Dear All
I need help in troubleshooting mossy fiber recordings. All my PhD and Post-doc experience so far was in SC-CA1 recordings (Both field and whole-cell recordings). For a new project, I am trying to record mossy fiber-evoked responses in CA3 cells with a low current stimulus strength (20-60uA). However, I am seeing a lot of polysynaptic activity in the recordings. I initially started the stimulation with a cluster bipolar electrode (which I frequently use for SC-CA1 recordings). However, most of the mossy fiber recording papers used a glass electrode for stimulation. So, I realized I might be stimulating a large set of fibers and hence changed the stimulating electrode to a monopolar glass electrode filled with recording aCSF (3-5MOhms, same pipettes used for CA3 recordings as well). But I still see a lot of polysynaptic activity and spontaneous activity.
Recording Conditions
Cutting and incubation in half sucrose and half NaCl-based solution. The hippocampal slices were cut at 10 degrees magic-cut whole hemispheres, both based on protocols from Peter Jonas's lab.
Recordings were obtained in a regular aCSF with 100uM PTX (No NMDAR blocker as I am clamping at -70mV). I do not have a fixed place for stimulating electrode position, I keep varying it from the inner DG to Hilus to proximity to the recording electrode. All positions gave me similar results.
I do start evoking within 3 minutes of breaking in. Should I wait for a longer period like 10 minutes?
I appreciate it if anyone could help me to get rid of the polysynaptic activity in these recordings.
Some suggestions and hope these will support your recordings (My experience is also in SC-CA1, but similar problem also occured before).
1. move the membrane test to the near end of each sweep (I see now it is presented just before the electric stimulus);
2. maybe you can try a smaller input stimulation even than 20uA? Usually I prefer trying to find the threshold which is sufficent to induce a very single minimum PSC.
3. Do you add QX-314 into the intracelluar solution?
4. If the problem remain when the hold-current (leaking current) of the cell
Jinming Zhang
Thanks for your suggestions
I tweaked some of the parameters today and was able to get a good response in one slice. I need to get consistently like this.
I made a parasagittal cut at 350um and reduced the PTX concentration to 10uM which largely reduced the polysynaptic activity. Moreover the responses obtained today have the characteristic features i.e. sharp AMPAR EPSCs with 1-1.5 ms rise time, 4-5ms latency after stimulation, 3 times facilitation ratio, a good number of synaptic failures. However in the same cells, I also got a paired-pulse depression, is it strange or common to see occasional PPD?
Raghava Jagadeesh Salaka
These sweeps look good.
Theoretically you should always see PPF when recording EPSC in my view, but I also got PPD in my previous experiments. I have no idea about this issue.