Data from: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches
Data files
Mar 05, 2026 version files 28.17 MB
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1_Motif_Data_Siblings.zip
775.63 KB
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2_Number_of_FMvsHStack_notes.zip
296.71 KB
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3_Linearity_Consistency_Stereotypy.zip
16.30 KB
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4_Fathers_vs_Siblings_Analysis.zip
117.24 KB
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5_Ave_DARPP_counts_X_LMANsh_13-1-2025.csv
1.28 KB
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5_DARPP32_counts_Siblings.zip
983.48 KB
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5a_Vol_X_HVC_RA_LMAN
0 B
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5a_Vol_X_HVC_RA_LMAN.zip
9.95 KB
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6_All_data_Average_Puncta_Counts_13-1-2025.csv
460 B
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6_Analysis_of_Puncta_6_birds_CSV_27-1-2026.zip
686.46 KB
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6_Full_length_WB_7-1-2024.tif
25.27 MB
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7_IntroNotes_per_motif_and_Motifs_per_Bout_13-1-2025.csv
932 B
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README.md
12.14 KB
Abstract
Delta-opioid receptors (δ-ORs) are known to be involved in associative learning and modulating motivational states. We wanted to study whether they were also involved in naturally occurring reinforcement learning behaviors, such as vocal learning, using the zebra finch model system. Zebra finches learn to vocalize early in development, and song learning in males is affected by factors such as the social environment and internal reward, both of which are modulated by endogenous opioids. Pairs of juvenile male siblings (35-day-old) were systemically administered a δ-OR-selective antagonist, naltrindole or vehicle (controls) for 10 days. The acoustic structure of songs differed across treated and control groups at adulthood (120 days). Naltrindole-treated birds had a significantly lower pitch, mean frequency, and frequency modulation than controls, whereas there was no difference in the number of songs in naltrindole-treated and control siblings. Since the opioid and dopaminergic systems interact, we decided to study whether blocking δ-ORs during the sensitive period led to changes in dopaminoceptive neurons in Area X, a song control nucleus in the basal ganglia. Interestingly, compared with controls, naltrindole-treated birds had higher numbers of DARPP-32-positive medium spiny neurons and potentially excitatory synapses in Area X. We show that manipulating δ-OR signaling during the learning phase resulted in alterations in the acoustic features of song and had long-term effects on dopaminergic targets within the basal ganglia in adulthood. Our results suggest that endogenous opioids regulate the development of cognitive processes and the underlying neural circuitry during the sensitive period for learning.
https://doi.org/10.5061/dryad.x69p8czsh
Description of the data and file structure
The data provided here has been organized according to the order of the figures in the paper:
Figure 1
(1) 1_Motif_Data_Siblings.zip; Subfolder '1 Motif Data Siblings'
Data from n = 9 sets of birds is provided. For each set of male zebra finch siblings (control or naltrindole-treated), files contain the analysis for each motif segmented and analyzed using Sound Analysis Pro (SAP) [(SA+), Ver.-1.02].
The Wiener entropy was set at 3.6 for all birds. This was followed by setting the amplitude for each bird, such that the software recognized individual notes in the songs while ensuring that background sounds were not included. We did not smooth any feature of the songs during our analysis. The ‘minimum stop duration’ were set to 7ms and ‘bout ends’ were set to 100ms. These settings were used uniformly throughout our experiments.
The files include:
1) Data for each bird with the bird ID, age and song:
1. Bout ID
2. Syllable Duration (ms)
3. Syllable Start
4. Mean Amplitude*
5. Mean pitch
6. Mean FM
7. Mean AM^2*
8. Mean Entropy
9. Mean Pitch goodness
10. Mean Mean frequency
11. Variance Pitch**
12. Variance FM**
13. Variance Entropy**
14. Variance pitch goodness**
15. Variance mean freq**
16. Variance AM**
* Mean AM and Mean AM^2 were not used for our analysis since this measure can be affected by distance from the microphone, which can vary across different birds and also between different motifs sung by individual birds.
** Other data included in each file pertaining to variance pitch, variance FM, variance entropy, variance pitch goodness, variance mean frequency and variance AM was not used for our analysis.
2) Files ending in 'With ISI' for each bird:
Intersyllable intervals (ISI) are calculated by using the formula:
Motif Duration - Sum (all syllables)/number of intersyllable intervals
The IDs (column B) of individual syllables is labelled '0' and motif duration is labelled '1'.
