment, the subject was anesthetized with 3– 4 mL/kg of a pentobarbital/ chloral hydrate cocktail similar in composition to Equithesin. A small hole was opened over the stereotaxic coordinates for NCM (from 0.7 to 1.2 mm anterior to the bifurcation of the central sinus; 0.3 mm wide and centered on 0.25 mm lateral to the central sinus), the hole was covered with sterile bone wax, and a stainless-steel head post was attached to the skull with dental cement (Grip Cement, L. Caulk Co.). Each bird was returned to its parents’ care and allowed to recover for at least 40 h before the recording session. On the day of electrophysiological recording, the subjects were restrained in a cloth jacket and placed in a custom stereotaxic apparatus (H. Adams, Caltech Central Engineering). NCM was located by its stereotaxic coordinates. Each subject was isolated in the dark, in a tabletop anechoic chamber (Tracor, Inc.). A microelectrode (glass or lacquercoated platinum/iridium, FHC, Inc.) was lowered into the brain with a hydraulic microdrive (FHC, Inc.). Each experiment lasted 6 to 8 h. Subjects were monitored for signs of stress, but typically remained very still throughout the experiment, and were offered water from an eyedropper every 1 to 1.5 h. Recent analysis of electrophysiological responses to auditory stimuli in other regions of the zebra finch telencephalon (HVC and RA) has revealed that the responses may be greatly enhanced when the animals fall asleep. We did not continuously monitor the sleep/wake state of the animals in our study and this remains a caveat to the interpretation of our results. However, the door to the anechoic chamber was opened on almost an hourly basis to offer water to the bird and during these times the birds were awake. Auditory responses observed while the door was open or shortly afterward did not appear diminished or in any other way different than at other times during the experiment. Thus, although we cannot rule out effects of the animals’ sleep/ wake state in our experiments, we did not see evidence that such factors were of significant influence on our results.

Auditory Stimuli All auditory stimuli were presented using custom-designed software (Roy Stripling, LabView programming environment, National Instruments, Inc.) for a Power Macintosh 8500. Auditory stimuli were selected to facilitate comparison to previous studies of zenk and electrophysiological responses to song by NCM neurons. Whenever possible, the

Response Development in Zebra Finch NCM stimuli used were the same as those used in Stripling et al. (1997), or were designed to match their acoustic characteristics. Song stimuli were obtained from the collection of Dr. Susan Volman, or were recorded from adult birds in our aviary (Beckman Institute). The white noise burst was 2 s long and included 50 ms rise and fall times. In the ascending tone set, each tone burst was 300 ms in length with 50 ms rise and fall times and 300 ms intertone intervals. This stimulus stepped upward in 500 Hz increments, with the first tone at 1.5 kHz, and the last at 4.0 kHz. A second ascending tone set, beginning at 1.75 kHz and ending at 4.25 kHz was used as a search stimulus (see below). At each unit, test stimuli included four novel conspecific songs (chosen from a collection of ⬇30 songs), one of these songs played backwards (reversed song), one heterospecific song [a white crowned sparrow song—the same heterospecific song stimulus used in Stripling et al. (1997)], the ascending tone set, and the white noise burst. All stimuli were presented at an average sound pressure level of 70 dB as measured from the location of the bird’s head. Each stimulus was presented once every 10 s, for 10 consecutive presentations, but in a different order at each recording site.

Characterization of Responses to Auditory Stimuli Neural activity was amplified with an AC amplifier (XCell3, FHC, Inc.). Auditory responsive units in NCM were discriminated on-line with a window discriminator (slope/ height window discriminator, FHC, Inc.). Two arbitrarily chosen conspecific songs and one ascending tone set (stepping from 1.75 to 4.25 kHz) were used to search for units. These conspecific songs were not used later in experimental trials unless very few or no other songs tested were able to elicit a response. Systematic recordings of responses to a stimulus set were made wherever a single, responding unit could be isolated [n ⫽ 56 in d20 juveniles (36 in males and 20 in females); n ⫽ 64 in d30 juveniles (38 in males and 26 in females)]. The timing of unit action potentials was stored on the computer to an accuracy of 1.0 ms. Once a suitable site was identified, the single unit activity was recorded during presentation of a variety of stimulus types as described above. Electrophysiological activity was measured by recording the number of spikes produced during the 1 s of silence before each stimulus onset (basal activity) and during the stimulus presentation itself (see Figs. 1 and 2 for reference). Each stimulus was presented for a block of 10 consecutive repetitions. The summed response to these 10 playbacks was used to create a peristimulus time histogram (PSTH), which represented that unit’s response for that stimulus. Qualitative discriminations in response characteristics can be identified from the PSTHs (e.g., Figs. 1, 2, and 3). Quantitative comparisons of response magnitudes for different stimuli (Fig. 4) were based on the mean spike frequency (spikes per second) during 10 consecutive presentations of each stimulus. At each unit we measured the response to four novel conspecific songs (selected from a collection of ⬇30 songs), and used the mean spike rate for

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these four songs as that unit’s response to conspecific song. To control for differences in the level of basal activity and to limit the influence of one or a few very active units, we normalized each unit’s stimulus response, as reported in Stripling et al. (1997). Briefly, each unit’s basal activity rate (b) was subtracted from the spike frequency generated during the stimulus presentation (a), and then this value was divided by their sum.

