Extracellular Recordings - Science method

I am afraid that is not ideal. Using a single electrode to record in vivo local field potentials (LFPs) in the hippocampus is generally not recommended because: 1. Lack of Reference Point: LFP recordings measure the summed electrical activity of populations of neurons. Without a separate reference electrode, it’s hard to distinguish between true neural activity and background noise or artifact. A single electrode does not provide a way to differentiate local changes in electrical potential from broader shifts in the electrical field. 2. Volume Conduction: The hippocampus is embedded in a complex neural network, and the electrical signals recorded by a single electrode can include not just local activity but also signals that are conducted from other brain regions. This "volume conduction" effect makes it difficult to isolate hippocampal activity with a single electrode. 3. Spatial Resolution: A single electrode does not provide information about spatial variations in the neural activity across different regions of the hippocampus. This is especially problematic because the hippocampus has distinct anatomical and functional regions (e.g., CA1, CA3, dentate gyrus) that can have different activity patterns. Multi-electrode arrays or tetrodes allow for simultaneous recordings from multiple locations, improving spatial resolution and allowing for more precise mapping of hippocampal activity. 4. Difficulty in Signal Interpretation: LFPs represent a mixture of signals from excitatory and inhibitory neurons, synaptic potentials, and even glial cell activity. With only a single electrode, it is much harder to interpret which sources are contributing to the recorded signal, making the analysis less reliable. 5. Signal-to-Noise Ratio (SNR): Single electrodes are prone to picking up noise, which can be difficult to filter out when there is no comparison or control signal from nearby regions. Using multiple electrodes allows for better noise cancellation and more accurate isolation of the signal of interest. In summary, using a single electrode for LFP recordings in the hippocampus is limited because of issues with reference point clarity, signal contamination from volume conduction, reduced spatial resolution, and difficulties in interpreting the resulting signal. Multi-electrode setups help overcome many of these issues, providing a more accurate representation of hippocampal activity.

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However, it's still technically possible if necessary for specific experimental constraints. Here is a draft protocol: 1. Preparation - Anesthetize the animal with an appropriate anesthetic (e.g., isoflurane or ketamine/xylazine) and place it in a stereotaxic frame to secure the head. Ensure that the animal is fully anesthetized by monitoring vital signs (e.g., absence of the pedal reflex). - Shave the scalp and sterilize the area with ethanol and iodine. Make a midline incision to expose the skull. - Use a stereotaxic atlas to determine the precise coordinates for the hippocampus. Mark the area on the skull. 2. Craniotomy and Electrode Placement - Carefully drill a small hole in the skull over the target location using a micro-drill. - Insert the reference electrode in a distant brain region (e.g., the cerebellum or a peripheral muscle). Fix the ground/reference electrode securely. - Lower the recording electrode: Using the stereotaxic manipulator, slowly lower the single recording electrode into the hippocampus. Advance the electrode slowly to avoid damaging the tissue. 3. Signal Amplification and Recording - Attach the recording electrode to a pre-amplifier and connect the reference electrode to the ground of the system. - Adjust filtering parameters: Set up the amplifier to filter the signals between 1 Hz and 300 Hz (the typical frequency range of LFPs). Adjust the gain of the amplifier to ensure the signal is visible but not saturated. - Monitor the signal: Observe the incoming LFP signal in real-time on the data acquisition system. Adjust the electrode depth if necessary to optimize the quality of the signal. LFPs are typically visible as slow oscillations in the frequency range of 0.5 to 100 Hz, depending on brain state. 4. Recording - Once the electrode is properly placed, begin recording LFP data. Record for a suitable time period depending on your experimental question (e.g., several minutes to hours). - Monitor physiological parameters: Ensure the animal remains properly anesthetized throughout the procedure and check for any signs of discomfort or distress. Continuously monitor vital signs. 5. Post-recording - Electrode removal: Once recording is complete, carefully remove the electrode without damaging the tissue. - Close the craniotomy: Clean the exposed area of the skull and apply dental cement to cover the craniotomy. Use sutures to close the scalp. - Postoperative care: Administer analgesics (e.g., buprenorphine) and monitor the animal until it recovers from anesthesia. Place the animal in a warm recovery area. 6. Data Analysis - Filtering: Apply additional digital filtering if necessary to isolate LFPs from noise. - Artifact removal: Identify and remove artifacts from movement, heartbeat, or respiration. - Spectral analysis: Perform frequency-domain analysis (e.g., power spectral density) to examine oscillations like theta (4-8 Hz), gamma (30-80 Hz), or sharp wave-ripples (~100-200 Hz). - Signal-to-noise analysis: Evaluate the quality of the recording and signal-to-noise ratio.