3) Data pertaining to Syllables ending with 'syllable'
4) Data pertaining to Motifs ending with 'motif'.
Naming Conventions for file names in this folder:
- In the file "Motif data siblings", files are named Set# (1-9) followed by the Band numbers of Control and Treated siblings
- B: blue; Y: Yellow are used for color of bands used to identify different zebra finches followed by a two- or three-digit number.
- In folders for each bird, different CSV files have been identified for each bird used in this experiment, and days on which they were recorded (70-120 days) are also mentioned. Since zebra finches initially sing more than one motif, these notations have also been mentioned. For example, for the file:
- B56 80 days dir song ABCB'_B56 80d song ABCB
(i) The file name indicates that this song was sung by B56 at 80 days post hatch.
(ii) 'Dir' (wherever mentioned) indicates that the song was female-directed, although all song data in this study pertain to directed songs.
(iii) ABCB and ABCB': B56 sang two slightly different versions of his directed song each of which had 4 syllables. One was ABCB (where B was repeated twice in the 2nd and 4th positions), whereas there was a slight variation in the second version of the song with a B' sung at the 4th position.
(iv) In some folders, 'type1, type1a or type2' has also been used as a part of the file names, indicating different versions of the songs sung by experimental birds.
(v) If the file name contains 'i' preceding syllables (such as 'iBC'), then 'i' stands for 'introductory note', followed by syllables B and C.
Other examples:
CDEFGH': Bird's song with six syllables (C, D, E, F, G, H')
ABCDD'EF': Bird's song with 7 syllables (A, B, C, D, D', E, F'). In this song, D and D' are syllables wherein the spectral structure is very similar but not identical.
Figures 2 and 3
(2) 2_Number_of_FMvsHStack_notes.zip
The analysis was performed using the SA+ (Ver.-1.02) software on Harmonic Stack (HStack) and Frequency Modulated (FM) notes.
Each file contains the following data for control birds and their naltrindole-treated siblings:
1. Syllable Duration (ms)
2. Syllable Start
3. Mean Amplitude*
4. Mean pitch
5. Mean FM
6. Mean AM^2*
7. Mean Entropy
8. Mean Pitch goodness
9. Mean Mean frequency
10. Variance Pitch*
11. Variance FM*
12. Variance Entropy*
13. Variance pitch goodness*
14. Variance mean freq*
15. Variance AM*
*As mentioned above, Mean Amplitude, Mean AM^2 and the 'Variance' measures were not used for our analysis.
A CSV file containing the numbers of FM versus Hstack syllables sung by control and naltrindole-treated birds is provided in this folder (2_Number of FMvsHstck notes).
The medians, means and standard error for FM and Hstck notes is also provided for creating graphs.
Figure 4
(3) 3_Linearity_Consistency_Stereotypy.zip
1) Contains the folder "Analysis LinConsSter" with the analysis of the note order of control and naltrindole-treated birds' songs using the Songinator program (.txt files).
2) A CSV file "Compiled Results LCS" containing measurements of Linearity, Consistency and Stereotypy in control and naltrindole-treated birds which were used for analysis. Gaps separate data from different sets of birds at different ages (80d, 90d, 100d, 110d and 120d).
Figure 5
(4) 4_Fathers_vs_Siblings_Analysis.zip
The zip file contains a folder 'Fathers Songs CSVFiles' with subfolders containing the songs of the fathers of different sets of siblings used for our experiments.
The files in the subfolder contain:
1) CSV files with Bird IDs ending with 'Analysis' contain:
1. Syllable Duration (ms)
2. Syllable Start
3. Mean Amplitude*
4. Mean pitch
5. Mean FM
6. Mean AM^2*
7. Mean Entropy
8. Mean Pitch goodness
9. Mean Mean frequency
10. Variance Pitch*
11. Variance FM*
12. Variance Entropy*
13. Variance pitch goodness*
14. Variance mean freq*
15. Variance AM*
*Mean Amplitude, Mean AM^2 and the 'Variance' measures were not used for our analysis.
2) File with Bird IDs ending with ISI calculation
Intersyllable intervals were calculated for each father as mentioned above.
