Alterations in ZENK Expression in Zebra Finches Reared by a Single Parent

Tareq Yousef, Jordan Fisk, Tara S. Perrot, and Leslie S. PhillmoreDepartment of Psychology and Neuroscience

Introduction

Dalhousie University, Halifax, NS Canada

Conclusions

Future Directions

References

Acknowledgments

Bolhuis JJ, Zijlstra GCO, Den Boer-Visser SM, Van der Zee EA. (2000). Localized neuronal activation in the zebra finch brain is related to the strength of song learning. Proc Natl Acad Sci USA 97:2282-2285.MacDougall-Shackleton SA, Hulse SH, Ball GF. (1998). Neural bases of song preferences in female zebra finches (Taeniopygia guttata). NeuroReport 9:3047–3052.Maney D. & Pinaud R. (2011). Estradiol-dependent modulation of auditory processing and selectivity in songbirds. Frontiers in Neuroendocrinology, 32:287-302.

Results

Methods ZENK-ir - Effects of Having a Single Parent

Song Learning Song Learning:• Having a single father did not

compromise song learning in male

offspring.

• Having a single mother did not reduce

similarity to father’s song to zero,

indicating there must be a minimal

level of similarity among all male

songs.

• All birds sang consistently.

ZENK-ir in NCM and CMM:• CMM, important for conspecific

recognition in males and song

preference in females, had the

greatest ZENK-ir in birds reared

without their father in response to

hearing playback of their father’s song

for the first time. Birds reared with a

father present (biparental or single

father) were familiar with father’s song,

possibly affecting ZENK-ir.

• In NCM, rearing group had no effect on

ZENK-ir in males, but appears to affect

ZENK-ir in females.

NCM and Song Learning:• Amount of ZENK-ir was positively

correlated with how well father’s song

was copied (Bolhuis et al. 2000), but

only when birds were reared by both

parents.

Determine if other factors, such as

number of male siblings, may affect

amount of ZENK-ir in finches reared

without a father.

Finches paired and allowed to breed

Biparental Father or mother removed 4-5d post hatch

Allow birds to mature in natural nest groups

Single female broods separated from other groups with adult males

Record directed song of adult male

offspring ZENK-ir – Song Learning and Response in NCM

Song Analyses

As part of a larger project examining the effects of having a single parent in a normally biparental species, we wondered how rearing offspring with only a father or mother might affect song learning in males and neural response to father’s song in males and females.In this study we removed one parent of a brood and examined:

•How well a son copied their father’s song•Whether zenk protein immunoreactivity (ZENK-ir) to playback of

father’s song was correlated with strength of learning in males (as in Bolhuis et al. 2000)

•Whether ZENK-ir to father’s song varied in females reared with one or both parents

• Recorded father’s directed song before breeding

• Recorded offspring’s directed song after PHD 90

• Used Sound Analysis Pro (Tchernichovski) to assess:

• Similarity to father’s song (% of song copied)

• Of the song copied from father, how accurate that copy was

• Similarity to self (stability)

Sons with single fathers did not have lower song similarity or accuracy to father’s song than sons in biparental nests.Sons without fathers had lower accuracy copying father’s song, and a trend toward lower similarity (p=0.055).

Despite differences in song quality, all offspring sang their own song consistently, and were not different from fathers’ consistency.

The authors wish to thank Ashley Lockyer, Dominique Shephard, and Jaya Wadhawan for help with bird care, video recording, and project management.

*

Recording of fathers and male offspring were collected by exposing birds to live females and vocal recordings after isolating the male overnight in a sound-attenuated chamber. IHC for zenk protein (SC-189) followed standard protocols after playback of father’s song to offspring. Images captured at 20x and ROI placed as in Maney et al. 2011.

Fathers Biparental Single Mother Single Father0

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Playback father’s song to male and female offspring Isolation overnight, 30 min playback, sac 60 min after end playback,

Record responses to playback.

Section brain tissue (coronal) and process in immunohistochemistry for zenk. Count number of labeled cells within ROI placed in NCM and CMM.

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Analyzing all male offspring together, there was no correlation between ZENK-ir and similarity to father’s song (Spearman’s rho (14)=0.02, p=0.29) or accuracy of copy (rho(14) = 0.04, p=0.89).

Males reared by both parents: ZENK-ir and similarity were positively but not significantly correlated (rho(4)=0.32, p=-.68); same for ZENK-ir and accuracy (rho(4)=0.95,p=0.051).

Males reared by a single mother: ZENK-ir was negatively but not significantly correlated with similarity (rho(3)=-0.50, p=0.67).

Males reared by a single father: ZENK-ir was negatively but not significantly correlated with similarity (rho(7)=-0.23, p=0.61).

Biparental Single Mother Single Father0

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NCMImage captured at 4x, coronal section, ROI placed within NCM.

A group x sex ANOVA showed no differences between rearing groups or between sexes. A group x sex interaction F(2,15) = 3.58, p=0.054) indicated a trend that ZENK-ir differed between males and females in the single mother group but did not differ between males and females in the other groups.

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**

A group x sex ANOVA showed no difference between sexes nor a group x sex interaction. However, there was a significant effect of group F(2,15)=6.59, p = 0.01; offspring reared by single mothers had greater ZENK-ir to playback of their father than birds reared with their father.

CMMImage captured at 4x, coronal section, ROI placed within CMM.

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R² = 0.000526485506956442

Number of ZENK-ir Cells in NCM

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R² = 0.00171002121608044

Number of ZENK-ir Cells in NCM

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R² = 0.0382433076513931

Number of ZENK-ir Cells in NCM

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R² = 0.0167905797980418

Number of ZENK-ir Cells in NCM

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R² = 0.0380518104969466

Number of ZENK-ir Cells in NCM

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Biparental Single Mother Single Father