(response_index) ⫽





共a ⫺ b兲 共a ⫹ b兲

(1)

This resulted in a response index whose values are constrained between ⫾1, and where values greater than zero indicate net excitation , values less than zero indicate net inhibition, and values close to zero indicate little change from basal activity. In our quantitative analysis of responses to auditory stimuli, the ascending tone stimulus was assayed over its entire duration rather than independently quantifying each tone within the stimulus. This was done to facilitate comparisons of tone responses to the other auditory stimuli used in this study, and because similar sequential tone stimuli have been used as a negative control stimulus in other studies of the zenk gene response in NCM (Mello et al., 1992; Jin and Clayton, 1997). In those studies, such tone stimuli did not induce the zenk gene. Electrophysiological habituation was quantified using the same methods reported in Stripling et al. (1997) for “response modulation.” In this analysis, we first calculated the total spike production during each individual presentation of a stimulus, then divided this value by the stimulus length to produce a presentation-by-presentation spike rate. Because spike rates can vary 30-fold across the population of units, the time course responses were normalized to facilitate population comparisons. For each unit, the initial response to a specific stimulus was divided into itself and all subsequent response values for that stimulus and then multiplied by 100. This yielded a “percent initial response” time course for each unit’s response to each stimulus (for example, see Fig. 5). For conspecific songs, where responses to four novel songs were recorded at each unit, a unit mean time course for conspecific song was first calculated by averaging the individual conspecific song time courses together, then normalizing as above.

Statistical Methods for Electrophysiology In all statistical analyses, probability levels of below 0.05 were considered necessary to demonstrate significance. Normalized mean response magnitudes (presented in Fig. 4) were first tested for their difference from the basal activity rate. In this case, one-sample Student’s t tests were made within each specific age and stimulus group (e.g., response to conspecific song by NCM neurons of d20 birds vs. the associated basal activity rate, which is normalized to zero using the response index defined above). Next, we analyzed this data set for differences related to age, sex, and stimulus type using a

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Figure 1 Typical response patterns of juvenile zebra finch NCM units during presentation of white noise and ascending tone bursts. In each panel, a raster plot (top), peri-stimulus time histogram (PSTH; middle), and the stimulus amplitude waveform (bottom) are shown aligned in time. Time shown is seconds from stimulus onset. (A) and (B): Responses by a single NCM unit from a d20 (A) and d30 (B) juvenile zebra finch during 10 consecutive presentations of the white noise burst. (C) and (D): Responses by a single NCM unit from a d20 (C) and d30 (D) juvenile zebra finch during 10 consecutive presentations of the ascending tone set (see Methods for description of this stimulus). split-factor ANOVA with two between group factors (age and sex) and one within group factor (stimulus type). When statistical differences were detected, pair-wise posthoc contrasts were run (as recommended by Kirk, 1992) to determine where the specific differences were located. To test for significant levels of habituation in the presentation-by-presentation time courses (Figs. 5 and 6), a split-factor ANOVA with two between group factors (age and sex) and two within group factors (stimulus type and repetition number) was employed. Again, when statistical differences were detected, appropriate pairwise posthoc contrasts were run (Kirk, 1992) to determine where the specific differences were located. Two-sample Stu-

dent’s t tests were employed where comparisons were made between the present set of juvenile data and the adult data set first presented in Stripling et al. (1997). Such comparisons were made for the response magnitudes of the heterospecific songs, and for the initial rate of response habituation during conspecific song presentations.

Anatomical Analyses To identify recording sites, small electrolytic lesions (8 ␮A for 8 s) were made on one to three penetrations in

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Figure 2 Diversity of robust responses to song by single units in the NCM of juvenile zebra finches. (A–D): Responses to the same conspecific song by four different neurons, two from d20 [(A) and (C)] and two from d30 [(B) and (D)] birds. In each panel, a raster plot (top), and a peri-stimulus time histogram (PSTH; bottom) are shown for action potentials recorded during 10 repetitions of the song stimulus. Abscissa: time (in seconds) from stimulus onset. (E) and (F): Amplitude waveform of the song stimulus shown time-aligned with the unit responses in (A) and (C), and (B) and (D), respectively. (G): Sonogram of the song stimulus expanded for greater clarity. each animal. Images of sections containing the lesion sites were digitized using a CCD video camera system (LX-450A, Optronics Engineering) and image capturing

software (Scion Image, version 1.59, Scion Corp.) and were used to create composite maps representing the recording sites in each group of juvenile bird. Recordings

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Figure 3 Responses to forward versus reversed song by two single units in the NCM of juvenile zebra finches. Responses are shown for one unit from a d20 bird [panels (A) and (C)], and one unit from a d30 bird [panels (B) and (D)]. Each panel includes an aligned raster plot (top), PSTH (middle top), stimulus amplitude waveform (middle bottom), and a sonogram, which has been expanded for clarity (bottom). Abscissa: time (in seconds) from stimulus onset. Approximately 10% of all cells recorded in NCM of both age groups showed this type of stimulus selectivity. (A–B): Responses to a conspecific (forward) song. (C–D): Responses to the same song [(A) or (B), respectively] reversed.