3) File with Bird ID and ending _With ISI
Compiled data of acoustic features with Averages
Analyzed Data:
4) CSV file "Av Similarity" containing averages of scores for Similarity Scores, Percentage Similarity, Mean Accuracy, Sequential Match, Kolmogorov-Smirnov (KS) distance for comparison between control and naltrindole-treated siblings, Fathers versus controls and Fathers versus treated birds used for statistics. Column A lists the IDs of the control and experimental birds and Column B lists the IDs of the fathers. Gaps (columns) are used to separate each set of data.
5) CSV file "Fathers vs Sons Data for Similarity" containing data for Similarity Scores, Percentage Similarity, Mean Accuracy, Sequential Match, Kolmogorov-Smirnov (KS) distance for comparison between control and naltrindole-treated siblings, Fathers versus controls and Fathers versus treated birds. Column A lists the IDs of the fathers and their progeny. Gaps (columns) are used to separate each set of data.
Figure 6
(5) 5_DARPP32_counts_Siblings.zip
Each subfolder contains counts of DARPP32 in different song control nuclei [Area X, HVC, LMAN (core and shell) and RA] for 6 sets of control and naltrindole-treated siblings.
For each song control nucleus, the following Stereoinvestigator files for 6 sets of control and naltrindole-treated siblings are provided:
Different files contain the following:
1. Summary
2. Parameters
3. Counts by Site
4. Coefficient of Error (CE)
5. CE Variance Details
6. Section Details
7. Z Depth
8. Z Depth Details
9. Smoothness
10. Planimetry
11. Z Order
12. Raw Report
The associated CSV file '5_Ave_DARPP_counts_X_LMANsh_13-1-2025.csv' contains averages of counts of DARPP32-positive neurons in Area X and LMANshell, which were used for statistics, since there was a significant difference between these measures in control and naltrindole-treated birds.
(5a) 5a_Vol_X_HVC_RA_LMAN.zip
Major song control nuclei (LMAN, Area X, HVC and RA) in control and naltrindole-treated birds (n = 9 sets) were outlined in their entirety, using the Stereoinvestigator software (MBF Bioscience). The Cavalieri estimator probe was used to estimate the volume of each nucleus in mm3. Of the 5 series of cryosections (40 μm) obtained from these birds, volume estimation was performed on the first section, which was stained with Nissl. A grid spacing of 10 μm used to place grids generated by the software over the song control nuclei, which were observed at a magnification of 40X. Points in the grid overlying the song control nuclei in each bird were then marked. The volume of each song control nucleus was given by the formula:
Volume of a structure = Total number of points marked × Distance between points in XY × Distance between points in Z (wherein X, Y and Z represent the three axes).
1. Data for each song control area is provided in CSV files ending as 'DATA' which was obtained from the Stereoinvestigator software.
2. Averages are provided for different sets of control and naltrindole-treated birds (Bird IDs in Column A) in files ending with 'Arranged' used for statistics.
Figure 7
1) 6_Full_length_WB_7-1-2024.tif
Full Length Western blot for PSD-95, Synaptotagmin and DARPP32 (TIF File)
2) Puncta data
The number of synapses (points of co-localization) was counted in naltrindole-treated birds versus their vehicle-treated siblings (n = 5 sets of siblings). A confocal microscope (LSM 510 Meta, Carl Zeiss, Germany) equipped with an Argon laser and a HeNe laser and was used to image synaptotagmin and PSD-95-positive puncta using a Plan-Apochromat 63x/1.4 NA oil immersion lens. Alexa Fluor 488 was visualized using a 505–550 band pass filter whereas Alexa Fluor 594 was captured in the 560 band pass range.
The method for counting puncta is provided in Ippolito and Eroglu (2010). Z-stacks (stacks consisted of fifteen 0.33 μm thick optical sections) at 512 × 512 pixels were captured for further analysis. The Image J plugin, Iterative Deconvolve 3D, was used to deconvolve the images, which was followed by quantifying puncta using the Puncta Analyzer plugin. Three sections were merged into one maximum intensity projection (MIP). In this way, 5 MIPs were generated from each stack with each MIP being ~ 1 μm in thickness. An ROI of 0.34 x 0.34 pixels was defined and the plugin was used to calculate the number of synapses within this square. 5 such ROIs were defined for each of the 5 MIP images in each stack with each MIP being ~ 1 μm in thickness. The 5 ROIs were then averaged to yield a single value for one MIP image. Thus, one stack resulted in 5 data points corresponding to the 5 MIPs. Six such stacks were analyzed per bird.
