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).

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 (&#62;90 days post hatch).
1. Construction of Operant Conditioning Chamber
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place bird seed and water next to each of the perches.</li>
	<li>Place a speaker at each end of each chamber.</li>
	<li>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.</li>
	<li>Connect the speaker to an audio amplifier and connect the audio amplifier to the sound output on a computer.</li>
</ol>
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).
<ol>
	<li>Install a digital I/O card (Table of Materials), its drivers, and the associated program &#39;Measurement and Automation&#39;. 
	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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#8211;30 V input and must be connected to an AC converter (e.g., a 120 V to 12 V converter).</li>
	<li>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.</li>
	<li>Select &#39;Device Pinouts&#39; option to determine the port and line number that correspond with the inputs in 2.3.</li>
	<li>Verify that the I/O detects when the IR beam is broken using the Test Panels&#8230; 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.</li>
</ol>
3. Install Software and Hardware to Count Perch Landings
<ol>
	<li>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.</li>
	<li>Open SAP recorder.exe.</li>
	<li>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).</li>
	<li>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.</li>
	<li>In the Main window for the SAP recorder, hit Train to activate the sensors for the appropriate channels. 
	NOTE: A yellow button should appear.</li>
	<li>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&#39;s identification in the Name field.</li>
	<li>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.</li>
</ol>
4. Isolate Pairs of Birds and Collect Male Song
<ol>
	<li>Isolate a male and a female finch in a sound-attenuation chamber for 24&#8211;48 h. During this cohabitation, collect the male song, which is referred to as the &#39;partner&#39;s song&#39;. 
	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&#8211;48 h.
	<ol>
		<li>Use a microphone connected to an audio amplifier to capture the partner&#39;s song.</li>
		<li>Acquire the song using Sound Analysis Pro 2011 and store the song on a hard drive.</li>
		<li>To test for the effect of the neurotransmitter systems on female song preference, give the female a 50 &#181;L 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&#8211;24 h (see Figure 1B).</li>
	</ol>
	</li>
	<li>Create songs for playback in the operant conditioning cage.
	<ol>
		<li>Create a .wav file that plays &#39;silence&#39; approximately the same length as the song playbacks. 
		NOTE: This can be done using any sound-editing software (e.g., Audacity).</li>
		<li>Cut a representative song (2&#8211;3 motifs) from the partner male.</li>
		<li>Cut a representative song (2&#8211;3 motifs) from an unfamiliar male. 
		NOTE: Use the same unfamiliar male song for all experiments.</li>
		<li>Filter the songs from 300 Hz&#8211;10 kHz.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Testing Song Preference in Paired Females
<ol>
	<li>Determine the female&#39;s side chamber preference.
	<ol>
		<li>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.</li>
		<li>In Sound Analysis Pro, select the &#39;Playbacks&#39; tab and then the &#39;Sounds&#39; button in the main window.</li>
		<li>Select the audio .wav file to play &#39;Silence&#39; from chamber 1 and chamber 2.</li>
		<li>Go back to &#39;Main&#39; and hit &#39;Reset&#39; on the top of the boxes on the right.</li>
		<li>After the acclimation period, press &#39;Start&#39;.</li>
		<li>At the end of the session, press &#39;Stop&#39;. 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).</li>
	</ol>
	</li>
	<li>Determine song preference.
	<ol>
		<li>Repeat the steps in section 5.1, but choose the partner&#39;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&#39;s song is played from the side chamber with the fewest triggers during the playback of silence (the non-preferred side).</li>
		<li>Press &#39;reset&#39; prior to starting the 1 h with song playback.</li>
		<li>Calculate the chamber and song preference by dividing the number of triggers on the side chamber playing the partner&#39;s song by the total number of triggers.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>McGraw, L. A., Young, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prairie+vole:+an+emerging+model+organism+for+understanding+the+social+brain.">The prairie vole: an emerging model organism for understanding the social brain.</a> Trends in Neuroscience. 33 (2), 103-109 (2010).</li><li>Young, K. A., Gobrogge, K. L., Liu, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+pair+bonding:+Insights+from+a+socially+monogamous+rodent.">The neurobiology of pair bonding: Insights from a socially monogamous rodent.</a> Frontiers in Neuroendocrinology. 32 (1), 53-69 (2011).</li><li>Young, L. J., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+pair+bonding.">The neurobiology of pair bonding.</a> Nature Neuroscience. 7 (10), 1048-1054 (2004).</li><li>Aragona, B. J., Liu, Y., Curtis, J. T., Stephan, F. K., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+nucleus+accumbens+dopamine+in+partner-preference+formation+in+male+prairie+voles.">A critical role for nucleus accumbens dopamine in partner-preference formation in male prairie voles.</a> Journal of Neuroscience. 23 (8), 3483-3490 (2003).</li><li>Lippert, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+assessment+of+stimulus-evoked+regional+dopamine+release.">Time-dependent assessment of stimulus-evoked regional dopamine release.</a> Nature Communications. 10 (1), 336 (2019).</li><li>Lack, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ecological adaptations for breeding in birds. , (1968).</li><li>O'Connell, L. A., Hofmann, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vertebrate+mesolimbic+reward+system+and+social+behavior+network:+a+comparative+synthesis.">The vertebrate mesolimbic reward system and social behavior network: a comparative synthesis.</a> Journal of Comparative Neurology. 519 (18), 3599-3639 (2011).</li><li>O'Connell, L. A., Hofmann, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes,+hormones,+and+circuits:+an+integrative+approach+to+study+the+evolution+of+social+behavior.">Genes, hormones, and circuits: an integrative approach to study the evolution of social behavior.