Dear Paulo,

can you expand a bit more on the two types of experiments which your students performed? What did they observe and what did they measure?

I usually refer to input-output when I discuss the spike rate versus injected current for a neuron, but you may mean something else here.

I think that the speed of the flow rate can influence fEPSP amplitude. You may believe that your flow rate is constant between conditions, but if your system is gravity fed, it could be that the flow rate varies depending on the height of the solution.

Apostolos' idea about reference electrode is worth considering, but I believe that changes between reference (ground) and recording electrode will influence the absolute baseline values, but not the amplitude of the fEPSP.

The record appears to have a very slow time base, if I am reading it correctly (5 secs per horizontal box). The electrode resistance is very low. What is the solution in the pipette? If you're still using your internal solution from whole cell recording, my guess is that l you're looking at junction potential fluctuations due to bath solution and pipette solution intermixing at the solution interface at the pipette tip. Depending on what you aim to record, you might consider changing electrode types and amplifier.

Hi, thank you for the rapind response, but i'm looking for references for spherical correction, these links introduce psychopy, the third link talks about mouse clicking responses and i'm talking about the animal mouse

Thank you very much for your fast reply and valuable suggestion:) Yes, I guess it is definitely worth it to adapt glass electrodes and see.

Hi Fatemeh Shariftabar ,

There are no fixed biophysical characteristics (spike amplitude/magnitude of response, firing rate and frequency, response delay rate and the ratio of onset or offset of the responses, etc.) for the firing or responses that one may record during LFP after the stimulation of various brain regions or fibers.

The shape, size, delay... of the neuronal responses vary greatly depending on the type of the fibers that you would stimulate and the neurons that you would record from (e.g. compare the responses from Purkinje cells in cerebellum with those of the pyramidal neurons in the CA1, or even in hippocampus itself we have granular neurons in DG that would show different response characteristics than pyramidal neurons. This will even become more tricky about the cortical layers where there are thick-tufted and fast spiking neuorns vs other subtypes).

LFP signals may be theoretically larger in magnitude (and the slope of the EPSP) compared to APs because they are mainly recorded as the postsynaptic responses but these might be either excitatory or inhibitory and even with different characteristics about STDPs...

So, I would recommend that you would not think of a fixed formulated scheme or pattern about the shape of the LFPs, EPSPs, APs,... as a standard single-shaped response, if you are stimulating and recording in hands-on experiments on rats/mice.

For a pilot experimental study, first you should have more info regarding the region of interest and where in the brain you would like to stimulate and record neuronal activity. Then you can sort your stimulating parameters (current magnitude to be injected, single or train, interstimulus intervals, stimulation time and periods, recording times, ...). Frequency and even other parameters of a signal (EPSP, AP, IPSP, STDP,...) is a dependent variable in relation to the type of neuron, region and the aim of your study. For example, different parameters are used for induction of LTP and LTD!

But if you are aiming for modelling a specific type of neuron, firing after let's say a train of injected currents with fixed and defined characteristics, then it would be different and you can use Matlab toolboxes or Python to simulate a neuronal activity.

If you would need more help, please provide more details about the mentioned points and the circuit that you would like to design.

Yes, for example, look at the website for BlackRock microSystems, as well as TBSI. I think some labs as well might have designed their own devices.

Dear Jacci,

welcome,

Sue i came late to your forum but my answer may help all colleagues who have troubles with interference on their electrodes. The problem with such electrodes is that they have high impedance loops and therefore they are subjected to the pick up of high frequency nearby fields. 

So, you have to isolate and shield them very well. 