Response Development in Zebra Finch NCM made outside of the boundaries of NCM were not used in any of these analyses.

zenk Induction and in Situ Hybridizations Juvenile (d20, n ⫽ 5; and d30, n ⫽ 9) and adult (n ⫽ 11) zebra finches were presented with playback of either a novel conspecific song, a heterospecific song (white crowned sparrow), or no song at all (silent controls). Prior to this, adult and d30 juvenile birds were individually isolated for 24 h in an anechoic chamber (Tracor, Inc.). d20 birds (who were not yet weaned from parental care) were isolated with their mothers in the chambers for 24 h prior to song stimulation. Songs were presented for 30 min at a rate of one 2.5 s song bout every 10 s. Immediately after the stimulation period, each subject was sacrificed. Their brains were rapidly dissected, frozen in Tissue Tek, and stored at ⫺80°C for later processing. Parasagittal sections were cut at 10 ␮m in a cryostat, thaw mounted onto TESPA-treated slides, and then fixed in 3% paraformaldehyde. Methods of in situ hybridization were the same as reported in Jin and Clayton (1997) for digoxigenin labeled tissue. We report levels of zenk mRNA expression in NCM as the number of digoxigenin-labeled cells within the NCM’s dorso-ventral and rostro-caudal boundaries (Fig. 9, see Results for justification). This approach could lead to erroneous conclusions if the densities of cells within NCM (labeled and unlabeled) were also changing during development. However, we measured total cell densities within NCM in 18 birds (six adult, six d30, and six d20) and found no differences in cell density with respect to age (Fig. 8). Cells were counted on digitized images scanned at 20X magnification using the Microcomputer Imaging Device (MCID) software system (Imaging Research, Inc.) in combination with a Zeiss Axiovert microscope and a Hamamatsu 10-bit CCD camera. The MCID system identifies all labeled targets that exceed a user-specified threshold. To determine a suitable, independent threshold, we first scanned the overlying hippocampus (which consistently shows a very low level of labeling and thus serves as an independent measure of background) and created a histogram of the pixel values in this region. We then used the value of the 99th percentile of this Poisson distribution as the threshold for positive digoxigenin label in NCM. After identifying labeled NCM targets using this threshold, the system corrects for targets with overlapping boundaries by utilizing a mean cell size, which is established by the user (without this correction, cells whose labeled signals overlapped would be counted as a single cell). We empirically found that a mean cell size between 130 to 180 ␮m2—with all targets smaller than 30 ␮m2 excluded—provided the most reliable “estimated counts.” Manual confirmation of accurate estimates for overlapping targets were made for each scanned image. This was accomplished by identifying at least 20 multiple cell targets in each image and manually counting the number of cells in that cluster and comparing our counts to those estimated by the MCID software system.

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All cell counts were conducted by an individual blind to the experimental conditions. The product of this effort was an estimated cell count per unit area, reported as the number of “labeled cells per mm2” in Figure 9(A), and used in statistical analyses of the zenk response. The Kruskal-Wallis test was used to assess significance in all of the zenk values reported. To facilitate comparison to previous related work, we also present labeled cell density values normalized to the age-matched basal expression levels (obtained from the silent controls). These values are presented in Figure 9(B) and serve as a qualitative confirmation of the validity of the cell counting approach.

RESULTS We began by characterizing the responses of NCM neurons in unanesthetized, restrained juvenile zebra finches exposed to various auditory stimuli, using single-unit recording methods as in our previous analysis of adult responses (Stripling et al., 1997). This analysis of juveniles included both males and females, as at d20 and d30 sex determination could be made only by postmortem inspection. We found no electrophysiological properties exhibiting a sex difference (see details below), and thus this report focuses on potential age differences. Previous studies of zenk expression and electrophysiological responses by NCM neurons focused on a representative set of auditory stimuli. Studies of zenk expression explored responses to a single conspecific song, a single heterospecific song, white noise bursts, and a tone stimulus made up of several simple tones presented sequentially (Mello et al., 1992; Jin and Clayton, 1997). Electrophysiological studies in adult zebra finches included these stimuli, but also measured responses of NCM neurons to several different conspecific songs, the bird’s own song, and conspecific songs played backwards (Chew et al., 1995, 1996; Stripling et al., 1997). The primary goal for the experiments reported here was to search for developmental changes in these genomic and electrophysiological responses. Consequently, we focused on auditory stimuli that were the same as or similar to those previously used to facilitate comparison of the data sets. The data presented here should, therefore, be viewed with the caveat that they do not provide a comprehensive characterization of NCM responses for all types of auditory experiences.