</a> Frontiers in Neuroendocrinology. 32 (3), 320-335 (2011).</li><li>Banerjee, S. B., Dias, B. G., Crews, D., Adkins-Regan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+paired+zebra+finches+have+higher+dopamine+levels+and+immediate+early+gene+Fos+expression+in+dopaminergic+neurons.">Newly paired zebra finches have higher dopamine levels and immediate early gene Fos expression in dopaminergic neurons.</a> European Journal of Neuroscience. 38 (12), 3731-3739 (2013).</li><li>Gil, D., Gahr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+honesty+of+bird+song:+multiple+constraints+for+multiple+traits.">The honesty of bird song: multiple constraints for multiple traits.</a> Trends in Ecology &amp; Evolution. 17 (3), 133-141 (2002).</li><li>Tomaszycki, M. L., Adkins-Regan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+alteration+of+male+song+quality+and+output+affects+female+mate+choice+and+pair+bond+formation+in+zebra+finches.">Experimental alteration of male song quality and output affects female mate choice and pair bond formation in zebra finches.</a> Animal Behaviour. 70 (4), 785-794 (2005).</li><li>Woodgate, J. L., Mariette, M. M., Bennett, A. T. D., Griffith, S. C., Buchanan, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+song+structure+predicts+reproductive+success+in+a+wild+zebra+finch+population.">Male song structure predicts reproductive success in a wild zebra finch population.</a> Animal Behaviour. 83 (3), 773-781 (2012).</li><li>Zann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+in+a+Zebra+Finch+Colony+in+South-eastern+Australia:+the+Significance+of+Monogamy,+Precocial+Breeding+and+Multiple+Broods+in+a+Highly+Mobile+Species.">Reproduction in a Zebra Finch Colony in South-eastern Australia: the Significance of Monogamy, Precocial Breeding and Multiple Broods in a Highly Mobile Species.</a> Emu. 94 (4), 285-299 (1994).</li><li>Klatt, J. D., Goodson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin-like+receptors+mediate+pair+bonding+in+a+socially+monogamous+songbird.">Oxytocin-like receptors mediate pair bonding in a socially monogamous songbird.</a> Proceedings of the Royal Society of London B: Biological Sciences. 280 (1750), 20122396 (2013).</li><li>Pedersen, A., Tomaszycki, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+antagonist+treatments+alter+the+formation+of+pair+relationships+in+zebra+finches+of+both+sexes.">Oxytocin antagonist treatments alter the formation of pair relationships in zebra finches of both sexes.</a> Hormones and Behavior. 62 (2), 113-119 (2012).</li><li>Adkins-Regan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroendocrinology+of+social+behavior.">Neuroendocrinology of social behavior.</a> Ilar Journal. 50 (1), 5-14 (2009).</li><li>Goodson, J. L., Kabelik, D., Kelly, A. M., Rinaldi, J., Klatt, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+dopamine+neurons+reflect+affiliation+phenotypes+in+finches+and+are+tightly+coupled+to+courtship.">Midbrain dopamine neurons reflect affiliation phenotypes in finches and are tightly coupled to courtship.</a> Proceedings of the National Academy of Sciences the United States of America. 106 (21), 8737-8742 (2009).</li><li>Iwasaki, M., Poulsen, T. M., Oka, K., Hessler, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+dimorphic+activation+of+dopaminergic+areas+depends+on+affiliation+during+courtship+and+pair+formation.">Sexually dimorphic activation of dopaminergic areas depends on affiliation during courtship and pair formation.</a> Frontiers in Behavioral Neuroscience. 8 (210), (2014).</li><li>Gess, A., Schneider, D. M., Vyas, A., Woolley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+auditory+recognition+training+and+testing.">Automated auditory recognition training and testing.</a> Animal Behavior. 82 (2), 285-293 (2011).</li><li>Woolley, S. C., Doupe, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+context-induced+song+variation+affects+female+behavior+and+gene+expression.">Social context-induced song variation affects female behavior and gene expression.</a> PLoS Biology. 6 (3), 62 (2008).</li><li>Day, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D2+dopamine+receptor+activation+induces+female+preference+for+male+song+in+the+monogamous+zebra+finch.">D2 dopamine receptor activation induces female preference for male song in the monogamous zebra finch.</a> Journal of Experimental Biology. 222, (2019).</li><li>Hahn, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+of+male+black-capped+chickadee+songs:+relationship+between+acoustic+preference+and+performance+accuracy.">Discrimination of male black-capped chickadee songs: relationship between acoustic preference and performance accuracy.</a> Animal Behaviour. 126, 107-121 (2017).</li><li>Leit&#227;o, A., ten Cate, C., Riebel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-song+complexity+in+a+songbird+is+meaningful+to+both+male+and+female+receivers.">Within-song complexity in a songbird is meaningful to both male and female receivers.</a> Animal Behaviour. 71 (6), 1289-1296 (2006).</li><li>Day, N. F., Hobbs, T. G., Heston, J. B., White, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Critical+Period+Learning:+Striatal+FoxP2+Affects+the+Active+Maintenance+of+Learned+Vocalizations+in+Adulthood.">Beyond Critical Period Learning: Striatal FoxP2 Affects the Active Maintenance of Learned Vocalizations in Adulthood.</a> eNeuro. 6 (2), (2019).</li><li>Chen, Y., Clark, O., Woolley, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Courtship+song+preferences+in+female+zebra+finches+are+shaped+by+developmental+auditory+experience.">Courtship song preferences in female zebra finches are shaped by developmental auditory experience.</a> Proceedings of the Royal Society B: Biological Sciences. 284 (1855), 20170054 (2017).</li><li>Heston, J. B., White, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior-linked+FoxP2+regulation+enables+zebra+finch+vocal+learning.">Behavior-linked FoxP2 regulation enables zebra finch vocal learning.</a> Journal of Neuroscience. 35 (7), 2885-2894 (2015).</li><li>Adkins-Regan, E., Tomaszycki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monogamy+on+the+fast+track.">Monogamy on the fast track.</a> Biological Letters. 3 (6), 617-619 (2007).</li><li>Young, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+transcriptomic+profiles+underpin+monogamy+across+vertebrates.">Conserved transcriptomic profiles underpin monogamy across vertebrates.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2019).</li></ol>

