We describe a technique to evaluate song preference in zebra finches. Females are placed in a two-chambered cage and song preference is measured by the number of times she triggers the playback of one song by landing on a perch within one chamber, compared with triggering a different song in the second chamber. Perch landings are counted using infrared sensors.
An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting opening and indicate their preference for a song by landing on a perch within each chamber. By interrupting the infrared beam from a photoelectric sensor above each perch, the bird activates the playback of a song through a speaker located on each side of the cage. Freely available software is used to trigger the song playback from each perch. To determine the song preference of each animal, her chamber preference is first identified by triggering no song playback when she lands on each perch. This chamber preference is then compared to her song preference. A minimum activity threshold is set to ensure the preference is real. Using this method, we show that paired females prefer the song of their partner. This method was used to understand the contribution of dopamine to the formation and maintenance of song preference.
One of the fundamental questions in biology is how animals form affiliative bonds. In particular, what are the neural mechanisms by which these bonds are made and are these basic mechanisms conserved across vertebrate species? The prairie vole has given clues to some of the neurotransmitter systems that are important for pair-bond formation1,2,3. In particular, dopamine acting through receptors in the nucleus accumbens can induce partner preference in both male and female voles4,5. It is unclear whether the general principles underlying partner preference formation and maintenance are evolutionarily conserved.
Monogamy is more common in birds than in mammals6. Therefore, comparing mechanisms of affiliative behavior in birds to other species is critical to understanding conserved neural foundations7,8,9. In general, across many species of songbirds, male song is thought to serve as an honest indicator of male fitness10,11. Male zebra finch songbirds sing to attract mates and influence the formation of monogamous pairs12,13. Thus, song can be used to determine partner preference in these birds.
The mechanisms by which females form a preference for their partner’s song is unknown. Mesotocin, the avian homologue of oxytocin, appears to play a role in pair-bond formation in finches14,15. In addition, dopamine has also been shown to play a role in pair-bond formation9,16,17,18. For example, dopamine levels are higher in the nucleus accumbens of paired versus non-paired finches9.
To study the role of neurotransmitter systems on pair-bond formation and maintenance, we measured song preference using an operant testing paradigm equipped with infrared sensors to trigger song playbacks19. The ratio of song playbacks identified the female’s preference for a male’s song. Prior to the operant conditioning task, each female was isolated for up to 48 h in an anechoic chamber with her partner for ‘paired females’ or with an unfamiliar male, for ‘unpaired females’. To test the effect of dopamine on song preference, unpaired females were treated twice with a D2R dopamine agonist while isolated with the unfamiliar male. This behavioral paradigm is based on previous studies19,20 and is amenable to research by undergraduate students.
All experiments were approved by the Institutional Care and Use Committee of the W.M. Keck Science Department, in accordance to NIH guidelines. All animals used were adults (>90 days post hatch).
1. Construction of Operant Conditioning Chamber
2. Connection of Sensors
NOTE: There are two sets of IR sensors, each with an emitter and receiver. The emitter has a cable with four wires (brown, blue, black, and white). The receiver has a cable with three wires (brown, blue, and black). The white wire of one emitter is connected directly to input #1 on the digital I/O board. The white wire of the second emitter is connected directly to input #9. The AC power input has two wires (usually red and black).
3. Install Software and Hardware to Count Perch Landings
4. Isolate Pairs of Birds and Collect Male Song
5. Testing Song Preference in Paired Females
Following the protocol, we found that paired females preferred their partner’s song (Figure 2A). There was a significant difference between the side chamber preference during silence to that during song playback (t-test corrected for multiple comparisons; p = 0.004; t = 3.35, df = 16). Thus, the female preferentially triggered the song of her partner in comparison to the song of an unfamiliar male.
Females that were paired with an unfamiliar male for 24–48 h and given saline injections did not have a preference for his song over the song of another unfamiliar male. That is, there was no difference between the side chamber preference when landing on a perch that triggered silence compared to triggering song (t-test corrected for multiple comparisons; p = 0.726; t = 0.357, df = 16). Therefore, females housed with a male for less than 48 h did not form a preference for his song.
After showing that we could use the behavioral paradigm to test the song preference of female finches, we used the assay to test the influence of dopamine on formation of song preference. In this case, a naive female was housed with an unfamiliar male for 24–48 h and given two subcutaneous injections of the dopamine 2 receptor agonist quinpirole (Figure 1B,2B). Overall, females given quinpirole preferred the song of the male she was with for only 24–48 h (t-test corrected for multiple comparisons; p < 0.001; t = 5.25; df = 18). In the example shown, two of the females did not show a preference for her partner’s song (Figure 2A). In one case the female was older (>5 years). It is not clear why the second female did not form a preference. One possibility is that the female may not have been given a complete dose of the drug.
