These different findings could be reconciled by a model in which HVCX neurons accumulate feedback information slowly (hours to days) and where feedback-driven changes in these cells first appear as a subtle modification of synaptic input, rather than changes in

action potential output. Testing this model requires a way of longitudinally monitoring synapses on identified Lumacaftor solubility dmso neurons before and after manipulation of auditory feedback changes song output, a goal currently impractical to achieve using electrophysiological methods. In vivo, multiphoton imaging of fluorescently labeled neurons can resolve individual dendritic spines, which are postsynaptic components of excitatory synapses in the vertebrate brain (De Robertis and Bennett, 1955 and Palay, 1956), and this method has been used in a variety of longitudinal studies to measure experience-dependent changes to synapses (for reviews, see Alvarez and Sabatini, 2007, and Holtmaat and Svoboda, 2009). Recently, this method has also been used to show that auditory experience of a vocal model stabilizes

and enlarges HVC dendritic spines in juvenile songbirds over a period of days (Roberts et al., 2010), advancing it as a suitable method for detecting relatively slow feedback-related changes to synapses in the HVC of adult songbirds. Here, we used longitudinal in vivo two-photon imaging of dendritic spines in deafened adult zebra finches to test the idea that synapses on HVC PNs are ABT-199 ic50 sensitive to changes in auditory

feedback. To label and identify HVC projection neurons for in vivo imaging, a GFP-lentivirus was injected into HVC, and differently colored retrograde tracers were injected into the two downstream targets of HVC, the striatal region Area X and the song premotor nucleus RA, in young adult male zebra finches (Figure 1A and see Figure S1A available online; 80 to 150 days posthatch (dph), mean age was 97 ± 5 days, all reported errors are SEM unless otherwise noted). Birds were maintained on a reverse day-night cycle and imaging sessions were conducted during the birds’ subjective nighttimes, to minimize interference with singing behavior Phosphoprotein phosphatase (2 sessions per night separated by a 2 hr interval). Images were obtained through a cranial window and collection of imaging data was restricted to neurons with dendritic spines, because both populations of HVC PNs are spinous (Mooney, 2000). Neurons were identified as either HVCX or HVCRA cells by the presence of blue or red retrograde label or, in the absence of retrograde label, by the measurement of soma size, which differed significantly for the two PN types (Figures 1A and S1B). After collecting 1–2 nights of baseline imaging data, birds were deafened by bilateral removal of the cochleae, and data collection was continued as long as possible (13 birds were imaged for an average of 7.2 ± 4.1 nights postdeafening).