The directed song is sung within the proximity of the female and is thought to be the mating song. The undirected song is used alone and when in society driven environments where the male will sing to himself and possibly towards a potential mate, but not for territory. In roughly 90 days following the beginning of song development, the finch has a fully functional song that is ready to be tested in either scenario. There also appears to be notable physical differences in the behavior of the male when in company of a female. Zann’s previous experiments show that during the undirected song, the male will change posture and maintain no particular form. However during the directed song, the male usually goes into a full display form. This postures continues while the male tries to stiffen up and stick out his chest as much as possible. The songs are both regulated by gonadal hormones. The frequency of songs are correlated with levels of testosterone, but undirected songs have higher thresholds. Since the song is only sung by the male and the song is the same is both situations, the Z. Finch makes for a perfect specimen in our experiment.

It has been observed that specific brain regions process different information from the stimuli that is sent through the ear. The L2 area of the Z. Finch processes auditory stimuli. NCM then recognizes the stimulus. Caudo-medial nucleus, NCM for short, is the dominant area we are observing for ZENK. This auditory processing is important because of the ZENK response after the bird is subjected to either a novel song or a habituated song.

ZENK is an immediate early gene response seen in the auditory regions of the brain. Research suggests that the ZENK response may be a protein level response to auditory memories. For our research we will be using ZENK as a marker to reflect a difference in song discrimination. ZENK is labeled using antibodies that will bind to the protein and dye brown when introduced to an immunocytochemistry. We should expect to see large levels of ZENK labeled in the NCM area of the avian brain.

As previously shown by Mello 95 that when a Z. Finch is exposed to the same song there is a decrease in ZENK production in the NCM. He concluded that hearing a novel song after being habituated did indeed produce more ZENK in the brain region. For our experiment we are trying to detect if the female Z. Finch can distinguish between an undirected song and a directed song. If the female can, we shall look for a difference in ZENK response in the NCM to represent this behavior.

For our experiment we are using 3 adult female Zebra Finches that have been kept adequately fed and watered. The birds have been placed in cages that are being held in sound proof chambers specifically designed to house live animals. We have installed a speaker for each of the three chambers, and will be playing either a directed of undirected song to each female.

The compact discs contain 30 minutes of playback consisting of 15 seconds of song followed by 45 seconds of silence. Prior to beginning the experiment, the birds were placed on a time schedule of 14 hours of daylight and 10 hours of darkness. The birds were allowed to adjust to this cycle for several days before playback began. Over the course of 3 days each, the birds were subjected to the song playback 30 minutes after lights on. After the birds listened to 2 days of the same song, we divided the birds into a habituation, novelty and experimental groups. The habituation was played the same song and the other two heard either a undirected novel song or a directed novel song.

Independent Variables

After this final day we waited for a few hours to give time for he ZENK response to reach its peak before overdosing the Z. Finches with an anesthetic. We perfused the finch with 4% paraformaldehyde for preservation. We then dissected the brain into two hemispheres, followed by two sagittal slices. The brain was bound onto the microtome base using super glue. The brain was fresh, and was cut in saline. Slices measuring 40 micrometers each were cut ensuring we collected the NCM area of the tissue. We removed each slice with a state of the art painter’s brush and placed them into more saline solution for further storage. At room temperature we prepared sections to be free floating while quenching them in endogenous peroxides with hydrogen peroxide to give the first antibody stain something to bind to. We used 5% normal goat serum solution. The primary antibody was left overnight egr-1 antibody (1:1000 dilution) purchased from Santa Cruz Biotechnology. The next day we did secondary antibody reaction tissue with biotinylated antirabiit antibody solution. The brain tissues stayed for one hour before rinsed with saline 3 times for 3 minutes each. We then reacted the tissue with avidin-biotin peroxides solution. We dyed the peroxides with 0.05% 3, 30-diaminobenzidine solution. After rinsing with saline 3 additional times, the tissue samples were mounted on slides with a coverslip. We used a microscope with stage and camera connected to a computer running Microbrightfield Stereo Investigator Software, 1000X magnification with Differential Interference Contrast optics; our super expensive toy we like to use to look at 3 sections of NCM counted per bird. An Optical Fractionator probe was used to generate random site on the NCM where stained spots of ZENK were counted and labeled. This generated a total number of ZENK labeled cells per section. We calculated the density of ZENK labeled cells by dividing the total number of section by the volume of the three sections.