To ensure there was no difference in total activity of females between treatment groups that could account for the difference in song preference we compared the total number of perch triggers (Figure 2B). The total number of perch triggers is the number of times a female landed on both perches during the 1 h testing period for either silence or song playback. The total activity of the birds in any treatment was similar (ANOVA, p = 0.436).
Figure 1: Schematic of operant conditioning cage and timeline of the experiment. (A) The cage has two chambers of equal size with an opening connecting the two side chambers. An opposed pair of photoelectric sensors is placed above each perch that spans the width of the chamber. When a bird lands on a perch she breaks the IR beam from the sensors, which triggers the song playback from the speaker nearest the perch. The dimensions of the cage are shown, in addition to the placement of the perches. (B) Timeline of the experiment. This figure was modified from Day et al.21. Please click here to view a larger version of this figure.
Figure 2: Representative data of female song preference. (A) The ratio of perch triggers during operant testing indicates the preference for songs produced by conspecific males. Each female’s song preference was compared to her side chamber preference in which no song (silence) was triggered when she landed on a perch. In each case the partner’s song was played when she landed on a perch in the non-preferred chamber. Connected points are preferences for each individual. This figure was modified from Day et al.21. (B) The total number of perches on each side chamber of the cage for females in all conditions. Please click here to view a larger version of this figure.
We describe a method to test the song preference of zebra finches. We used this assay to test the preference of a paired female for her partner’s song. In addition, the assay was used to test the effect of dopamine on induction of song preference in naive females. This method is relatively inexpensive and was designed and used by undergraduate students, making it an excellent training tool for students. Several other studies have tested song preference in several song species, making this method useful to many investigators studying auditory preferences in birds22,23.
This behavioral paradigm is based on an auditory recognition testing system, ARTsy, first described by Gess et al.19. These authors used a similar system to test song discrimination in birds. We modified the system to test the preference of a female for one song over another. We adapted the protocol described in Gess et al.19 to use SAP2011 to play song and collect the data, instead of custom scripts in MATLAB. We found this change for data acquisition easier to use and more accessible to undergraduate students. Operant playbacks can also be triggered using the perches as switches24.
We found the protocol described here results in data that are easier to analyze than using similar methods. For example, Woolley and Doupe20 measured the length of time a female spent on each side of a cage during passive playbacks of different song types to identify song preference. Quantifying the time in one chamber compared to another requires filming the birds while they behave, which is not required for the protocol described here. Similarly, others have counted the number of female calls in response to male song as a measure of her preference25,26. This requires using manual or semiautomated counting of female calls. The quantification of song preference described here is much simpler as the software automatically keeps track of the number of perch landings.
Using the protocol described here we found that paired females consistently chose to listen to the song of their partner. The task does not test the female’s partner preference, only her song preference. Future experiments should examine a partner preference as well as song preference13,16,27. This protocol can be used to test the influence of other neurotransmitter systems on formation of song preference to compare the conserved evolutionary mechanisms underlying social behavior network shared by many vertebrates7,28. For example, future experiments can target directly specific areas in the brain while animals are learning the task to gain access to the neural circuits underlying social behaviors.
The authors have nothing to disclose.
We thank Petra Grutzik for construction of the behavioral cage, Dr. Thomas Borowski for help designing the experiment, and Dr. Stephanie White for her generous support. Funding from an HHMI undergraduate summer research grant (HHMI #30052007536 to D.S. and L.H).
(-)-Quinpirole | sigmaaldrich | Q102 | |
acoustic foam | Coulbourn Instruments, Allentown, PA | H10-24A | |
audio amplifier – 2 channel | Amazon | Pyle PCAU46A | any small audio amplifier should work |
Banner Engineering Q08 Series, emitter. SO60-Q08 | https://www.alliedelec.com | Stock #:70659809 | photoelectric sensor |
Banner Engineering Q08 series, receiver. EO60-Q08-AN6X | https://www.alliedelec.com | Stock #:70699384 | photoelectric sensor |
Car stereo speakers | Amazon | Pioneer TS – F1643R | |
Digital I/O card | National Instruments | PCI-6503 or USB-6501 | |
LED lights – under-counter | Amazon | ||
multifunctional ribbon cable | National Instruments | 180524-20 | |
sound pressure level meter | Amazon | ||
terminal block | National Instruments | 777101-1 |