I've been working with field electrophysiology in the hippocampus of mice for 10+ years. I recently started electrophysiology in rats and for the life of me cannot create reproducible LTP! This is by either 2 trains 100Hz, 1 train 100Hz, or theta burst stimulation. I am beyond frustrated. If I tell you what I'm doing, perhaps you could shed some light on what I may be doing wrong?
-Cervical decapitation on awake Sprague Dawley rats (2 to 12 months)-no anesthesia.
-Brain removed within 30-60 seconds after decapitation into ice-cold oxygenated sucrose cutting solution.
-Brain chills for approximately 30-45 seconds before cutting 400um horizontal sections on Vibratome while remaining chilled.
-Dissected hippocampi rest at RT 50/50 Oxygenated ACSF/Cutting.
I thought it was my ACSF recipe since my d-glucose concentration was significantly higher than most other publications, but that hasn't changed even with lower glucose:
ACSF: (in mM) 125 NaCl, 3.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 10 d-glucose, 2.5 CaCl2, 1.3 MgCl2
-Slices mounted on interface chamber by Automate Scientific (https://www.autom8.com/bsc1-interface-submerged/) for 1 hour 20 minutes to 2 hours. ACSF is oxygenated and heated by TCU to 34 C (temperature of the slices ends up being 30 C by the time it flows around the interface netting). This chamber allows for a humid environment to reduce drying out the slices. Flow rate is ~1.5 ml/min by peristaltic pump.
-Stimulating with bipolar nichrome formvar electrodes at the edge of CA3
-Recording at CA1 with ACSF-filled recording electrodes (I use pClamp software for signal acquisition).
-Stable and strong signals are achieved before running input-output curve.
-Stimulus intensity level determined by 1/2 max fEPSP attained from I/O curve.
-Record for 20 minutes before initiating high-frequency stimulation (either 100Hz or TBS).
It should be noted that I get reproducible LTD with chemically-induced LTD with DHPG (haven't tried LFS).
-My signals will either remain the same intensity, reduce, or completely disappear during or after HFS.
Some slices will dry out before I even record.
Perhaps my electrode position is wrong? I usually look for optimal fEPSP slopes at about 5 ms past stimulation. This is what I normally do for mice. Perhaps the distance needs to be greater?
I would love some assistance with this. I have aged animals that need electrophysiology done now, but I cannot euthanize them knowing that they will not produce LTP.
Thanks in advance!
You can try to perfuse your animals in a chilled choline chloride solution before removing the brain and also cut your slices in the same solution! For me coronal slices are better than dissecting the hippocampus! And I never used an interface chamber! Maybe you should try a regular chamber where the slices are submerged! And keep the rest of the slices in a bath at RT! concerning atimulation I will go for a 3x 100Hz! Good luck!
I have been trying to induce LTP in 4-5 month old Wistar rats for last 3-4 years. I had done more than 50 animals and nearly 100 slices and tried all different protocols. To my surprise I could get LTP (>40%) very rarely, may be once in 25-30 slices. As Paula Parra suggested, we do cold aCSF perfusion under isofluorane anesthesia but it just improved the EPSPs quality but not LTP induction. I believe this is because of the strong GABAergic population in adult animals. We dont use any GABAergic inhibitor for our studies. Are you using Picrotoxin or Bicuculline ? Our results with 100nM picrotoxin is, the induction of LTP (succes rate ) has improved compared to just aCSF but the percentage of LTP is not so high (around 40%). Current we are repeating the experiments with Picrotoxin and Bicuclline.
Note: With less than 2 months old animals, I used to get good succes rate in inducing LTP ( 4/5 out of 10 slices).
Yes I do use Picrotoxin! And I make a cut between CA3 and CA1. I use a Chilled Choline Chloride instead of ACSF for perfusion and cutting. I seems to be better for older animals (2M+). Also check the stabiloty of your perfusion system, It can be very important for fields LTP.
Paula Parra and Raghava Jagadeesh Salaka : your use of GABAa antagonists intrigues me. I am a bit nervous to try this as the genetic model I’m preparing to use is known for GABAa Beta subunit 3 deficiencies which can lead to seizure. Haven’t tried them since I was not yet successful with control Sprague Dawley rats. I’m nervous to induce overexcitation with PT or Bicc. What are your thoughts?
In that case better not to use PTX!!!! But because your having problems eliciting LTP I should try it with PTX in one control and in one of your experimental animals and see how it looks like! You can eventually use physiological CaCl2 and MgCl2 (1.2mM each) to have less excitability....
I know that it will not help you immediately with your problem, but I think when you have time this lecture would help you:
https://videocast.nih.gov/summary.asp?Live=13678&bhcp=1
I have heard from many other labs about the problems with eliciting LTP (so, I think you are not alone on this). The lecture gives an overview of problems in the field. If my memory serves correctly, Dr. Nicoll divulges that there is actually no agreed upon definition of LTP. Anyway, I think it would be worth your time to take an hour and sit down and watch it.
Cheers,
Ted
Melinda Peters and Paula Parra I too working in epilepsy field. I could not use GABAergic blockers in my experiment since Epilepsy is all about imablance between excitation and inhibition and I dont want to block one of these components. We started using PTX to verify why there are so many failures in LTP induction in adult rats. Our lab here at NIMHANS, India has published several paper in LTP with good succes rate in young adults (
here is my recipe for choline chloride cutting and slicing if you want to try it.
First of all try 8 weeks old mouse with anesthesia i hope it would be an ideal combination even we have recorded 8 month old mouse hippocampus activity , 2nd thing that we are using 2.5 kCL instead of 3.5, and 0.2 MGSO4 instead of MgCl2. Recording through MEA chips and our slice recording success ratio is almost 90% to100%.
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