DOI: 10.5061/dryad.ghx3ffbtt
title: Social experiences shape song preference learning independent of developmental exposure to song

description: Communication governs the formation and maintenance of social
 relationships. The interpretation of communication signals depends not
 only on the signal’s content, but also on a receiver’s individual
 experience. Experiences throughout life may interact to affect behavioral
 plasticity, such that a lack of developmental sensory exposure could
 constrain adult learning, while salient adult social experiences could
 remedy developmental deficits. We investigated how experiences impact the
 formation and direction of female auditory preferences in the zebra finch.
 Zebra finches form long-lasting pair bonds and females learn preferences
 for their mate’s vocalizations. We found that after two weeks of
 cohabitation with a male, females formed pair bonds and learned to prefer
 their partner’s song regardless of whether they were reared with
 (“normally-reared”) or without (“song-naïve”) developmental exposure to
 song. In contrast, females that heard but did not physically interact with
 a male did not prefer his song. In addition, previous work has found that
 song-naive females do not show species-typical preferences for courtship
 song. We found that cohabitation with a male ameliorated this difference
 in preference. Thus, courtship and pair bonding, but not acoustic-only
 interactions, strongly influence preference learning regardless of rearing
 experience, and may dynamically drive auditory plasticity for recognition
 and preference.
description: <em>Animals.</em> All zebra finches (n=86 females and
 24 males, average 9 months old, range 3-26 months) were maintained on a
 14:10 light:dark cycle, given ad libitum access to seed, grit, and water,
 and weekly supplements (e.g. lettuce, egg). We raised females in one of
 two conditions. One set of females (‘normally-reared’) was raised to 60
 days post hatch in a cage with both parents and siblings. A second set of
 females (‘song-naïve’) were raised in sound-attenuating chambers
 (‘soundboxes’; TRA Acoustics, Cornwall, Ontario) with only the mother and
 siblings. Specifically, fathers were removed within five to seven days
 post-hatch, prior to the period when females memorize the father’s song
 [1] and male siblings were removed at 30-40 days post-hatch, prior to
 producing stereotyped song [2]. After 60 days, normally-reared-females
 were housed either in same-sex group cages in our mixed-sex colony (n=23)
 or in same-sex group cages in an all-female colony (n=21), and song-naïve
 females (n=38) were housed in same-sex group cages in an all-female
 colony. All males used for social cohabitation or song recordings were
 normally-reared and housed in same-sex group cages in the mixed-sex colony
 prior to use. All procedures adhered to Canadian Council on Animal Care
 guidelines and the protocol approved by the Animal Care Committee of
 McGill University. <em>Cohabitation
 conditions.</em> To assess the degree to which female preference is
 shaped by specific social experiences in adulthood, females were either
 housed in a cage with a male, thus providing them with an opportunity to
 mate (‘opposite-sex paired females’), or housed in a cage with another
 female (‘same-sex paired females’) (Figure 1A). The cages containing
 opposite-sex paired (OSP) and same-sex paired (SSP) females were both
 located within the same soundbox but separated from each other by an
 opaque barrier. This meant that all birds could hear each other, but only
 see and physically interact with their cage mate. Both cages were provided
 with nesting material weekly and remained together for two
 weeks. <em>Video recording. </em>We filmed
 each group when the male was first introduced (‘Week 0’), and after the
 first (‘Week 1’) and second (‘Week 2’) week of cohabitation using a GoPro
 Hero or Hero 5 camera.  All recordings lasted for at least 2 hours and
 were recorded at the same time of day within each group. In a few cases
 (n=18) we recorded birds continuously over 2 weeks. For these birds, we
 analyzed 2h of video from the three time points listed above.
 <em>Preference testing.</em>  To assess preferences
 for male songs, females were given a two-choice active choice task as
 described previously [3]. Briefly, females were placed in a cage
 containing two strings that, when pulled, would each trigger the playback
 of song from a single male zebra finch through an adjacent speaker (e.g.,
 song of male A for one string and song of male B for the other string).
