Effects of lentivirus-mediated downregulation and upregulation of FoxP2 on incorporation of new song elements in adult male canaries

Christopher Thompson, postdoctoral student

Adult male canaries incorporate new song elements into their repertoires at a significantly higher rate in the fall when circulating levels of T are undetectable and expression levels of FoxP2 in Area X are elevated (1). One way to test if FoxP2 plays a role in the incorporation of new song elements in adult birds is to block the activity of FoxP2; this will be achieved using RNAi. The Scharff lab has engineered a lentivirus that selectively knocks down the expression of FoxP2 mRNA and have successfully used it reduce the ability of juvenile zebra finches to copy a tutor’s song (2). I will inject the FoxP2-knockdown or a control virus into the Area X of both hemispheres in adult male canaries while exposed to breeding conditions, transfer these birds to nonbreeding conditions by withdrawing T and photoshifting them to SD, and continually monitor singing behavior in order to monitor the incorporation of new song elements into each bird’s repertoire.

In addition, if reducing the activity of FoxP2 leads to decreased incorporation and/or production of new song elements, then upregulating FoxP2 expression may increase the variability of singing behavior. Overexpression of FoxP2 in Area X may sufficiently drive synaptic plasticity to allow greater incorporation of new song elements under conditions when adult male canaries are least likely to change their repertoires. I will develop a virus which overexpresses FoxP2 and use it increase the activity of FoxP2 in adult male canaries held under breeding conditions. I hypothesize that overexpressing FoxP2 mRNA in Area X will lead to an increase in the variability of singing behavior, including increased incorporation of new song elements.

T-withdrawal and upregulation of FoxP2 expression in Area X of male canaries

FoxP2 expression in Area X appears to be inversely correlated with circulating levels of T (1). A direct link between circulating T and FoxP2 expression in Area X is unknown, however. I propose to test the link by manipulating circulating levels of T in adult male canaries with subcutaneous Silastic capsules filled with T as well as castration. I hypothesize that a rapid withdrawal of circulating T will upregulate FoxP2 expression in Area X. I will rapidly transition adult male canaries from breeding to nonbreeding conditions in order to determine how rapidly FoxP2 expression is upregulated. I will bring the birds into breeding condition by exposing them to long day photoperiod (20:4; LD) and implanting subcutaneous T-pellets, which are necessary because LD alone tends to be insufficient to induce an increase in circulating levels of T in birds held under unnatural, laboratory conditions. After 28 days, I will remove the T-pellets, castrate the birds, and rapidly shift them to short day photoperiod (8:16; SD). I will sacrifice three groups of birds 0, 7 and 20 days after T-withdrawal and photoshift. These time points are based on results from my dissertation on seasonal regression in white-crowned sparrows in the Brenowitz lab (3); they may be modified depending upon preliminary results for this specific aim. I will examine levels of FoxP2 mRNA in Area X with in situ hybridization and/or real-time PCR.

Do the metabolites of T, DHT and E2, mediate the upregulation of FoxP2 expression in vitro?

It is well established that T can be metabolized into two different sex steroids: estradiol (E2) through the activity of aromatase, and the nonaromatizable androgen 5-dihydrotestosterone (DHT). Neurons in HVC express both androgen receptors (AR) and estrogen receptors (ER), and aromatase is highly expressed in brain areas near HVC. Aromatase inhibitors decreases the volume of HVC, and concurrent estrogen replacement reduces the decrease (4). Administration of either non-aromatizable androgens or estrogen alone induce growth of the song control system, though the growth is less than that seen with the administration of T alone (5). It should be noted, however, that in canaries that social context can influence the effects of T on song control nuclei volume (6). In addition, T induces enhanced singing behavior and growth of HVC with 11 days, whereas administration of DHT or E2 is not as effective as T in male canaries (7).

It is unknown if T, DHT, or E2 modulates the expression levels of FoxP2 in Area X. I will assess the contributions of each of these hormones to FoxP2 expression using an established in vitro protocol for songbird brain long-term cultures (8, 9). I will block steroid hormone receptor with drugs such as flutamide for AR and tamoxifen for ER. Each slice will be evaluated for expression levels of FoxP2 mRNA using in situ hybridization and/or real-time PCR.  I hypothesize that I will observe downregulated expression of FoxP2 in Area X in sections exposed to T and DHT and observe high levels of FoxP2 in sections treated with T + flutamide, DHT + flutamide, and sections not treated with hormones or antagonists.  I also hypothesize that sections treated with E2, and E2 + tamoxifen will have upregulated FoxP2 expression levels because there are no reports of ER in Area X, thus FoxP2 expression is not likely to be affected by estrogens.

Does targeted photolysis of HVC neurons block the seasonal upregulation of FoxP2 in ipsilateral Area X?

T may act directly on Area X neurons to modulate FoxP2 expression. Yet there are good reasons to believe that seasonal changes in FoxP2 expression may depend upon afferent input from HVC. An intracranial T-implant placed near HVC induces an increase in the volume of ipsilateral HVC, RA, and Area X in male white-crowned sparrows; an implant near RA does not induce growth of ipsilateral song system nuclei (10). In addition, lesions of HVC block the growth of ipsilateral Area X in male birds given subcutaneous T-pellets (11). It is unknown if afferent input from HVC is necessary for changes in FoxP2 expression levels in Area X. The Scharff lab is ideally suited to test this hypothesis because they can selectively kill neurons that project to Area X and leave the rest of the neurons in HVC intact using targeted photolysis (12). I will expose male canaries to LD and implant them with T-pellets. 21 days later, I will inject chlorin e6–conjugated rhodamine-labeled nanospheres into Area X in one hemisphere, which will be retrogradedly transported to the somas of HVC neurons that project to Area X. The contralateral hemisphere will receive a sham injection. After 21 days, I will use a laser to release the chlorin and selectively kill neurons that took up the nanospheres. Seven days after targeted photolysis, I will sacrifice one group of males while still exposed to LD and high levels of circulating T and shift another group to nonbreeding conditions. The second group will be sacrificed at either 7 or 20 days after T-withdrawal and photoshift, depending on which time point has maximal FoxP2 expression based results obtained in Specific Aim 3. In every group, I will inject a retrograde tracer into Area X bilaterally in order to assess the effectiveness of targeted photolysis. I will compare ipsilateral Area X to the contralateral, so each animal serves as its own control.


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       Proc Natl Acad Sci U S A 104, 15520 (Sep 25, 2007).
 (4)  K. K. Soma, A. D. Tramontin, J. Featherstone, E. A. Brenowitz,
       J Neurobiol 58, 413 (Feb 15, 2004).
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       Neuron 25, 481 (Feb, 2000).