Reference: Dominic Ippolito and Cagla Eroglu: Quantifying synapses: an immunocytochemistry-based assay to quantify synapse number. JoVE (Nov 16, 2010)
1. 6_Analysis_of_Puncta_6_birds_CSV_27-1-2026.zip: CSV files with the analysis of the number of excitatory synapses in Area X for 3 sets pf control and naltrindole-treated birds. The points of co-localization of the presynaptic marker Synaptotagmin and postsynaptic marker PSD-95 were analyzed using plugins from Image J.
2. A CSV file "6_All_data_Average_Puncta_Counts_13-1-2025.csv" provides the averages of counts of puncta for 5 sets of control and treated birds, used for statistical analysis.
Supplementary Figure 2:
A CSV file '7_IntroNotes_per_motif_and_Motifs_per_Bout_13-1-2025.csv' provides calculations of (i) Introductory Notes per Motif and (ii) Motifs per bout.
Code/software
CSV files, any software for viewing TIFF files.
Animals
All experiments were approved by the Institutional Animal Ethics Committee at the National Brain Research Centre (NBRC), Manesar, in accordance with the guidelines laid down by the Committee for the Control and Supervision of Experiments on Animals (CCSEA), India (protocol number: NBRC/IAEC/2013/85). All birds used in this study were obtained from the Animal House at NBRC, Manesar. Nine pairs of juvenile male zebra finches (35dph) were used for this study, each pair consisting of male siblings/clutch-mates. Birds were housed within the aviaries with their parents during the course of the experiment to ensure normal social interactions. The aviaries housed twelve pairs of adult birds (>120dph) with three to four offspring per pair. At 50dph, experimental birds were removed from the aviary and housed in separate cages in auditory and visual contact with their parents. Beginning from 80dph to 120dph, female-directed songs (FD songs) were recorded from both control and treated birds during the first half of the day at ten-day intervals. Throughout the duration of the experiment and until 120dph, the experimental birds were situated in an enriched social environment in auditory and visual proximity with approximately 50 other birds in the aviary.
Treatment paradigm
The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1mg/ml). The drug dose was calculated according to the weight of the birds, and the stock was diluted to a volume of 100µl for injections. In each experimental set, one bird was administered naltrindole intramuscularly (10mg/kg body weight) once every day for 10 days, starting at 35dph to 45dph, whereas its control sibling was administered saline (vehicle) for the same duration. Birds were observed for 30 minutes after the injections for any signs of stress or trauma.
Behavioral Recordings
Behavioral recordings were performed in a cage (dimensions: 12 × 15 × 11 inches; length × breadth × height) placed in a sound-attenuated chamber. Female birds were housed in a separate cage placed in visual and auditory proximity to that of the male birds. Software, namely Goldwave (Version 5.10 Goldwave Inc.) and Audacity (Version 3.14-beta), was used to record songs and calls. Video recordings were performed using a handycam (Sony; DCR-SR67E E37).
Song Analysis
Songs recorded between 80 and 120dph were isolated using Sound Analysis Pro (SAP) [(SA+), Ver.-1.02]. Song quality was assessed through temporal features such as syllable duration and intersyllable interval (ISI), and through frequency-based/spectral features including mean pitch [henceforth termed pitch], mean frequency (MF) and mean pitch goodness, amplitude and amplitude modulation (AM), FM, and Wiener entropy. Spectro-temporal features were also compared across tutors and tutees (aged 120 dph). We analyzed 15-20 songs/bird for each developmental time point. Songs were randomly chosen and only excluded if cage noise or female calls overlapped the song. Furthermore, stereotypy was analyzed by averaging sequence linearity (the order of syllables within a motif) and consistency (the predominant transitions for a particular syllable). We used the online program, ‘the Songinator’ (http://bottjerlab.usc.edu/songinator.html; Zevin J.) to calculate linearity, consistency, and stereotypy in the song. All variations in song were included, whereas introductory notes were excluded for this analysis. We also counted the number of introductory notes, motifs, and motif variants.
Histology:
Tissue sectioning
Transcardial perfusion was performed after birds were deeply anesthetized with an overdose of intramuscular injections of ketamine (100mg/kg body weight) and xylazine (1 mg/kg body weight), until they were areflexive to hard toe pinches. After initial perfusion with saline (0.74%), all birds used in these experiments were perfused with 4% paraformaldehyde (PFA) after the last song recordings were obtained (at 120dph). Serial coronal sections (5 series; 40μm thick) were cut using a cryostat (Leica, CM 3050 S). The first series of sections from each brain was stained with Nissl (Thionin) to identify various brain regions. The remaining sections were used for immunohistochemistry (IHC) using different antibodies. Whole brain lysates from adult male zebra finches sacrificed by an overdose of halothane were used to test the specificity of the antibodies used for IHC for zebra finch brain tissue.