After counting the ZENK labeled cells in the NCM, we used the computer to estimate the density of ZENK in each of the 3 brain portions. The novel group showed an expectedly high level of ZENK density. The habituation results were about half of that of the novel as exposure leads to lower ZENK production. Finally we analyzed the experimental group that revealed a response that resembled the habituation group very closely. This evidence is shown in the figures where the dark spots represent ZENK labeling.

These are the graphed results of our ZENK measurements.

Experimental

Habituation

Novel

From the results we collected, the female bird does not recognize the directed song as a novel song from sound stimulation alone. This may mean that the undirected and directed songs are closely perceived by the female and no difference can be recognized from sound alone. Also the recordings of the songs may be corrupt from that of the real life repetition done by the male’s presence. The albums played back 15 seconds in a pattern that may not be sufficiently timed to the male’s timing. The female may have also become habituated to the male’s song and pattern after the 2 day exposure and the mild change in timing for the directed song was not enough to trigger a novel sound recognition within the female.

No males were in contact with the birds during the recording process as to ensure that only song playback was stimulating the finch. Therefore the normal sexual behavior the male displays while in the mating song may strongly affect how the female perceives the males song. Since the females could not get to a male, the female probably just suspected that the male’s call was not a directed but instead just the same habituated undirected song. Without the visual stimulation of a male, there may be no recognition of directed or undirected.

The male performs a series of physical acts that are viewed every time a male is singing in the directed form. This may be an evolutionary cue to the female that the male is not singing his song to another mate, but is in fact available to reproduce. When the female spots the male and becomes aroused, she may then be recognizing the song as directed instead of undirected. Little evidence is known if there is a difference in the recoding time of the two song types, or if the difference is complimented by a timing sequence designated by the male in response to the reception of a female.

When the female is hearing the undirected song, she may be hunting for a potential partner. When she captures the attention of a partner, pheromones may released affecting the male’s behavior sexually. Perhaps this stimulates the male to introduce himself to the female and mate. If the male responds to the females signal, than he will begin directing his song to her where he would be causing the female to have a hormonal fluctuation. This fluctuation may be a possible correlation with the female recognizing a male’s song as directed or novel.

It is my recommendation that the next study should use a male to deliver the female’s last day of directed song rather than the recordings. Perhaps using the same bird will keep the song the same, but produce the timing and personal touch of the male delivery. In comparison the cold speaker is delivering the message while the hidden female is being held away from the potential mater’s calling song. The visual stimulation of a male may be essential in the recognition of a novel song and implementing this in the next study would test for validity.

As an alternative to using the male’s presence, hormonal injections could be administered to the female to check the response of just gonadal levels. If the bird feels the way she does when male is normally present making a song directed, will she interpret the song as novel? Pheromones may be ruled out by keeping the male from sight while still reproducing hormone levels of an excited female.

In conclusion, the Zebra Finch female does not show a ZENK level response that would suggest that she is capable of distinguishing between directed and undirected songs from the same male after being habituated to the male’s undirected song. Our statistics show that the ZENK response in the experimental Z. Finch matched that of the habituated bird. More studies incorporating the use of male presence will be necessary in the future to draw further conclusions about what is necessary for females to interpret a song as being directed. The experiment was a success as we were able to collect all of our data from the tissue samples, and interpret our results into scientific evidence.

Mello, Claudio et al (1995). Repeated Exposure to One Song Leads to a Rapid and Persistent Decline in an Immediate Early Gene’s Response to That Song in Zebra Finch Telencephalon. Journal of Neuroscience. Rockefeller University New York, NY.

Mello, Claudio and Sidarta Rebeiro (1998). ZENK Protein Regulation by Song

in the Brain of Songbirds. Journal of Comparative Neurology. Rockefeller University New Work, NY.

Zann, Richard (1996). The Zebra Finch: A Synthesis of Field and Laboratory Studies. Oxford University Press. Oxford.