 Females were trained to associate pulling strings with sound playback
 using either conspecific and heterospecific songs or calls from unfamiliar
 females. Each test consisted of two two-hour sessions. To control for side
 bias, contingencies switched for the second session of every test. For
 both sessions, the session started once a female pulled each string three
 times. Each female was tested on at least three stimulus sets: familiar
 male vs. unfamiliar male, courtship vs. non-courtship songs from a
 familiar male, and courtship vs. non-courtship songs from an unfamiliar
 male. The order of stimulus presentation was randomized within and across
 testing days for each female. Females were tested on 1-2 stimulus sets per
 day for a maximum of five consecutive days, including acclimation time. If
 females did not complete the necessary tests after five days, they were
 housed in small same-sex groups for at least 2 days before resuming
 testing. We found that these brief pauses in testing improved motivation
 to hear song and complete the tests. Females that did not complete any of
 the three tests after two attempts each were excluded from further
 analysis (4 normally-reared and 10 song-naive). 
 <em>Song Stimuli.</em> Songs were recorded as
 previously described [4–6]. Briefly, males unrelated to the experimental
 subjects were recorded in sound attenuating chambers with omnidirectional
 microphones (Countryman Associates, California) using a sound-activated
 recordings system (Sound Analysis Pro, SAP; 44.1kHz)[7]. Unfamiliar
 females not involved in the experiment were presented to the male in a
 separate cage for brief intervals to collect female-directed courtship
 song. Songs selected for stimuli were a representative sample of the
 male’s typical variation in song duration, number of motifs, and
 introductory notes. All songs were free of noise and female calls. Each
 stimulus set consisted of 8-15 songs from each male. When females were
 tested on the songs of two different males, males with similar durations
 and numbers of motifs were selected as stimulus sets. For familiar male
 vs. unfamiliar male tests, we used a yoked design: each male’s song served
 as the familiar song for one set of females (one OSP and two SSP, see
 Cohabitation conditions), and the unfamiliar song for a different set of
 females. Therefore, the same stimulus set is used for two sets of females
 whose experience with the two males differ. All stimulus songs were
 bandpass filtered (300–10 kHz), normalized by their maximum amplitude, and
 saved as wav files (44.1 kHz) using custom written code in Matlab
 (Mathworks, Natick, MA).
 <strong>Analyses</strong>
 <em>Pair Behaviors.</em> The three 2h samples of
 video taken at Weeks 0, 1, and 2, were quantified by individuals blind to
 the rearing condition of the females. We recorded the duration (in
 seconds) and number of occurrences of a number of individual and pair
 bonding behaviors: courtship singing, non-courtship singing, clumping,
 allopreening, pecking, bill fencing, and nesting behaviors [5,8–11].
 Courtship singing by the male was defined by a display of at least two of
 the following behaviors during singing: orienting towards the female,
 fluffing of body feathers while flattening feathers on the head, and
 courtship dancing [5,6,11,12]. We calculated the percent of time spent on
 each behavior out of two hours for each pair. 
 <em>Preference tests.</em> From each test, the total
 number of playbacks for Stimulus A and Stimulus B during each session was
 determined. In addition to quantifying the number of pulls for each
 stimulus, we also performed a bootstrap with replacement (10,000
 iterations) to attain a normalized metric of preference strength
 (‘preference index’) with 95% confidence intervals (CIs). Females were
 considered to have a significant preference if the mean and 95% CIs were
 above or below the chance threshold (0.5). We used mixed-effects models
 with bootstrapped preferences as the dependent variable and cohabitation
 experience, rearing, and the interaction of experience and rearing as
 independent variables and individual ID as a random variable. For each
 rearing and cohabitation experience condition, we also tested whether the
 distribution was significantly different from chance (0.5). Finally, we
 also coded responses as either a significant preference or no preference
 based on the bootstrapped preference and CIs. Preferences were coded as 1
 (above 0.5) or -1 (below 0.5) if the CIs did not cross 0.5, and as 0 if
 the CIs did cross 0.5. We analyzed variation in the pattern of significant
 preferences across groups using nominal logistic models with rearing,
 cohabitation experience, and their interaction as independent
 variables. We investigated the correlation between
 preference strength and individual pair bonding behaviors in two ways.