Immunohistochemistry
Sections mounted on gelatin-coated slides were incubated in blocking solution containing 5% Normal Goat Serum (NGS; S-1000, Vector Laboratories, Burlingame, CA) and 1% bovine serum albumin (BSA; A-7906, Sigma-Aldrich) which was followed by incubation in a solution containing a combination of anti-PSD-95 (1:200; ab9708, Abcam; polyclonal; a marker for post-synaptic scaffolding proteins in excitatory neurons and anti-Synaptotagmin antibody (1:200; MAB5200 Millipore, monoclonal; a calcium-sensitive pre-synaptic marker 3% NGS, 0.3% Triton X-100 and 1% BSA). The slides were rinsed with PBS, after which they were incubated in a secondary antibody solution containing Goat Anti-Rabbit IgG Alexa Fluor 488 (1:300; A-11008, Invitrogen), and Goat anti-Mouse IgG, Alexa Fluor 594 (1:300; A-11005, Invitrogen). Sections were rinsed in PBS and mounted with VECTASHIELD Anti-fade mounting medium containing DAPI (H-1200, Vector Labs). Fluorescence images of the sections were captured using a confocal microscope (LSM 510 meta, Zeiss). For performing western blots and immunostaining for DARPP32, we followed the protocols detailed in Singh and Iyengar (2019).
Stereology
All song control nuclei were outlined using the Stereoinvestigator software (MBF Bioscience). The Cavalieri estimator probe was used to estimate the volume of each nucleus in mm3. Volume estimation was performed on the first Nissl-stained section in a series of four sections, using a grid spacing of 10µm at a magnification of 40X. The number of DARPP32-positive cells in Area X was counted in sections at the level of the anterior forebrain, stained using immunohistochemistry (n = 5 sets of randomly selected sibling pairs). Every second section in the series of four sections was used for counting DARPP32+ve cells. The optical fractionator probe was used by viewing sections under the 100x objective and using a counting frame size of 150μm x150μm. The thickness of the section was measured at each counting site of the optical fractionator probe.
Puncta Analysis
To quantify the changes in synapses in the naltrindole-treated birds versus their vehicle-treated siblings (n = 5 sets of randomly selected siblings), we counted the number of synapses in Area X (cf.). Briefly, 0.33μm thick optical sections were captured as z-stacks, each of which consisted of 15 sections. Three sections were merged into one maximum intensity projection (MIP). In this way, 5 MIPs were generated from each stack, with each MIP being ~1μm in thickness. The Image J plugin, Iterative Deconvolve 3D, was used to deconvolve the images, which was followed by quantifying puncta using the Puncta Analyzer plugin.
Statistical Analysis
All statistical tests were performed using software from SigmaPlot (versions 12 and 15; Systat Software Inc., USA). Two-Way Repeated Measures analysis of variance (Two-Way RM ANOVA) was used to test the interaction between developmental age, treatment, and values of several songs, spectral and temporal features of control birds, and their naltrindole-treated siblings. Of these, we found significant differences only in the spectral features. This was followed by post-hoc analysis using the Bonferroni method. We also used the Benjamini-Hochberg (B-H) correction with an FDR (false discovery rate) of 0.05 for this data. While using this correction, p-values for sets of comparisons at each age were compared with their critical B-H value, obtained by the formula (i/m)Q, where ‘i’ = rank of individual p-value, m = total number of correlations, and Q (FDR) = 0.05.
Furthermore, changes in the number of FM and H-stack syllables in the songs of control and treated birds, changes in linearity, consistency, and stereotypy, changes in the number of DARPP-32-positive neurons, and differences in the number of synapses in Area X between control and treated birds were analyzed using the Student t-test and the Mann-Whitney Rank Sum test (MWU) was used wherever data were not normally distributed. Bar graphs represent mean values, and error bars represent standard deviation. Each point in the scatter plots represents the average valueof the data for one bird. One-way ANOVA was used to compare spectral and temporal features between siblings and tutors. The Holm-Sidak method was used for pairwise multiple comparisons to test for statistical significance.