Description of Electrophysiological Responses in Juvenile NCM We ultimately recorded from 56 single units in d20 birds (36 in males and 20 in females) and 64 single

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Figure 4

Response Development in Zebra Finch NCM

Figure 5 Response habituation to conspecific song in d20, d30, and adult zebra finches. Data represent normalized population means for all single units responding to 10 consecutive presentations of conspecific song [at d20 n ⫽ 56 units, at d30 n ⫽ 64 units; data for adults (n ⫽ 118 units) adapted from Stripling et al., 1997]. In adults, the greatest change occurs between the first and second stimulus presentations. Even by the 10th song presentation, responses from juvenile NCM units have not matched this initial habituation observed for adults.

units in d30 birds (38 in males and 26 in females), all within the general confines of NCM as defined by the area of maximal zenk gene induction following song repetition that lies behind the lamina hyperstriatica. In both d20 and d30 juvenile zebra finches, the majority of single units changed their firing rate in response to one or more types of auditory stimuli, and most responded to a broad range of stimuli including noise bursts, multiple pure tones, and birdsongs (see Fig. 4 for detailed numerical breakdown of the units’ auditory responsiveness). Representative PSTHs illustrating typical response patterns for nonsong stimuli are shown in Figure 1. Responses to white noise bursts

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[Fig. 1(A,B)] showed maximal firing during the onset of the stimulus, followed by elevated, but diminishing excitation throughout the rest of the stimulus. In contrast, neurons that responded to tones often showed onset or offset excitation to multiple frequencies, while their firing was generally inhibited during the body of the stimulus [Fig. 1(C,D)]. The majority of single units in the NCM of d20 and d30 juvenile zebra finches responded robustly to conspecific songs. However, the patterns of action potential generation varied greatly from cell to cell. Song responses could not be readily segregated into discrete categories, as they appeared to more closely approximate a continuum of responses. This observed range of responses is illustrated by the sample PSTHs in Figures 2 and 3. At one end of the continuum, very active units generated action potentials throughout the song stimulus [Fig. 2(A,B)]. Such units would often fire during all song elements and often during pauses between these elements as well [Fig. 2(A,B)]. Nevertheless, these units did show some discrimination, firing more strongly during some song elements than during others, and firing more strongly during the song elements than during some of the pauses in between. Other robustly active units exhibited a greater selectivity for song elements over the silence between each element [Fig. 2(C,D)]. Such units typically fired intensely during each and every song element, but still showed greater excitation to some song elements than to others. At the other end of the continuum were units tightly synchronized to specific song elements or series of elements. This type of unit appeared to be sensitive to the specific temporal organization or dynamic frequency characteristics of the stimulus, as is illustrated by the PSTHs of the two units in Figure 3. These units fired robustly during selected elements of the song stimuli when it was played normally [Fig. 3(A,B)]. However, when the song was played backwards, little or no response above basal activity could be detected [Fig. 3(C,D)]. Of the 120 single units

Figure 4 Distribution of response magnitudes to various auditory stimuli. The histograms show the number of units (ordinate) plotted against the normalized index of their rate of firing (abscissa) (see Methods) during 10 presentations of conspecific songs (top row), reversed conspecific songs (second row), the heterospecific song (third row), white noise bursts (fourth row), and the ascending tone set (bottom row). Results from d20 subjects are presented in the left column; results from d30 subjects are presented in the right column. Positive values of the index indicate a net increase in firing during stimulus presentation, and negative values indicate a net decrease, relative to basal activity. The mean of the response index ⫾S.E.M. are shown in each panel. Asterisks denote response index that differs significantly from basal activity (i.e., differs significantly from a response index value of zero); in each case p ⬍ .001 (one-sample Student’s t test).

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Figure 6 Assessment of habituation to other auditory stimuli. (A) Data show responses to repeated presentations of conspecific song and other auditory stimuli, for all 120 units recorded from juveniles. Only conspecific song responses show a consistent habituating response time course. (B) Probability values (ordinate) plotted against the split-factor ANOVA posthoc, pair-wise comparisons, indicated on the abscissa. Differences between the first stimulus presentation and any subsequent presentations are considered significant when they fall below ␣ ⫽ 0.05 (dashed line). Changes from their initial response are significant for conspecific song by the third presentation, for the heterospecific song at the fourth, and seventh through tenth presentations, for reversed conspecific song in the fifth and tenth presentations, and for the white noise burst in the ninth presentation only.

sampled in both d20 and d30 birds, 13 units exhibited both this level of element selectivity and preference for the forward orientation of the song. Because these

units had low basal rates and were generally selective for a restricted set of auditory stimuli, they were difficult to detect. The true relative number of such

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units, therefore, is likely to be higher than our sample suggests.