 First, we performed a correlation analysis for each rearing condition and
 time point between the preference score and each of the pair bonding
 behaviors. To gain a greater sense of how combinations of behaviors relate
 to preference, we also used a principal components analysis (PCA) to
 investigate the correlations between pair bonding behaviors and partner
 preference. We ran separate PCAs for each time point (Week 0, 1, 2) and
 rearing condition and included all pair bonding behaviors and the
 bootstrapped preference score. We selected the component (in all cases,
 the first or second PC), with the highest loading of preference. We then
 compared the loading matrices for each of these components across time and
 rearing condition. All statistical analyses were completed using JMP
 Statistical Processing Software (SAS, Cary, NC, USA) or custom-written
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description: # Social experiences shape song preference learning independent of
 developmental exposure to song
 [https://doi.org/10.5061/dryad.ghx3ffbtt](https://doi.org/10.5061/dryad.ghx3ffbtt) In this study, we examined social behavior and auditory preferences in zebra finches. In the Pair Bonding dataset, we quantified the amount of time male-female cohabitating pairs spent on a variety of behaviors as a percent of time out of two hours at three timepoints (see methods). In the Preference dataset, we quantified the number of times females triggered the playback of male song in an active choice assay (pulls for Stimulus A and pulls for Stimulus B) on three test categories and calculated a normalized metric of preference strength. ## Description of the data and file structure **Pair Bonding Data Structure:** *Pair:* identification codes (leg band color and numbers) for the male and female recorded during two-weeks of cohabitation *Female ID:* identification code for the opposite-sex paired (OSP) female paired with the male for two weeks and tested on the preference tests *Rearing:* rearing experience condition for females from hatch until 60 days post-hatch (normally-reared or song-naive). Females are either raised with both parents and siblings (normally-reared), or raised with only the mother and siblings (song-naive). For the song-naive rearing condition, fathers were removed within five to seven days post-hatch, prior to the period when females memorize the father’s song, and male siblings were removed at 30-40 days post-hatch, prior to producing stereotyped song. *Housing:* housing condition for females from 60 days post-hatch until cohabitation with a male (colony or nursery). After 60 days, normally-reared-females were housed either in same-sex group cages in our mixed-sex colony (colony) or in same-sex group cages in an all-female colony (nursery), and song-naïve females were housed in same-sex group cages in an all-female colony (nursery). *Preference:* normalized metric of preference strength ('preference index') based on the number of pulls for each stimulus in the preference test for the familiar male the female was paired with vs. an unfamiliar male (between 0 and 1, where 0 is a preference for the unfamiliar male, and 1 is a preference for the familiar male). *Eggs:* whether or not eggs were observed during the two week period cohabitation (yes or not observed) For the following columns, we recorded the duration (in seconds) and number of occurrences of a number of individual and pair bonding behaviors: courtship singing, non-courtship singing, clumping, allopreening, pecking, bill fencing, and nesting behaviors. We calculated the percent of time spent on each behavior out of two hours for each pair. Each behavior has three columns that represent the three timepoints we sampled: week 0, week 1, and week 2. Example: Clumping_0 refers to the percent of time the male and female pair spent clumping during 2-hour sample on week 0 (when the male and female are first introduced). *CourtshipSong_0, CourtshipSong_1, CourtshipSong_2:* percent of time (0-100%) out of two hours that the male spent courtship singing on week 0, week 1, and week 2 *NonCourtshipSong_0, NonCourtshipSong_1, NonCourtshipSong_2:* percent