Any suggestions to get morphology of neurons in the superior colliculus?

01 January 2020 3 7K Report

Hi everyone,

I need to get the morphology and do reconstruction of neurons in the superior colliculus following patch clamping. I tried DAB staining for several times, but I couldn't get a satisfied one.

I can see that the soma is intact and clear, but I can't see many dendrites or some dendrites seems to be chopped off .. The tricky thing is I also patched one neuron in visual cortex, but the cortical neuron looks good. Is there anyone having experience for labelling neurons in superior colliculus? Can people give me some suggestions? Many thanks!!!

The following is the protocal I use:

1. Record neurons with intracellular solution containing 0.5% biocytin, fill the neuron for 20mins, pull out the pipette slowly, and then keep the slice in the bath for 20mins.

2. Fix the slice with 4% PFA (overnight, in the fridge)

3. Wash in PBS (PH=7.38) for 3 times (10 mins each time), put the slice in 1% H2O2 for 15mins, and then wash in PBS for 3 times

4. Add PBS+1%BSA+0.3%Triton to the slice, leave for 1 hr. (I also tried 2% Triton, but still remain the same)

5. Suck the solution off the slice and add the ABC solution (1dropA+1dropB+PBS+1%BSA+0.3%Triton), leave overnight at room temperature.

6. Wash in PBS for 3 times.

7. Add DAB solution (I can see the slice getting very dark).

8. Stop reaction by washing in PBS, and then mount the slice.

Thanks,

Yoyo

Antonio Viñuela

Good morning. You can used other tracer like the FG (fluorogold). It´s retrograde tracers.

you can injected in varios nuclei that innervate SC.

Best regard

Aaron G Roseberry

Have you tried cutting your slices in a different orientation or recording from cells deeper in the slice? The fact that you got a good fill of a cortical neuron suggests that you are cutting off all of the dendrites of your cells in the SC. Alternatively, try recording for a longer period of time to allow the biocytin to diffuse more. I doubt that is the cause, but those are 3 options to try.

Shuang Jiang

Thanks for your suggestion, Aaron. I've tried recording deeper neurons in the slice, but still didn't look good. I will try other 2 options you mentioned. Aaron G Roseberry

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