Quantitative Analysis of Stimulus Selectivity To test for developmental changes in the magnitude or selectivity of NCM’s electrophysiological responses, we quantified the relative firing rates of NCM single units during presentations of a variety of auditory stimuli. As observed in adults (Stripling et al., 1997), we found that units in the NCM of juvenile zebra finches varied over 30-fold range in the absolute rate of spike production; therefore, to avoid overly biasing our quantitative measurements toward units with high firing rates, we report on data only from well isolated single units, and normalize these data to a combination of the basal activity rate and the response magnitude of each cell (see Methods). With this approach, we first observed that the mean population response in NCM was significantly different from the basal activity for stimuli, including forward and reversed conspecific songs, heterospecific song, and white noise bursts (Fig. 4; p ⬍ .001 for each, one-sample Student’s t test). There was a small effect of age on the weak response to the tone stimulus; at d20 responses to the ascending tone set were not different from the normalized basal activity (p ⬎ .10, one-sample Student’s t test), whereas d30 birds exhibited a modestly enhanced activation relative to their basal rate (p ⬍ .001, one-sample Student’s t test). The mean population response in NCM did not differ significantly by age (comparing d20 and d30) for forward and reversed conspecific songs, heterospecific song, or for white noise bursts (Fig. 4; d20 vs. d30: forward conspecific songs p ⫽ .956; reversed songs p ⫽ .931; heterospecific song p ⫽ .729; and white noise p ⫽ .159, split-factor ANOVA). There were no sex-based differences in the magnitudes of electrophysiological responses for any stimulus or age (p ⬎ .10, split-factor ANOVA). There was a slight but significant difference in the tone response between birds at d20 and d30 (index ⫽ 0.030 ⫾ 0.021 at d20 vs. 0.104 ⫾ 0.026 at d30; p ⫽ .01, split-factor ANOVA). At both ages, however, we did observe a significantly greater response to conspecific songs than to heterospecific songs, white noise, or the ascending tone set (p ⬍ .001 in all cases, split-factor ANOVA). In our previous study, using the same conspecific and heterospecific stimuli, we observed no preference for conspecific over heterospecific songs in adults (Stripling et al., 1997). In those experiments, the heterospecific stimulus elicited a mean response index of 0.395

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⫾ 0.039, whereas here in the juveniles, we recorded a mean response index of 0.278 ⫾ 0.021; this difference is significant (p ⫽ .004, two-sample Student’s t test). Thus, juvenile NCM neurons exhibit an adult-like selectivity for songs over simpler auditory stimuli, but also show a greater relative preference for conspecific over heterospecific songs.

Habituation of Electrophysiological Responses The results so far demonstrate that NCM neurons in d20 birds have electrophysiological responses to song that are as robust and at least as selective as d30 birds—the age where zenk induction is first observed. We next set out to determine whether juvenile NCM neurons also exhibit the property of response habituation. This property was previously observed in adult NCM neurons, where it was the electrophysiological response property most strongly correlated with zenk induction (Stripling et al., 1997). First, we analyzed responses to consecutive repetitions of the same conspecific song, quantifying the rate of spike production (Fig. 5). Mean response intensities recorded during playback of four different novel conspecific songs were calculated on a presentation-by-presentation basis for each cell (with each individual song being presented for 10 consecutive presentations). These results were then normalized by dividing by the initial mean response for each cell to generate a percent initial response value for each presentation. The normalized data were then combined to calculate the population mean time courses for d20 juveniles and d30 juveniles. In juvenile birds of both ages, habituation of electrophysiological responses during conspecific song presentation was observed (Fig. 5), but there were no sex or age differences in this response property (p ⬎ .60, split-factor ANOVA). The rate of habituation in juveniles was notably less, however, than in adults. Stripling et al. (1997) used the difference between response intensities during the first and second song presentations as a simple measure of the habituation. In that study, the initial decline in adults was 15.31 ⫾ 2.21%, whereas, in the juveniles, we observed an initial habituation rate of only 4.54 ⫾ 1.00% (p ⬎ .001, two-sample Student’s t test). Although habituation was less pronounced in juveniles than in adults, this dynamic aspect of the response was nevertheless specific for song, as shown by the analysis in Figure 6. Figure 6(B) graphically depicts the corresponding probability significance values (␣, or type I probability) that result from pairwise, posthoc comparisons of the first response versus each subsequent response, for each type of stimulus.

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Figure 7 zenk mRNA labeling in juvenile and adult zebra finches. Representative parasagittal sections (0.25 mm from the midline) after in situ hybridization with digoxigenin labeled ZENK antisense RNA probe. Adult and d30 juveniles were tested with conspecific and heterospecific songs, d20 juveniles were tested with conspecific song, and all age groups were compared to unstimulated controls (silence). Schematic of the visible anatomical features is shown in the center left panel. Hp, hippocampus; NCM, caudomedial neostriatum; CMHV, caudomedial hyperstriatum ventrale.

Habituation was more apparent and consistent in juveniles for forward conspecific song than for any other stimulus, including reversed conspecific song or heterospecific song. This is somewhat different from adults, in which both conspecific and heterospecific songs induced a similar rate of habituation (Stripling et al., 1997).

