Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches

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.

Methods
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.