II. Recording dissociated pyramidal neurons

Uninfected control neurons

1. Refer the protocol to make the primary neuron culture (Lab web).

2. Recording is possible in the range of DIV 6~14 for whole-cell recording.

3. Div 7-12 is best for recording A-type currents.

4. Neurons located in the middle of cover slips are weaker or worse than those located closely to the boundary.

5. Groups clustered with two or three neurons are better than a single neuron. But groups clustered with many neurons are not better than a single neuron.

6. Neurons should have a volumed cell body (SCJ ver. Find the dark shadow of cell body line, The shadow indicates that cell shape is likely a ball). Gently push the cell body with recording pipettes to check if neurons have volume.

7. Parameters to determine good neurons.

• Whole-cell Capacitance;

In the range of Div 7-14, Div 7 usually shows 8-12 pF whole-cell cap. and Div 14, 15-23 pF. If neurons show higher capacitance (in case of Div 7 over 15 / Div 14 over 25), don’t use them. The membrane of neurons are too weak to make a whole-cell.

• Membrane potential right after breaking whole cell;

Div 7-14 neurons generally show -50 ~ -60 mV. Don’t use if neurons shows higher Vm (>-50 mV). Don’t try to find neurons showing lower Vm than -60 mV (It will waste much time)

• Membrane potential change during recording;

Don’t use neurons if they are showing +/- 5 mV changes of Vm during normal recording.

* These conditions were used for Kim et al., 2007, Neuron paper.

Infected neurons

1. The infection protocol can be referred in the Lab website.

2. In all case of Kv4.2 and Kv4.2W362F, don’t use infected neurons more two days after infection (Best is only one day after infection according to SCJ ver.).

3. Infected and healthy neurons should show beautiful green spines in the soma and dendrites (Kim et al., 2007, Neuron). However, younger cells cannot show many spines. Therefore, don’t care spines if you use Div 6-10 neurons. Otherwise, you should find green-infected neurons showing green spines.

4. Waste much time to find green neurons on a cover slip. Sometimes only one infected neurons can be found in a whole cover slip, and it can be so healthy.

5. All parameters for good neurons are same with uninfected neurons.

6. Brightness of Kv4.2 and Kv4.2W362F is weaker than that of eGFP. Too brightened neurons after infection mean “I am not alive” or “Virus is not working” – Absolute law.

7. Don’t be excited if you see too many green neurons on a cover slip. It means “something is wrong”.

SCJ ver. External solution for Dissociated neurons

pH 7.3 (with NaOH)

• For A-current recording, Add 0.5 uM TTX.

• For NMDA current by using Picospritzer, add TTX 0.5 uM, strychinin 2 uM, bicuculine 10 uM.

• For chemical LTP, CaCl2 2.5mM can be used and Glycine 200 min for 5 min can be applied for the induction of chemical LTP.