Hi Luca,

are you doing whole cell of field recordings? What brain area? Have you changed really nothing in your experimental design? Why would you suspect the animals are more stressed now than when you first made the experiments? Your answers may help me help you.

Chronic psychisocial stress has been shown to adversely affect early and late phase hippocampal LTP in rodents depending on the location. Please see papers below:

 I would add that tbis should not suppress LTP completely so it is unlikely that stress from the animal facility is hr cause. Especially if they've been acclimated for at least few days before. It is likely method related. Check the equipment and the electrodes. Also for recording field potentiala  if the electrodes are functioning properly then keep in mind that the longer you stimulate or try to induce LTP the less likely you will be able to get a response in an animal. If you don't find it within 20 min it becomes very difficult to find the response in my experience. Good luck

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Hi Luca,

I believe it is unlikely to be stress, unless you are experimeting in a war zone or there is intense drilling or noise in your facility. Check with the facility manager any significant changes lately? Also check your animals if they look totally agitated, restless trying to escape or relaxed? Even so I would still expect to see some residual LTP. I think you should check your equipment, connections and batteries if you are using 9V batteries in your stimulator. Make sure everything is in good working order.

best wishes,

Refik

Hi guys,

thanks for your advices. I'll try to explain what the situation is at the moment. I'm recording fEPSPs in the CA1 of the hippocampus. In my set-up the only thing that i modified was the perfusion system. Now i'm using a peristaltic pump with 2 head for perfusion and aspiration of the ACSF. All the other stuff are the same respect the last experiment. I have very big problems in finding good responses. I remember that after few hours from the slicing procedure i was able to put stimulating and recording electrodes and to find very quickly good responses with very good amplitude and shape (4/5 mV amplitude with population spikes also). Now, nothing, very few responses of 1 mV. I tried to put in my acsf also bicuculline (in CA1 is not necessary anyway) but nothing. I tried also in DG with bicuculline but nothing at all. I tried several stimulation protocols (100 stimulai@250 Hz, 100 stimulai@100Hz, 100stimulai@100Hz x 4) but nothing! I don't know what is happening. 

Thanks so much for the help guys

Dear Luca,

 I recommend that you check everything (solutions, connections, bath flow, cellular viability) one by one and eliminate all the culpirits. You should also check your tissue, whether the cells are healthy or not. For example, if your vibratome for some reason is not cutting good-healhy tissues, you will not get good responses. I would fix and counterstain couple of sections at the end of the experiment if the cells are intact and that will also allow you to check all the anatomical landmarks. Also check your flow rates with your new system and see if the tissues are getting enough bubbled solution.

Good luck and all the best,

Refik

Thanks for the advices Refik. Yesterday i check visually, with my 40X, the quality of pyramidals neurons and they looks like pretty nice. The acsf was ok (one of the firts things that i changed), maybe there is a little bit of noise ora the flow rate is too low. I'll try to increase the flow and to solve the noise. Yuo know what is the best blade angle of the vibratome to make best coronal hippocampal slices? I'm using 10°

Hi Luca,

Sounds good. The flow rate should be around 4 ml/min and also make sure that the incoming solution and the suction are at opposite poles of the bath, and that your harp does not prevent fresh solution directly hitting your tissue. My vibratome is manually adjusted, so I guess around 10 to 20 degrees angle should be good. Are you removing the duramater and brain membranes before you cut sections?

Well, I'm cutting with 10° angle, so seems to be ok. Before cutting i just remove the brain that i put in ice-cold cutting solution and after few minutes i cut cerebellum and a little bit of prefrontal cortex before to do coronal sections

I would go and check the stimulator, in case you are using a battery operated one, and the wiring. This sudden failure to induce proper responses may be simply down to either used up batteries or bad connections to your electrode...

I'd agree with Paulo that it sounds like a stimulator issue. I haven't much slice experience (all in vivo LTP here) but checking your connections and having a supply of replacement batteries is essential with electrophys. What works today might not work tomorrow and the usual culprit is a crocodile clip that isn't making good contact or a battery has gone dead because the unit wasn't turned off after the last use.

Thanks Paulo and John, 

I'll check all the connections of my set-up but This is not a problem of batteries because my isolation unit is directly linked to a power supply connected to the electricity with no batteries :)

Thanks a lot

Dear Luca, besides all the excellent points mentioned before it might be worth checking for an infection the animals might suffer from.

How about to check your isolated stimulator?

Sometimes it goes wrong without any reason.

Try again with another one.

It may help you

Dear Luca, if the slices look OK visually it is probably not a problem of vibratome or blade angle. The fact that you don't see usual field EPSPs may be due to a failure of stimulation or a failure of transmission. Do you see the afferent voley and is it normal? If yes, then this would mean that you ability to stimulate is not the problem (the stimulator is OK, the presynaptic fibers are not depolariized and so on). Then, the problem is either the release or the postsynaptic neurons. If the Afferent voley is fine, I would rather think that all the cells in the slices are ok and thus that your postsynaptic CA1 pyralidal cells are not depolarised or dead either. If you or a colleague can patch some CA1 neurons in your slices, you will be able to have a better idea of what's going on (value of the resting membrane potential, input resistance, AP threshold, frequency of EPSPs...). You said that you changed your perfsusion system. Are you sure that the new tubes you are using are good ones, i.e. do not release something that blocks release or postsynaptic receptors. If you are using glue to seal the bottom or differrent parts of your recording chamber, some of those can also release dirty stuff that block some channels...