Operant Conditioning Task to Measure Song Preference in Zebra Finches

Behavior

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Summary

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.

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Coleman, M. J., Saxon, D., Robbins, A., Lillie, N., Day, N. F. Operant Conditioning Task to Measure Song Preference in Zebra Finches. J. Vis. Exp. (154), e60590, doi:10.3791/60590 (2019).

Abstract

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.

Introduction

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.

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Protocol

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

  1. Build a testing cage composed of two identical chambers, 15 x 15 x 17 inch3 each (Figure 1A). Make a small door (as an entrance to each chamber) on the side of the cage near the perches by cutting out a small 4.5 x 6 inch2 window that is 2.5 inchs away from the center of the cage between the chambers. Use a separate piece of the shelving to make a door that covers the window.
    NOTE: The cage is made from wire shelving for closets that can be obtained from any home improvement store. The hinge of the door can be made with zip-ties.
  2. Cut the shelving with wire cutters to make a 4 x 6 inch2 opening between the two chambers to allow the bird to move between chambers.
  3. In each chamber, place a perch 11 inches from the center of the cage and 6 in from the bottom of the cage. Use any perch that is suitable for the type of bird being tested and that will span the width of the cage.
  4. Place the emitter and receiver of the photoelectric sensor on the cage directly above each end of the perch. Tether the sensors to the wire above the perch using zip-ties.
    NOTE: Placing the sensors onto a rigid backing (e.g., a tongue depressor) can help keep the sensors from twisting on the wire cage and becoming misaligned. It is important to test the placement of the sensors prior to each experiment by manually breaking the infrared (IR) beam. If the sensors are not correctly aligned, a bird will not trigger a song, or landing on the perch may be counted as multiple events.
  5. Place under-cabinet LED lights on the top of the cage to provide enough illumination so that the animals will move freely between the two side chambers.
  6. Place bird seed and water next to each of the perches.
  7. Place a speaker at each end of each chamber.
  8. To reduce sound diffraction and audio and visual distractions, place anechoic foam on the perimeter of the entire cage, with holes left for the cage doors and water bottles.
  9. Connect the speaker to an audio amplifier and connect the audio amplifier to the sound output on a computer.

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).

  1. Install a digital I/O card (Table of Materials), its drivers, and the associated program 'Measurement and Automation'.
    NOTE: The digital I/O card used here requires a connector block and multifunction ribbon cable to complete installation. Alternatively, a USB I/O device requires no PCI card (or the required connector block and ribbon cable accessories), and can be run on a laptop.
  2. Connect the two blue wires of each emitter and receiver set together. Attach two small wires to the black wire of the power cable so that the black power wire can connect to the blue wires of each sensor set.
  3. Connect the black power wire and two blue wires to a common wire. Connect this common wire to either input #2 or # 9 on the connector block.
    NOTE: One set of sensors should be connected to input #1 and #2 and the other set of sensors should be connected to input #9 and #10.
  4. For both sensor sets, connect the brown wire from the emitter and the receiver together. For each sensor set, connect the two brown wires to the red AC power wire.
    NOTE: All four brown wires are connected to the red AC power wire. The photoelectric sensors are powered by a 10–30 V input and must be connected to an AC converter (e.g., a 120 V to 12 V converter).
  5. Within the Measurement and Automation program associated with the digital I/O card, determine the Device ID for the I/O card by selecting the Devices and Interfaces menu.
  6. Select 'Device Pinouts' option to determine the port and line number that correspond with the inputs in 2.3.
  7. Verify that the I/O detects when the IR beam is broken using the Test Panels… option in the program. Note the channel and port IDs for each set of sensors.
    NOTE: Green indicators will change color when the status of the beam is altered.

