Naspm

We pharmacologically manipulated RA in vivo in freely behaving and singing birds as previously described^{33 (link),34 (link)}. Adult (>100 days post hatch) male bengalese finches were implanted bilaterally with microdialysis probes (CMA) targeted to RA. Accurate placement in RA was confirmed during surgery by extracellular recording of RA’s characteristic spontaneous activity. After recovery from surgery, the birds were housed individually within sound attenuating chambers (Acoustic Systems) on a 14/10 h light/dark schedule with free access to food, grit, and water, and vocalizations were recorded with a microphone fixed to the cage ceiling. PBS was continuously delivered to RA at a rate of 0.1 μl/min via a fluid commutator connected to a syringe pump outside the bird’s isolation chamber. To manipulate inhibitory function within RA, we switched from PBS to either NASPM (Tocris) or midazolam (Sigma). Because the switch occurred outside the isolation chamber, the birds remained undisturbed and continued to behave and sing normally through the transition from PBS to drug. A total of >100 undirected song bouts were collected during both PBS and subsequent drug dialysis for each experiment.

All reagents were prepared fresh into concentrated stocks as indicated below, and stored frozen at −20 °C. The following compounds were used in pharmacological experiments: 1-Naphthyl acetyl spermine trihydrochloride (“Naspm”, 100 mM in PBS, Tocris Cat No 2766); (+)-MK 801 maleate (“MK-801”, 100 mM in DMSO, Tocris Cat No 0924); 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (“NBQX”, 100 mM in DMSO, Tocris Cat No 0373); D-2-amino-5-phosphonovalerate (“D-AP5”, 50 mM in PBS, Tocris Cat No 0106); Retigabine (100 mM in DMSO, Tocris Cat No. 6233). For all experiments on MEAs, and immediately before adding a compound to the cells, an aliquot of the concentrated stock was first diluted 1:10 in room temperature DPBS and vortexed briefly. Then, the appropriate amount of working dilution was added directly to wells on the MEA and mixing was primarily through diffusion into the (500 µl) cell culture medium. Of note, where relevant the DMSO concentration in the medium was always ≤0.05% v/v.