Dear All, I want to measure the fEPSP slope for LTP. But I met some problem I cannot solve.
First, my fEPSP is too small and just hold about 1 ms.
Second, whatever stimulating the slice by HFS or TBS, I cannot induce LTP.
Next, I sum up my protocol here. Thanks for your help.
1. My slicing buffer and ACSF.
Slicing buffer: 87 mM NaCl, 25 mM NaHCO3,25 mM glucose, 75 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5mMCaCl2 and 7 mM MgCl2
ACSF: 126 mM NaCl, 26 mM NaHCO3, 25mM, glucose, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, and 1 mM MgCl2. And adding 50µM PTX to block GABA receptor.
2. Conditions of anesthesia: pentobarbital sodium for 200 mg/kg
3. Slicing parameter:
Coronal section for hippocampus in 300-400µm. Incubate the slice for 30min in 34℃。
4. Stimulating and recording electrode
Stimulating electrode: < 1 MΩ, fulling with ACSF.
Recording electrode: 1-2 MΩ, fulling with 2 M NaCl.
Both two electrode made by glass capillary
Electrode position is in figure 1, and Stimulating electrode is in figure 2.
5. Flowing velocity in recording chamber is 1mL/min.
6. The waveform of fEPSP and the inducing condition.
The waveform is in figure 3.
Inducing condition: Stimulus pulses were delivered at 0.05 Hz. the stimulus intensity selected for baseline measurements was adjusted to yield about 40–60 % of its maximal amplitude
7. HFS and TBS
HFS: 3 trains of 100 pulses at 100Hz with inter-train interval being 10 s.
TBS: four trains of 5 bursts, with each burst having five pulses at 100 Hz, 200 ms between bursts, and 5 s between trains.
It doesn't seem to be a success fEPSP recording cause the extremely narrow wave (~1ms). Typically, a good fEPSP that can be observed in CA3-CA1 fiber has a width of ~20ms or larger, wheras the amplitude may various across Labs.
Some personal suggestions:
1. A metal stimulation electrode may be better than a glass one;-----Larger stimulation area.
2. Increase the primary gain in your 700B software, try to find a proper parameter. -----increase both the signal and noise.
3. put the recording electrode onto the stratum of the CA1 area for fEPSP recording. if you put the electrode in the cell line, mostly PS will be observed.
Here are some literatures may help:
doi:10.1101/pdb.top087262
doi:10.1101/pdb.prot091298
also my own protocol which you can find in:
doi: 10.1016/j.neuroscience.2020.03.016
doi: 10.1016/j.bja.2023.01.019
Best,
Jinming
- Badges
- Science topic
- Similar topics
- Engineering
- Electrical Engineering
- Electrodes