3. Install Software and Hardware to Count Perch Landings

  1. Download and install Sound Analysis Pro 2011 (SAP2011) and MySQL from http://soundanalysispro.com/.
    NOTE: This is freeware and comes with installation instructions and a user manual.
  2. Open SAP recorder.exe.
  3. To configure the sensor input so that SAP can detect perch landings and initiate song playbacks, click the Operant Devices tab in the SAP Recorder control window. Check the box Enable Operant Training (NI Card Installed).
  4. Select the appropriate Device ID (the Ports slider can be set to 3). For each detector, indicate the appropriate port and line from the information collected in step 2.6.
    NOTE: When the port and line are set correctly for each detector, the light will change from yellow to red.
  5. In the Main window for the SAP recorder, hit Train to activate the sensors for the appropriate channels.
    NOTE: A yellow button should appear.
  6. Record the name of the bird by selecting the channel number of interest (Press channel 1 or 2 tab on the left side of the screen), and in the Identification and Mode window, type in the bird's identification in the Name field.
  7. To ensure that sound is played to the speakers, manipulate the settings in the Output Selection tab to select the appropriate device connected to the computer (i.e., speakers) and the channel connected to the speaker.
    NOTE: Additional setup information can be found in the Sound Analysis Pro user manual.

4. Isolate Pairs of Birds and Collect Male Song

  1. Isolate a male and a female finch in a sound-attenuation chamber for 24–48 h. During this cohabitation, collect the male song, which is referred to as the 'partner's song'.
    NOTE: Paired birds are a male and female that have been housed together for at least 2 weeks. Unpaired females are placed with a male for only 24–48 h.
    1. Use a microphone connected to an audio amplifier to capture the partner's song.
    2. Acquire the song using Sound Analysis Pro 2011 and store the song on a hard drive.
    3. To test for the effect of the neurotransmitter systems on female song preference, give the female a 50 µL subcutaneous injection of saline (vehicle control) or quinpirole (1 mg/mL in 0.9% saline) when the pair is first put into the chamber. Repeat the injection after 6–24 h (see Figure 1B).
  2. Create songs for playback in the operant conditioning cage.
    1. Create a .wav file that plays 'silence' approximately the same length as the song playbacks.
      NOTE: This can be done using any sound-editing software (e.g., Audacity).
    2. Cut a representative song (2–3 motifs) from the partner male.
    3. Cut a representative song (2–3 motifs) from an unfamiliar male.
      NOTE: Use the same unfamiliar male song for all experiments.
    4. Filter the songs from 300 Hz–10 kHz.
    5. Adjust the volume so that the songs play at ~70 dB (average amplitude over the song duration from the speakers in the behavior chamber).
      NOTE: The volume of the song is tested using a sound pressure meter that is placed at the perch.

5. Testing Song Preference in Paired Females

  1. Determine the female's side chamber preference.
    1. Prior to the behavioral testing, place the female in the testing cage to allow her time to adjust to the cage for at least 1 h. Ensure the finch explores both side chambers by using an object to cause the bird to move from one chamber to the other through the opening.
    2. In Sound Analysis Pro, select the 'Playbacks' tab and then the 'Sounds' button in the main window.
    3. Select the audio .wav file to play 'Silence' from chamber 1 and chamber 2.
    4. Go back to 'Main' and hit 'Reset' on the top of the boxes on the right.
    5. After the acclimation period, press 'Start'.
    6. At the end of the session, press 'Stop'. Write down the number of triggers at each perch, which is displayed in the blue boxes.
      NOTE: The data are saved to a MySQL database. Have a minimum number of triggers to count the trial to ensure the female is active enough (e.g., 12 or more perches total).
  2. Determine song preference.
    1. Repeat the steps in section 5.1, but choose the partner's song to play from the side chamber with the fewest perch triggers (the non-preferred side), and an unfamiliar song from the side chamber with the most perch triggers (the preferred side).
      NOTE: The partner's song is played from the side chamber with the fewest triggers during the playback of silence (the non-preferred side).
    2. Press 'reset' prior to starting the 1 h with song playback.
    3. Calculate the chamber and song preference by dividing the number of triggers on the side chamber playing the partner's song by the total number of triggers.

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Representative Results

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
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
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.

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Discussion

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.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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).

Materials

Name Company Catalog Number Comments
(-)-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

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References

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