1. Understanding the Timeline of 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