To complement the answer please see the correct procedure for stimulation and recording in the paper at the link:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291724/

The authors gave the precautions to avoid interference from the environment.

Best wishes

If these are the probes you mean!?

Fully integrated silicon probes for high-density recording of neural activity

Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank

Thank you both for your help. It will help a lot

Hi Jacci Newel , sorry I misunderstood the way your microscope is set up. If your slice does not look at all like the one in the file attached, I think it may still be a contrast issue. It's hard to tell without seeing the rig but an easy way to improve your contrast would be to tilt your transmitted light source a bit to get some sort of 'oblique' effect. It could be as easy as putting a bit of tin foil to cover part of the light source. You could even 3D print a wedge in clear-ish plastic to get a gradient.

Dear Jerome,

I have never used copper wires for recording and I also never heard about someone using this metal for extracellular recordings. Tissue reactions to the electrode in contact with the CNS can be minimized if one uses electrode material (metal core and insulation material) which is biocompatible. I always differ between chemical and mechanical compatibility with the tissue. In case of the metal I would not recommend to use copper, because it is well known as a toxic material for biological systems. Silver and copper wires usually are proved to be the most toxic materials to brain tissue. For details see Geddes & Roeder (1).

Babb & Dymond (2) reported that tissue reaction to the implanted electrode can be minimized by proper selection of both electrode and insulation materials . The authors stated: “…Implanting in brain tissue for 2 months without passage of current showed platinum, platinum-8 percent tungsten, platinum-l0 percent rhodium, platinum-10 percent iridium, platinum-10 percent nickel, platinized platinum, a gold-nickel-chromium alloy, a gold-palladium-rhodium alloy, a chromium-nickel-molybdenum alloy (Vitallium), stainless steel, rhenium, gold and boron to be nontoxic (Dymond et al ., 1970) . Silver (Dymond et al ., 1970), silver chloride, and copper (Fischer et al ., 1961) are highly toxic…”

I understand that you are recording from an insect brain. I know from researchers using twisted wire tetrodes in insects made of 12µm diameter coated nichrome wires. (3) This might be a better choice. Please find the cited literature below:

1. Geddes LA, Roeder R. 2003. Criteria for the selection of materials for implanted electrodes. Ann Biomed Eng 31:879-90.

2. Babb MI, Dymond AM. 1975. Electrode implantation in the human body. Bull Prosthet Res:51-150.

3. Guo P, Pollack AJ, Varga AG, Martin JP, Ritzmann RE. 2014. Extracellular wire tetrode recording in brain of freely walking insects. Journal of visualized experiments : JoVE doi:10.3791/51337:51337.

I hope this information is helpful for you.

Best wishes, Dirk

Dear Agustin,

We use to analyze our extracellular data using the Clampex (Clampfit) program in which we usually do the patch clamp recordings. It already has most of these features as a built-in features.

Oh your drug then needs to change to not inhibit the response. Have you tried the hypnorm and midazolam cocktail? See this paper and I just emailed you a draft manuscript:

doi:10.1038/s41598-017-11349-z

Try Open Ephys.org for a flex drive core.

There is also a Jove video from Matt Wilson's lab that provides the code to print the 3D core of their drives. 

3D Systems, On Demand Manufacturing can print 25 3D cores for about $1000.  Make sure to use Accura60 for a polymer if you go this route. 

In both cases you still have to perform construction of microdrives of the array and make your own tetrodes out of Nickel-Chrome RO800, Redi Ohm 800 wire.

Your other option is to switch to silicon probes and use a Buzsaki style drive. Turn around on orders of silicon probes from NeuroNexus is pretty fast. The Buzsaki 32 is a nice array.

Hi J. do you mean like an organotypic slice culture?

Excessive pulsation is often the sign of tissue damage, oedema and changes in intracranial pressure and venous flow. It's important to prevent direct damage to the brain, excessive bleeding, or overhearing during drilling. Very small craniotomies can help, if applicable. After these are sorted, a layer of low gelling temperature agarose (4-10%) on the brain surface can be used to further reduce pulsation.