Electrophysiological Versus Genomic Response Development In contrast to the lack of electrophysiological differences observed here between d20 and d30 juveniles, conspecific song-induced zenk gene expression has been reported to differ at these ages: an inducible

response was not observed at d20, but began to emerge by d30 (Jin and Clayton, 1997). To confirm this difference, and to test further for the stimulus specificity of the genomic response to song, we quantified the zenk gene response to various stimuli in d20, d30, and adult zebra finches. For these experiments, d30 and adult birds were isolated from all other birds for 24 h, while d20 juveniles, who were not yet weaned from parental care, were isolated for 24 h with their mothers (who do not sing). After song playback (or silence for an equivalent amount of time), the subjects were sacrificed and parasagittal sections of NCM were analyzed for zenk mRNA expression by in situ hybridization (Fig. 7). Total cell densities (Fig. 8) and densities of positively labeled

Response Development in Zebra Finch NCM

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Figure 8 Assessment of overall cell density at different ages. Cell counts were made from a uniform field in each of three regions within NCM [n ⫽ 6 birds for each age (d20, d30, and adult)]. Cell density was calculated as the total number of neurons within each field as revealed by Cresyl violet staining, and was then plotted as a function of local region and developmental age. No changes in the density of cells for any subregion occurred as a result of development (p ⬎ .10 in each case, Kruskal-Wallis test).

cells [Fig. 9(A)] in NCM were established using an automated cell counting system (see Methods). The rationale for this quantification method is based on prior observations that individual cells are recruited in an “all or none” fashion (Mello et al., 1992; Jin and Clayton, 1997). Thus, a mean threshold for positive labeling of individual cells can be established and variations in cell labeling intensities above this threshold ignored. We confirmed that there were no confounding developmental changes in total cell density for any NCM region at the ages examined in this report (Fig. 8, p ⬎ .10 for each region, KruskalWallis test). Unlike the “fold-induction” measurements used in earlier studies of zenk expression, this approach does not require normalization to a basal expression level, which is now known to change with development (Jin and Clayton, 1997). However, to facilitate comparisons to the previous literature, we also show the fold induction over basal expression, normalizing each value by the mean density of labeled cells for the age-matched silent controls [Fig. 9(B)]. We also note that zenk expression is apparent in the neighboring region of CMHV, as has been reported previously for both adults and juveniles (Mello et al., 1992; Jin and Clayton, 1997). However, these previous reports quantified the zenk response only in NCM. All of the recordings analyzed in the present study were restricted to NCM as well. For these reasons, the values of zenk expression we report are likewise restricted to NCM proper. The data obtained from the adult birds in these experiments are consistent with earlier results based on autoradiographic densitometry (Mello et al., 1992; Mello and Clayton, 1994): basal expression was low

in controls exposed only to silence (mean ⫽ 107.3 ⫾ 33.5 cells/mm2), about fourfold higher for adult subjects who heard the heterospecific song (mean ⫽ 453.5 ⫾ 46.7 cells/mm2), and about twice as high again for the adult subjects who heard conspecific song (mean ⫽ 1009.1 ⫾ 181.7 cells/mm2). Differences between the conspecific response and heterospecific response, as well as between the conspecific response and silent controls, were significant (p ⬍ .002). The data obtained here from the juvenile birds also confirm the essential result of Jin and Clayton (1997): conspecific song presentation to d20 birds does not result in a detectable increase in zenk labeling in NCM, but by d30 an inducible zenk response to conspecific song presentation is emerging [Fig. 9(A,B)]. Moreover, the basal expression of zenk in d20 birds exposed only to silence was very high (884.9 ⫾ 105.7 cells/mm2), not statistically different (p ⬎ .20) from the levels achieved in d30 juveniles and mature adults only after song stimulation [Fig. 9(A)]. Indeed, the emergence of an inducible zenk response from d30 to adulthood is attributable solely to a decline in the basal expression level [Fig. 9(A)], and not to any increase in the maximal level following stimulation. This report also includes the first look at zenk responses to a heterospecific song in juvenile zebra finches. At d30, the age at which an inducible response to conspecific song is first evident, juvenile NCM neurons also exhibit a zenk response following playback of the heterospecific song. The induction is significantly different from birds kept in silence [p ⬍ .05; Fig. 9(A)], but is not different from birds hearing the conspecific song (p ⬎ .20). We also note

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Figure 9 Quantification of zenk mRNA response in NCM. An automated cell counting procedure (see Methods) was used to determine the density of digoxigenin-labeled cells in NCM from juvenile (d20 and d30) and adult zebra finches. (A) Absolute value of labeled cell density (per mm2). Note that the basal level of zenk expression (i.e., silent control) at d20 does not differ substantially from conspecific song-induced levels at d30 or adulthood. Also note the developmental decline in basal and heterospecific song-induced levels. (B) Same data normalized by dividing each value by the labeled cell density of the age-matched silent control, to yield a “fold induction” value, as used in prior X-ray film autoradiographic analyses. Note that either method shows no induction at age d20, an emerging response by d30, and a robust and selective response to conspecific song by adulthood. Con, conspecific song; Hetero, heterospecific song.

that the absolute number of cells responding to the heterospecific song was higher at d30 than in adults [p ⬍ .05; Fig. 9(A)]. Thus, with maturation, zenk gene expression in NCM comes to be more and more specifically associated with the sound of conspecific song.