of time (0-100%) out of two hours that the male spent non-courtship singing on week 0, week 1, and week 2 *Clumping_0, Clumping_1, Clumping_2:* percent of time (0-100%) out of two hours that the pair spent clumping on week 0, week 1, and week 2 *Preening_0, Preening_1, Preening_2:* percent of time (0-100%) out of two hours that the pair spent allopreening on week 0, week 1, and week 2 *Pecking_0, Pecking_1, Pecking_2:* percent of time (0-100%) out of two hours that the pair spent pecking on week 0, week 1, and week 2 *Fencing_0, Fencing_1, Fencing_2:* percent of time (0-100%) out of two hours that the pair spent bill fencing on week 0, week 1, and week 2 *Nest Building_0, Nest Building_1, Nest Building_2:* percent of time (0-100%) out of two hours that the pair spent nest building on week 0, week 1, and week 2 *TimeInNest_0, TimeInNest_1, TimeInNest_2:* percent of time (0-100%) out of two hours that the pair spent in the nest on week 0, week 1, and week 2 *other_0, other_1, other_2:* the percent of time (0-100%) out of two hours that the male-female pair spend on other behaviors not accounted for in the other categories listed, including eating, drinking, and sleeping separately Row 25 - the values in this row represent the mean percent of time for each behavior at each timepoint (aligned with their respective columns) for all pairs Row 26 - the mean percent of time for each behavior at each timepoint (aligned with their respective columns) for all normally-reared pairs Row 27- the mean percent of time for each behavior at each timepoint (aligned with their respective columns) for all song-naive pairs **Preference Data Structure:** *Female ID:* individual identification code (leg band color and numbers) for the female zebra finch tested in the preference tests *60 day + housing:* whether the female was housed in a same-sex all-female colony ('nursery') or a mixed-sex colony ('colony') from 60 days post hatch until cohabitation (same as in Pair Bonding data, see above) *rearing:* whether the female was raised 'normally-reared' or 'song-naive' (same as in Pair Bonding data, see above) *adult experience:* whether the female was paired with a male (opposite-sex paired, referred to here as 'mated'), or with a female (same-sex paired, referred to here as 'unmated') during two weeks of cohabitation *test:* indicates the category of preference test stimuli (familiar vs unfamiliar, familiar courtship vs non-courtship, or unfamiliar courtship vs non-courtship) *pulls_a:* the number of times the female pulled on the string that corresponded to Stimulus A. For example, for the familiar vs unfamiliar preference test, pulls_a indicates the number of times the female pulled on the string that triggered the playback of the familiar male's song. (see methods for details) *pulls_b:* the number of times the female pulled on the string that corresponded to Stimulus B. For example, for the familiar vs unfamiliar preference test, pulls_b indicates the number of times the female pulled on the string that triggered the playback of the unfamiliar male's song. *bootstrap:* normalized metric of preference strength ('preference index') based on the number of pulls for each stimulus (between 0 and 1, where 1 is a preference for Stimulus A, and 0 is a preference for Stimulus B). *ci_low:* the lower bound of the 95% confidence interval calculated with the preference index (bootstrap with replacement (10,000 iterations)) *ci_high:* the upper bound of the 95% confidence interval *preference code:* 1 for significant preference for Stimulus A, -1 for significant preference for Stimulus B, and 0 for no significant preference. Preferences were coded as 1 (above 0.5) or -1 (below 0.5) if the CIs did not cross 0.5, and as 0 if the CIs did cross 0.5. *total number of pulls:* the total number of times the female pulled the strings that corresponded to Stimulus A and Stimulus B ## Code/Software All statistical analyses were conducted in JMP Statistical Processing Software. All stimulus songs were bandpass filtered (300–10 kHz), normalized by their maximum amplitude, and saved as wav files (44.1 kHz) using custom written code in Matlab (Mathworks, Natick, MA).