II. Recording dissociated pyramidal neurons

Uninfected Control Neurons

1. Refer to the primary neuron culture protocol (lab website).

2. Recording range: DIV 6–14.

3. DIV 7–12 is optimal for A-type current recording.

4. Neurons near coverslip edges are healthier than those at the center.

5. Small clusters (2–3 neurons) are preferable.

6. Healthy neurons have a voluminous soma
   (SCJ ver.: visible dark shadow indicating spherical shape).

7. Criteria for healthy neurons

   • Whole-cell capacitance:
     DIV 7: 8–12 pF; DIV 14: 15–23 pF
     (exclude >15 pF at DIV 7 or >25 pF at DIV 14)

   • Membrane potential after break-in: −50 to −60 mV

   • Stable Vm within ±5 mV during recording

These conditions were used for Kim et al., 2007 (Neuron).

Infected Neurons

1. Refer to the infection protocol (lab website).

2. Do not use Kv4.2 or Kv4.2W362F neurons more than 2 days post-infection
   (best: 1 day; SCJ ver.).

3. Healthy infected neurons show green spines on soma and dendrites.

4. Finding infected neurons may take time.

5. Criteria are the same as for uninfected neurons.

6. Kv4.2/Kv4.2W362F fluorescence is weaker than eGFP.

7. Excessive numbers of green neurons indicate problems.

SCJ ver. External solution for Dissociated neurons

pH 7.3 (adjusted with NaOH)

• For A-type current recording, add TTX (0.5 µM).

• For NMDA current recording using a Picospritzer, add TTX (0.5 µM), strychnine (2 µM), and bicuculline (10 µM).

• For chemical LTP induction, CaCl₂ (2.5 mM) can be used, and glycine (200 µM) can be applied for 5 min.