Indeed, the finding that deafening alters the spontaneous action potential output of HVCX neurons OSI-744 ic50 in anesthetized birds hints that the output of this cell type may also be altered during singing. Although the exact role of HVCX neuron output in singing awaits full elucidation,
a recent study in Bengalese finches implicates the singing-related action potential activity of these cells in the encoding of syllable sequences (Fujimoto et al., 2011), a song feature that is sensitive to feedback perturbation, including deafening (Sakata and Brainard, 2006 and Woolley and Rubel, 1997). Ultimately, long-term measurements of the effects of deafening on the singing-related activity of HVCX neurons await the development of in vivo functional imaging techniques or improvements to single-unit recording methods in songbirds. All procedures were in accordance with a protocol approved by the Duke University Institutional Animal Care and Use Committee. See also Supplemental Experimental Procedures. Male zebra finches (85 to 150 dph) were anesthetized by isoflurane inhalation (2%) and deafened by bilateral cochlear removal. Complete removal of each cochlea was confirmed by visual inspection under a microscope. Standard parametric and nonparametric statistical analyses were used for all comparisons (alpha = 0.05); reported errors are SEM unless otherwise noted. Undirected
song was recorded continuously starting at least 2 days before deafening until at least 1 week postdeafening. To assess Palbociclib price song degradation, spectral features of song were quantified by measuring the Wiener entropy and entropy variance (EV) of each syllable in a bird’s song using Sound Analysis Pro (Tchernichovski et al., 2000). Thirty examples of each syllable were measured on each day of song, and values from two predeafening days were pooled to obtain a baseline distribution of entropy and EV values for each syllable. The onset of song degradation for each bird was defined as the day on which the distribution of values for either the entropy or EV of any syllable
differed significantly from the baseline distribution and remained significantly different on all subsequent days (one-way ANOVA). Syllable sequence changes were measured using sequence consistency (adapted from Scharff and Nottebohm, 1991). The onset of temporal change Endonuclease to song was defined as the first day on which the mean sequence consistency was less than the lower bound of the 95% confidence interval for the mean sequence consistency measured on the last predeafening day. Birds were anesthetized with isoflurane inhalation (2%) and placed in a stereotaxic apparatus. Injection sites were localized using stereotaxic coordinates and multi-unit neural recordings. A glass pipette attached to a Nanoject II (Drummond Scientific) was used to deliver GFP-lentivirus (eGFP expressed under the control of the Rous Sarcoma Virus LTR [FRGW]) to HVC or neuronal retrograde tracers to Area X and RA (lentivirus: 32.