DISCUSSION The electrophysiological data reported in this study represent the first observations made in the NCM for

any juvenile songbird. As such, we have made some attempt to provide a qualitative description of the auditory responses present in this region. The primary focus of this report, however, was to search for possible developmental changes in the electrophysiological and genomic responses that have previously been observed in the adult zebra finch NCM. Therefore, interpretation of the current data set was largely focused on such factors, and on placing the observed physiological responses in the larger context of their function in the behaving animal.

Response Development in Zebra Finch NCM

Significance of Electrophysiological Responses in NCM Electrophysiological responses to song and other auditory stimuli are as robust and complex in the juvenile NCM as previously observed in adults. As early as d20, NCM neurons exhibit response patterns to songs that range from broadly excitatory to highly selective (Figs. 2 and 3). Just like the adult NCM, the mean magnitudes of the juvenile responses in NCM show a clear preference for conspecific song stimuli relative to noise and tone bursts (Fig. 4). In addition, we find that at least 10% of neurons in the juvenile NCM exhibit a marked preference for forward versus reversed versions of conspecific songs (for example, see Fig. 3), while the mean population response to both forward and reversed song are equivalent (Fig. 4). Preference for the forward conspecific song stimulus may indicate sensitivity to frequency modulations or temporal order. It is also a common property of auditory responses in neurons of the song nuclei (Margoliash, 1986; Doupe and Konishi, 1991; Volman, 1993; Doupe, 1997). Chew et al. (1996) found no general preference for forward over reversed song in the NCM of adult zebra finches; however, it is not clear if they also looked for a forward song preference among any subpopulation of NCM neurons. Our previous study of adult NCM responses did not include a reversed song stimulus; however, we note that approximately 10% of the neurons showed a high degree of selectivity (Stripling et al., 1997), comparable to what we report here for the forward preferring neurons in juveniles. It is interesting to note that the proportion of neurons preferring forward song to reversed song in the juvenile NCM is comparable to that found in the juvenile song nuclei (Volman, 1993; Doupe, 1997). Such comparisons must be made with caution, however, as responses in the juvenile song nuclei were recorded from anesthetized subjects, whereas our data were collected from unanesthetized birds. Recent evidence indicates that the auditory responses of the song nuclei may be greatly diminished in awake, unanesthetized adult subjects (Capsius and Leppelsack, 1996; Dave et al., 1998; Schmidt and Konishi, 1998). Thus, more rigorous comparisons between NCM and song nuclei responses to song by juveniles must wait for song nuclei recordings to be made in unanesthetized individuals. Not all of the electrophysiological response properties of the juvenile NCM are identical to the adult. Where there are age-related departures in the auditory responses of NCM neurons, however, they are relatively subtle. We find that juvenile NCM neurons

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respond to the heterospecific song with less overall intensity than adult NCM neurons. While we tested responses to only one heterospecific song (a white crowned sparrow song), we note that this is the same stimulus used in our previous study of adult NCM responses. We also note that the response magnitudes for all other auditory stimuli tested remain constant across all of these ages. Taken together, these results suggest that juvenile NCM neurons have a more selective preference for conspecific songs than do adults. There are two possible explanations for this: it is either due to tuning properties intrinsic to the ascending auditory pathway; or it is the result of a top-down influence (e.g., attention), which might be more selective for conspecific song at these early ages. Although the mean intensity of responses to conspecific songs does not differ between juveniles and adults, we find that the rate of habituation for conspecific song responses does. The habituation rate in juveniles is noticeably slower than the adult rate (Fig. 5). It is tempting to speculate that the rates of response habituation in both juveniles and adults reflect the influence of arousal mechanisms. Juvenile zebra finches hearing a conspecific song may simply be aroused longer than adults, and thus their response intensities decline more slowly. Evidence exists for catecholaminergic innervation of the caudal neostriatum (Mello et al., 1998), which could mediate an arousal based enhancement of responses. Such a process could also account for the apparent preference for conspecific songs over the heterospecific song just described. It would be interesting to see whether or not the catecholaminergic systems are indeed active in NCM during conspecific song playback, and whether they discriminate between conspecific and heterospecific songs in juveniles but not in adults.

Delayed Emergence of an Inducible zenk Response Even though song stimulation induces robust electrophysiological responses in zebra finches at d20, it does not cause an increase in zenk gene expression in NCM neurons at this age. On the other hand, zenk levels in NCM are already so high in the unstimulated birds at this age as to be indistinguishable from the song-induced levels in older birds. This is consistent with either of two mechanistic hypotheses: either the induction mechanism is still undeveloped, or some other mechanism has already driven the gene to its maximal level of expression at this age. In either case it is interesting to note that this developmental effect is at least somewhat specific for NCM, as we did not observe noticeably higher levels of basal expression

