For DHPG perfusions the 8 mm wells which access the neurons were replaced with Hibernate A medium with B27, and the inlet wells were replaced with freshly prepared 2.5-5% BSA. After replacing media, chambers were incubated for >30 min at 37°C. The syringe and tubing were filled with HBS then connected to the chamber. The BSA solution was perfused for ∼5 min prior to treatment, then the solution in the center inlet well was replaced with the pharmacological agent or vehicle control diluted in Hibernate A with B27. We visualized the perfusion of the non-BSA solution using a brightfield tissue culture microscope (Figure 8A).