The authors have nothing to disclose.

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.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60590/operant-conditioning-task-to-measure-song-preference-in-zebra-finches">Operant Conditioning Task to Measure Song Preference in Zebra Finches</a>. An affiliation&#160;was updated.
One of the affiliations was updated from:
W.M. Keck Science Department, Clarmont McKenna College &#8211; Pitzer College &#8211; Scripps College
to:
W.M. Keck Science Department, Claremont McKenna College &#8211; Pitzer College &#8211; Scripps College

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60590/operant-conditioning-task-to-measure-song-preference-in-zebra-finches">Operant Conditioning Task to Measure Song Preference in Zebra Finches</a>. An affiliation&#160;was updated.

Erratum: Operant Conditioning Task to Measure Song Preference in Zebra Finches

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.

<table><tbody><tr><td>(-)-Quinpirole</td><td>sigmaaldrich</td><td>Q102</td><td></td></tr><tr><td>acoustic foam</td><td>Coulbourn Instruments, Allentown, PA</td><td>H10-24A</td><td></td></tr><tr><td>audio amplifier - 2 channel</td><td>Amazon</td><td>Pyle PCAU46A</td><td>any small audio amplifier should work</td></tr><tr><td>Banner Engineering Q08 Series, emitter. SO60-Q08</td><td>&lt;a href=&quot;https://www.alliedelec.com&quot; target=&quot;_blank&quot;&gt;https://www.alliedelec.com&lt;/a&gt;</td><td>Stock #:70659809</td><td>photoelectric sensor</td></tr><tr><td>Banner Engineering Q08 series, receiver. EO60-Q08-AN6X</td><td>&lt;a href=&quot;https://www.alliedelec.com&quot; target=&quot;_blank&quot;&gt;https://www.alliedelec.com&lt;/a&gt;</td><td>Stock #:70699384</td><td>photoelectric sensor</td></tr><tr><td>Car stereo speakers</td><td>Amazon</td><td></td><td>Pioneer TS - F1643R</td></tr><tr><td>Digital I/O card</td><td>National Instruments</td><td>PCI-6503 or USB-6501</td><td></td></tr><tr><td>LED lights - under-counter</td><td>Amazon</td><td></td><td></td></tr><tr><td>multifunctional ribbon cable</td><td>National Instruments</td><td>180524-20</td><td></td></tr><tr><td>sound pressure level meter</td><td>Amazon</td><td></td><td></td></tr><tr><td>terminal block</td><td>National Instruments</td><td>777101-1</td><td></td></tr></tbody></table>

