Hi Chenglong,

how do you load the FM into the SVs? Do you see a specific puncta-like labeling?

Natalia

Hi Natalia,

We use the same electrical stimulation for labeling and excytosis. We also tried to use weak protocol for labeling. But they all can not label very well as 70K. I can not see many puncta-like labeling by electrical stimulation. But I see it with 70K. Is it the problem of labeling?@Natalia

I understand now that 70K works continuously through perfusion to hold membrane potential at a high level, and duration of my electrical stimulation is shorter than it.

But some experts told me that our current protocol is strong enough (1200 pulses, 10Hz, 1ms duration). 

Anyway, thanks a lot.

Hi, Chenglong,

I guess the unreliable field stimulation might be due to the inadequate power of isolator Iso-flex (max Power could be estimated by the product of max V x max I). If you could observe the vesicle turnover of sypHluorin, that implies that at some area of the network the field potential is enough for triggering vesicle turnover, but not competent for elsewhere. I guess the pHluorin signal in your experiment might not be that strong either, maybe only several puncta flash in the AOI. I highly suggest a high-power isolator, such as WPI A385. Since the fluctuation of the surface of perfusing solution would cause dramatical change of resistance between Pt electrodes, I suggest using constant current mode instead of voltage mode. Usually the current I is set 50mA to ensure the vesicle turnover everywhere in the stimulating field. Furthermore, practically 600AP at 10Hz would cause similar FM staining as that under the induction of High K (40-50mM,

Hi Chenlong Yu,

FM dyes lable a lot (synaptic vesicles, lysosomes, Golgi, etc.) depending on conc. Ca/Mg ratio, and duration of staining.

In cultured hippocampal neurons we were very succesful with FM1-43 and FM4-64 when incubating cells for 1-2 min in 50 mM K solution (replacing the same conc of extracellular Na!) with 2 mM Ca and 0 mM Mg to maximize vesicle turnover. Then, extencive washing with 0 Ca ACSF is required to get rid of non-endocytosed FM dyes. If this procedure does not yield punctate staining of synaptic boutons in your preparation something might be wrong with the dye. I attach 2 papers from our lab, where the labeling is described in detail.

Kind regards, Volkmar

http://www.ncbi.nlm.nih.gov/pubmed/11689429

http://www.ncbi.nlm.nih.gov/pubmed/11746350

Hi Chenglong, 

usually, FM dye can be loaded only very string stimulus e.g. KCL stimulation or 10Hz, 1000APs. You can see a weak labeling with milder stimuli, but it is not sufficient for a good release. People are still trying to understand what is the mechanism behind it. Now you can release the FM very well with KCL, strong or mild stimuli, but in the last case, you need a very sensitive camera to detect the changes. I would rather say that you might have a problem with the detection. Are you sure that your camera is sensitive enough? We use around 50nA for our filed stimulation. There is not need to perfuse KCL, a pulse is usually enough. Good luck!

Field stimulation by its nature is 'unreliable' you can never be sure that cells are driven one-to-one by the stimulation. In addition the use of high voltages will generate (even with platinum) some electrochemistry and potentially other artefacts. Check that you really have good connections (electrical) and that current is flowing between the electrodes. Equally, K depolarization is nothing like natural stimulation (continuous) and if you have not carefully corrected for K:Cl ratio it can cause significant water flow and unwanted synaptic effects unrelated to depolarization. The short answer is to take results of both approaches with a pinch of salt. Lastly you do not say which cells you are using. Are you sure the cells are excitable? K+ will work on in-excitable cells when electrical stimulation will not.

So check connections and excitability (by another means).

...I would suggest to FM lable with high K and then try to release FM from the same cells with electrical stimulation, in first place. If this is not succesful make a positive control with high K also for release of FM.

Best, Volkmar

Dear Hang,

Thank you for your detailed information.@Hang Zhou 

You are right that release of pHluorin is not so many in my images (but obvious, I would say). I also worry about the fast decay of electricity in the field. We are buying a Grass stimulator, hope it can work. I tested the peak amplitude of our current isolator, which is ~90V. Maybe I need either increase that peak amplitude or find a way to keep it from fast decay.

Can you explain more about perfusion? I usually give a high speed of perfusion, will that be a problem for resistance? I tried current mode but not so obvious changes. I will try more with the amplitude you suggested.

Thanks for the detailed information. Let’s keep contact.

Best regards,

Yu

Hi Volkmar,

Thank you for the papers. I will read carefully. @Volkmar Lessmann

Do you mean to use 50mM KCl and 50mM NaCl in the ACSF? I will try that. I though about using different concentration of Ca/Mg, but not allowed by my supervisor. He do not allow me to increase concentration of Ca, although someone used 3mM. But I can try to use 0 Mg for test.

So, do you think labeling is the problem? Maybe I should work more on 50K labeling and electrical stimulation for exocytosis. I could trigger good release with high K, but somehow my supervisor want to use electrical stimulation for excytosis. I think he want the temporal resolution.

Thank you again for the paper. Maybe I can present them in our group meeting this week.

