Alexafluor conjugated dextran amines

To reversibly inactivate the eyeblink region(s) of the cerebellum (anterior interpositus nucleus and/or eyeblink region of the cerebellar cortex), 100 nl of 1 mm muscimol (Tocris) dissolved in artificial CSF [aCSF; containing the following (in mm): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 2 CaCl₂, 2 MgCl₂, and 12.5 dextrose; pH adjusted to 7.4 after addition of muscimol] was infused before the training session. The solution was loaded into a Hamilton syringe fitted with Tygon tubing and a sterile infusion cannula (Plastics One, 33 gauge cannula that projected 1.8 mm from the bottom of the guide) and manually pressure injected at a rate of 25 nl every 15 sec. The training session began 15 min after the infusion to allow the muscimol to diffuse. Mice were given 2–3 d of additional training sessions before receiving a control infusion of a 10% solution of AlexaFluor-conjugated dextran amines (488, 3000 MW, Invitrogen) dissolved in aCSF. The same procedures were used for control and muscimol infusions. Mice received a final training session on the day following control infusions.

The same procedures used to inactivate cerebellar regions were used for unilateral reversible inactivation of the mPFC or bilateral reversible inactivation of amygdala central nuclei (100 nl of 1mm muscimol manually pressure injected at a rate of 25 nl every 15 s, for each hemisphere in cases of amygdala inactivation). After 2–3 d of postmuscimol training sessions, these mice also received control infusions of a 10% solution of AlexaFluor-conjugated dextran amines dissolved in aCSF (AlexaFluor 488–10,000 MW in the side ipsilateral to the trained eye, and AlexaFluor 594–10,000 MW contralateral to the trained eye; Invitrogen).