operant conditioning task to measure song preference in zebra finches

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.

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&#8211;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&#8211;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&#8211;48 h (t-test corrected for multiple comparisons; p &#60; 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 (&#62;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60590/60590fig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60590/60590fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60590/60590fig2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60590/60590fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Operant Conditioning Task to Measure Song Preference in Zebra Finches at JoVE.com

Operant Conditioning Task to Measure Song Preference in Zebra Finches

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 ...

operant-conditioning-task-to-measure-song-preference-in-zebra-finches

Nanyang Technological University

Research

JoVE Journal

Behavior

6.4K Views.  Claremont McKenna College – Pitzer College – Scripps College. 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.

Video: Operant Conditioning Task to Measure Song Preference in Zebra Finches

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval1, juvenile interactions2, opposite and same sex interactions3,4,5, and territorial interactions6. For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders7,8,9, and more recently to understand mechanisms and evolution of vocal communication among vertebrates10. Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds11. The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal&#39;s preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup ...

Experience is Instrumental in Tuning a Link Between Language and Cognition: Evidence from 6-  to 7- Month-Old Infants' Object Categorization

At birth, infants not only prefer listening to human vocalizations, but also have begun to link these vocalizations to cognition: For infants as young as three months of age, listening to human language supports object categorization, a core cognitive capacity. This precocious link is initially broad: At 3 and 4 months, vocalizations of both humans and nonhuman primates support categorization. But by 6 months, infants have narrowed the link: Only human vocalizations support object categorization. Here we ask what guides infants as they tune their initially broad link to a more precise one, engaged only by the vocalizations of our species. Across three studies, we use a novel exposure paradigm to examine the effects of experience. We document that merely exposing infants to nonhuman primate vocalizations enables infants to preserve the early-established link between this signal and categorization. In contrast, exposing infants to backward speech &#8212; a signal that fails to support categorization at any age &#8212; offers no such advantage. Our findings reveal the power of early experience as infants specify which signals, from an initially broad set, they will continue to link to cognition.

At 3-4 months, listening to human and nonhuman primate vocalizations boosts infants&#39; cognition; by 6 months, only human vocalizations exert this cognitive advantage. We describe an exposure manipulation that reveals the powerful shaping role of experience as infants specify which sounds to link to cognition and which to tune out.

At birth, infants not only prefer listening to human vocalizations, but also have begun to link these vocalizations to cognition: For infants as young as ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Neuroscience

A Standardized Protocol for Preference Testing to Assess Fish Welfare

Animal welfare assessment techniques try to take into consideration the specific needs and wants of the animal in question. Providing enrichment (the addition of physical objects or conspecifics in the housing environment) is often a way to give captive animals the opportunity to choose who or what they interact with and how they spend their time. A fundamental component of the aquatic environment that is often overlooked in captivity, however, is the ability for the animal to choose to engage in physical exercise. For many animals, including fish, exercise is an important aspect of their life history, and is known to have many health benefits, including positive changes in the brain and behavior. Here we present a method for assessing habitat preferences in captive animals. The protocol could easily be adapted to look at a variety of environmental factors (e.g., gravel versus sand as a substrate, plastic plants versus live plants, low flow versus high flow of water) in different aquatic species, or for use with terrestrial species. Statistical assessment of preference is carried out using Jacob&#39;s preference index, which ranks the habitats from -1 (avoidance) to +1 (most preferred). With this information, it can be determined what the animal wants from a welfare perspective, including their preferred location.

A fundamental aspect of assessing the welfare of animals in captivity is to ask whether the animals have what they want. Here, we present a protocol to determine housing preference in the zebrafish (Danio rerio) with respect to the presence/absence of environmental enrichment and access to flowing of water.

Animal welfare assessment techniques try to take into consideration the specific needs and wants of the animal in question. Providing enrichment (the ...

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

Biology

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR).&#160;This technique allows investigators to study early stages of development that were previously difficult to access.

The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications.

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and ...