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Kirn Lab Poster Summer 2015
- 1. Understanding the Timelineof New Parvalbumin-Expressing Neuron Addition in HVC During the Zebra Finch Critical Period Jennifer Cummings, Kemal Asik, John Kirn* Neuroscience and Behavior Department, Wesleyan University Jennifer Cummings Wesleyan University Jacummings@wesleyan.edu Contact 1. Wilbrecht, L., Williams, H., Gangadhar, N. & Nottebohm, F. High Levels of New Neuron Addition Persist When the Sensitive Period for Song Learning Is Experimentally Prolonged. Journal of Neuroscience 26, 9135–9141 (2006) 2. Hensch, T. Critical period plasticity in local cortical circuits. Nature Reviews Neuroscience 6, 877–888 (2005). 3. Kirn, J. The relationship of neurogenesis and growth of brain regions to song learning. Brain and language 115, 29–44 (2010). References Songbirds give us great insight into the cellular processes underlying vocal development and critical periods because songbirds, like humans, are vocal learners. A cortical pre-motor region called the High Vocal Center (HVC) plays a key role in the sensory-motor processes important in both song learning and maintenance in songbirds. We hypothesize that the addition of new inhibitory interneurons in HVC expressing parvalbumin (PV), a calcium-binding protein known to be important for critical periods in other systems, will occur at a higher rate during the critical period than in adulthood and that it can predict the extent to which tutor song has been copied. Through immunohistochemical identification, the presence of newly added PV-expressing interneurons in zebra finch HVC at different points during the critical period is quantified in order to construct a clear timeline of PV-expressing neuron addition, an important indicator in understanding the neural mechanisms of plasticity and sensory-motor learning Abstract § Birds are injected with BrdU, a thymidine analog that is incorporated into cells forming on the day of injection, to label new cells at 20, 40, or 60 days, or in adulthood. § All birds are killed and perfused at 120 days with 4% paraformaldehyde § Brains are embedded in Polyethylene Glycol (PEG), sliced with a microtome into 6 µm thick sections, and mounted on slides. § Immunohistochemical processing is performed to label neurons, new cells, and PV-expressing cells in sections. § Slides are imaged for fluorescent labeling on a confocal microscope and stitched together using Adobe Photoshop. § Neurons are quantified manually. Methods and Materials § We have quantified a very small sample size of birds, so no conclusions can currently be made about the data. § In the future, we are investigating the fate of cells formed at 20, 40, and 120 days post-hatch, both in isolates and birds reared normally. § If our hypothesis is supported, we expect to see higher rates of PV- expressing neuron addition in juveniles during the critical period than in adult birds, a delay in the decline of PV-expressing neuron addition in isolates, and a positive correlation between PV-expressing neuron addition and tutor song copying. Discussion and Future Directions Song System Circuit Introduction § Songbirds, like humans, are vocal learners, meaning that they must form auditory memories of adult songs, engage in vocal motor practice, and receive auditory feedback throughout adulthood in order to perfect and maintain their songs. § Also like humans, songbirds possess early critical periods during which exposure to adult song is necessary for an individual bird to learn and replicate it. In zebra finches, most song learning is finished after 90 to 120 days post hatch, at which point the bird is considered an adult. § A cortical pre-motor region, the High Vocal Center (HVC), plays a key role in the sensory-motor processes important in both song learning and maintenance. § Neurogenesis to the HVC is high during the critical period. A drop in neurogenesis is associated with the closing of the critical period. It has been found that isolating birds so that they are not exposed to a tutor can extend the critical period and high rates of neurogenesis to HVC will persist. (Wilbrecht et al., 2006). § Our experiment looks at parvalbumin (PV), a calcium-binding protein expressed in inhibitory interneurons that is associated with plasticity (Hensch, 2005). § We hypothesize that in HVC, the addition of new interneurons expressing PV will be highest during the critical period and that there will be a positive correlation between PV-expressing neuron addition and tutor song copying. Results From Reiner et al., 2008 Timeline of Experiment. Needles indicate BrdU injection points. Microphones designate when birdsong was recorded and indicate that experimental birds’ songs were compared to tutors’. During the sensory period, the young bird is forming a memory of its tutor’s song, and during the sensorimotor stage, the bird is practicing and processing its auditory feedback in order to replicate the tutor song. Modified from Rochefort et al, 2007 HVC connects both directly, via what is referred to as the “motor pathway” (solid line), and indirectly, via the “anterior forebrain pathway” (dotted line) to the Robust Nucleus of the Arcopallium (RA). RA projects to regions of the brain stem that innervate respiratory and motor neurons (Kirn, 2010). In this image, HVC is surrounded by a dotted white line. Hu-labeled cells (blue) are neurons, BrdU- labeled cells (green) were born on the date of injection, and PV- labeled cells (red) are expressing the protein Parvalbumin. Two PV/ Hu double-labeled cells are designated with thin arrows. One triple-labeled cell, indicating a new PV-expressing neuron, is identified with a bold arrow. 50 µm BrdU/Hu/PV This graph compares average number of neurons expressing Parvalbumin (Hu/PV) and number of new neuronss expressing Parvalbumin (Hu/PV/BrdU) in birds that were injected at 60 days old, both isolates and birds raised normally. Preliminary results indicate that isolates have higher rates of parvalbumin expression in general and in newly formed neurons, as is consistent with our hypothesis. I would like to thank Professor John Kirn for giving me the opportunity to work in his lab and Kemal Asik for guiding me. I would also like to thank the McNair Foundation and especially Ronnie Hendrix and Suzanne O’ Connell for their continued support. Acknowledgements 0 5 10 15 20 25 30 Hu/PV Hu/PV/BrdU + Cells per Thousand HVC Cells Cellular Markers Expressed Neurons in HVC Expressing PV and BrdU WT (60dph) n=5 Isolates (60dph) n=3