Best regards,

Yu

Hi Natalia,

Thank you for your suggestion. @Natalia L Kononenko

I am little bit confused about the amplitude. 50nA is the amplitude from electrode or the one you applied to neuron? I mean, is it the amplitude you measured near the neuron? Or the original amplitude from the electrode. Is it the readout from patch? I think I need to measure in our system with patch.

But I think our camera is fine. We are using iXonEM DU897 from Andor. I admire your stimulation. Somehow we are using 1200 Aps, but still can not label very well as high K.

Hope we could have a good electrical stimulation as you.

Thank you again.

Best,

Yu

Hi Euan,

Thank you for the advises. @Euan Robert Brown

That is new knowledge for me. Can you explain more about K:Cl ratio and its effect on water flow? According to Hang, the fluctuation of ACSF may change the resistance between electrodes. Does it mean that something wrong with my ACSF? We use 150mM NaCl, 4mM KCl, 2mM MgCl2, 2mM CaCl2, 10mM Hepes and 10mM Glucose. Is that OK?

I am sure about the connection as the readout of oscilloscope is normal (~90V in max) between the two electrodes. But I have not test the real amplitude within the solutions. You are right about using another means, and I will check it by patch without neurons.

I use cultured cortical neurons. But I found they are not so excitable as hippocampal neurons which I tested previously. But I could not trigger FM release even in hippocampal neurons. I tried Ca image to check the response of my neurons. But not obvious response even under 70K.

Anyway, thank you for the advises.

Best,

Yu

Hi, Chenglong @Chenglong Yu

You can consider the solution between the electrodes as continuous and flexible Resistors in parallel (however the real case is more complicated, for example, the ionization would affect the resistance significantly), and then we can assume that the higher the liquid surface, the smaller the resistance (R), and vice versa. If the liquid surface is concave (convex), the resistance in the middle part will be bigger (smaller) than the part near the electrodes, thus it will allocate larger (smaller) voltage, leading to uneven distribution of potential between electrodes. This might cause the heterogeneous characteristics of elicited vesicle turnover, to avoid this, the voltage or current should be set much higher to ensure the constant AP eliciting. Anyway, keeping the liquid surface flat and stable is very important. The speed of perfusion itself might not observably affect the resistance, but the higher speed might cause the fluctuation of surface and subsequently affect the resistance distribution.

Let's talk about the isolator again. Firstly one knowledge should be clarified here, what is "Constant Voltage Source". Optimal constant voltage source is defined as a power source without internal resistance (i.e. Ri=0 Ohm) in electric engineering. However, it does not exist in real world. Thereby practically we take a power source with very small Ri as a constant voltage source (how small is Ri, that depends on the different design of the equipment). Knowing this, we can analyze two typical cases during field stimulation.

1) If the liquid surface in the chamber is very low, then the equivalent R of solution is very big.

(continue) @Chenglong Yu

(roughly R is ~500-1000 Ohm, but it depends). I could not find the design map of the Iso-flex from website, then the internal resistance Ri could not be determined. But for the Max Output Power (MOP), we can estimate based on two parameters: Voltage output: 0-90V, Current output: 0-10mA, thus MOP is roughly 90Vx10mA =900mW. If you set the output voltage 90V, the expected current through electrodes will be around 90V/1KOhm = 90mA, then the power between electrode will be 90Vx90mA=8100mW>>900mW, means the Load is too high for the MOP, or say it is overloaded. In this case, the actual voltage between electrodes would be much smaller than 90V, which might not be adequate for inducing vesicle turnover.

2) If the liquid surface is very high, then the equivalent R of solution in chamber is very small, let's say 100-300 Ohm (but it depends). In this case, we could not neglect the internal resistance Ri of the isolator. The actual voltage between electrodes depends on the ratio of 90V x R/(R+Ri), which might be much smaller than 90V.

It should be noted that in both cases the key problem is the OVERLOAD of power.

In addition, the Iso-flex can only give monophasic but not biphasic pulse, leading to the ionization of solution near electrodes during high-frequency repetitive stimulation, such as 1200APs 10Hz stimulus. This might cause the change of local R and subsequently affect the local vesicle turnover.

Finally, small tip: always double-check the input signal generated by the stimulator and make sure the voltage of signal is > 5V, otherwise one can not even trigger the TTL circuit of isolator and no (or very weak) output pulse will be elicited.

Good luck! 

Hello Hang,

Thank you so much for the detailed information. I will do some test and email you for the progress.

I tested the amplitude within our chamber during stimulation. It is 50mV. Is it strong enough to trigger AP? And we saw some release today with the new stimulator.

I will keep testing and let's keep in tough. 

Thank you again. I really learnt a lot.

Best,

Yu

I have loaded FM dye at the nerve terminals of NMJ using high K and then stimulated the phrenic nerve using 50 and 100 Hz stimulations to study exdocytosis.

Hi Bula,

Thank you. I would like to use high frequency, but somehow we do not want to cause plasticity changes. Am I safe by using 10 Hz?

I indeed saw many release of pHluorin with 100 Hz.

Best,

Yu