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in the overlying hippocampus. We note that birds at d20 were in a slightly different social circumstance (isolated for 24 h with their mothers prior to song playback) than were the older birds, and perhaps this contributed to their higher levels of basal zenk expression. However, basal zenk levels were also elevated in d30 birds, suggesting that high basal zenk expression is inherent in young juvenile zebra finches, independent of recent auditory or social experience. Jin and Clayton (1997) also reported elevated basal levels of zenk in d20 and d30 juveniles compared to adults, although in their study the highest level was observed at d30. Evidence for developmental regulation of IEG activities has also emerged from studies of other systems in which functional plasticity is limited to a critical period, in particular the neonatal period for development of binocular inputs to the visual cortex in mammals (Mower, 1994; Kaplan et al., 1995; Kaczmarek et al., 1999). In kittens, brief visual experience elicits a transient induction of the c-fos IEG in layer IV of the visual cortex (Mower, 1994), and in mice monocular deprivation (which induces cortical synaptic rearrangement) causes the activation of calciumand cAMP-dependent signal transduction pathways involved in IEG induction (Pham et al., 1999). In both these cases the inducible response is greater during the critical period than after it. Evidence also exists for a constitutive enhancement of IEG levels during the critical period (Beaver et al., 1993; Kaplan et al., 1995), perhaps more analogous to the high expression described here for juvenile zebra finch NCM. The induction of zenk following novel song presentation is a phenomenon that continues well into adulthood, however, and likely supports a process that continues to function into adulthood as well. In zebra finches of either sex, behavioral recognition of individual songs is not apparent at d25, but is emerging by d35 (Clayton, 1988), and continues well into adulthood (Miller, 1979). Additionally, young male finches acquire auditory models for their own song development beginning at a similar age, between d28 and d35 (Bo¨hner, 1990; Morrison and Nottebohm, 1993). Thus, the zenk gene undergoes a change in its pattern of regulation in NCM that approximately coincides in time with a fundamental change in the way birds respond behaviorally to song. The functional significance of this correlation is not yet clear. Review of the literature on zenk and other “immediate early” genes suggests the general hypothesis that high levels of expression increase the rate or efficacy of synaptic consolidation processes (Clayton, 2000). It is tempting to speculate that the high basal expression observed in young birds marks a period of open or relatively indiscriminant sensitivity to all acoustic pat-

terns encountered. This might support the development of a rich perceptual repertoire, but might also make the bird more susceptible to “memory overwriting”—such a phenomenon has been described in young male zebra finches (Slater et al., 1991; Jones et al., 1996). The eventual decline in basal zenk expression might help stabilize existing memories and restrict the synaptic modification process to specific, significant experiential contexts (Clayton, 2000). This model predicts that experimental manipulations leading to reduced zenk expression in young birds should stabilize song memories that would otherwise have been overwritten. Conversely, manipulations resulting in increased constitutive zenk expression in adult birds should destabilize new song memories.

What Role for NCM in Song Learning? Our data reveal a correlation between the onset of long-term song-memory formation and the shift to an inducible zenk response to song in NCM. These observations could be taken to suggest that long-term auditory memories are stored in NCM. For several other reasons however, we favor the hypothesis that the IEG-mediated neural plasticity resulting from song playback is directed towards the presynaptic projections of NCM. First, we note that NCM’s broad electrophysiological response properties (Leppelsack, 1983; Mu¨ller and Leppelsack, 1985; Chew et al., 1996; Stripling et al., 1997), and the similarity of adult and juvenile responses as shown here both suggest that long-term memories are not encoded in the underlying circuitry of NCM itself. Second, Chew et al. (1996) found that NCM neurons in adult zebra finches habituated in a stimulus specific manner to a large number of novel conspecific songs, but the habituated responses did not persist for longer than 2–3 days. Third, we also note that in adult zebra finches, assays of electrophysiological (Chew et al., 1996; Stripling et al., 1997) and zenk responses (Mello, 1993) in NCM fail to distinguish the song most familiar to the bird, the bird’s own song, from other novel conspecific songs. Bolhuis et al. (2000) found a correlation between the number of song elements incorporated from a tutor song and the strength of the zenk response to that song. However, in that study the zenk response was assayed far more laterally than in any other study of the zenk response in zebra finches, in a region of the caudal neostriatum that is a probable down-stream target of NCM proper (Vates et al., 1996). Furthermore, that study failed to control for cause and effect: was it a memory of the tutor song that correlated with the strength of induction, or was it the case that birds with a stronger zenk response were the better learners?

Response Development in Zebra Finch NCM

The emergence of the inducible zenk response to song occurs at a time coincident with the opening of the sensory phase for song learning in this species. It is tempting to speculate that NCM’s auditory representations are utilized in the acquisition of a sensory template for song learning. NCM’s downstream targets appear to include regions associated with song learning, such as the shelf of HVC (Vates et al., 1996) and Area X (Foster and Bottjer, 1998). The observations presented here, indicating that auditory responses in NCM are already robust and mature by d20, are consistent with such a role. For neural systems analysis, the interesting challenge will be to determine how NCM’s representations of specific auditory experiences (such as the sound of a tutor’s song) are selectively communicated to these other neural systems to elicit appropriate behavioral responses. We thank Bridget Carragher and Steve Rogers of the Beckman Institute Microscopy Suite for training and assistance with the MCID system.

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