Doing an I/O curve can induce by itself a slight potentiation of fEPSPs and in certain situations can damage afferents at maximal voltage/current levels, especially in the current stimulation mode. The first case causes a wrong estimation of the 60% and might be avoidable by using only few stimulation at greater intervals and the second case, will just lead to smaller fEPSP during baseline stimulation. Thus, avoiding stimulation at the maximum, or just doing it once, might be better. The 60-70% for LTD induction might be too high and can induce damage of afferents and subsequently get a nice “LTD”, thus the fiber volley responses needs to be analyzed and compared with before and after LTD induction, and stimulation voltage could be around 2V. Best. T

We have similar results in CA1, CA3 and amygdala though I only did IO in CA1. Thanks for reminding me about the complexity of these studies. Interesting thought, in my opinion the main effect here is a local anesthetic like pre-synaptic effect that inhibits the fiber volleys, increases the lag time of the response and reduces the postsynaptic response, the legend below summarizes my findings:

Thanks for the information provided.

Best Regards,

Grandhi V Ramalingayya

Hi Fading,

In addition to Michel recommendations, it is in general worth evaluating how much noise is acceptable for what you want to record. Even though it is definitively nicer to have as less noise as possible, you may realize that sometimes your noise level is not such an issue if you are recording large whole cell currents of nA amplitude. Arguably it is more critical when working with small currents or doing single channel recording.

This being said, make sure that everything is properly grounded. You should ground everything (including the Faraday cage) to a single point inside of the cage (you can use either copper wires or even better some mesh), and from there you go to the signal ground of your amplifier using a single wire. Also make sure you do not have cell phone around... Things like the air conditioning, or the compressor (if you use any) to bring air into your air table may induce spikes when turning on. So again make sure everything is properly grounded to minimize all of these external signals. One approach is to to assemble you rig with as less as possible equipment, check the noise, if everything is fine bring back one by one your different elements and see how it affects the noise level. That way you can identify if one particular element is causing this unnecessary noise. With time and method and will make it eventually without using any external noise silencer.

Hope this helps. Good luck!

Please read this nice Cohen and Miles 2000 paper

Also the wave shape of  LFP depends on recording electrode position

Hi Alba,

first thanks to Hugo for recommending our methods. However, please note that all the methods in SPIKY are designed to look for synchrony between two or more spike trains. In case you are mainly interested in properties of individual spike trains (frequency spectrum etc.) please check out this tutorial which includes examples of Matalb source codes:

All the best,

Thomas

Read literature and find the more appropriate one. This is an old procedure used before the discover of patch-clamp technique. In a library you can find many books about the so called "old-electrophysiological techniques".......Forgotten in the mind of younger electrophysiologists.....

Good luck

Hi Rita,

In addition to Alberto's suggestions you can try to halve your external K+ ion concentration to 1.5 to 2 mM.

best wishes, Refik

I was wrong my memory after more than 20 years was not so good. Kruger used them for cortex not for slices. This is a references where probably he describe the technique or refers to some other reference where it is described how electrodes were prepared. Independently on where he used, maybe it is an idea for your homemade preparation :). This is the complete reference :) 

J Neurophysiol. 1988 Aug;60(2):798-828.

Multimicroelectrode investigation of monkey striate cortex: spike train correlations in the infragranular layers.

Krüger J1, Aiple F.

The stimulus connector is simply used for commutation.  That is, one can either record from the electrode connected to the "Input" connector, or send the stimulus from the stimulus isolator connected to the "Stimulus" connector, to the same electrode.  The toggle switch "Mode" allows the commutation. You do need to use stimulus isolator (or stimulator), since AM 1700 does not have any stimulating circuit.  The positive and negative outputs of the stimulus isolator are connected to pins A and B of the "Stimulus" connector; in the stimulation mode, these outputs will be connected to the pins A and B of the "Input" connector and, therefore, to the electrode; the amplifying circuitry will be disconnected from the electrode and grounded.

Hi,

i'm doing multiple single-unit with tetrodes during auditory fear conditioning in rats. i can identify the following potential issues:

1/ The artefact from the shock will saturate your amplifier and you won't have a stable signal for some time (hundreds of ms). you will be 'blind' for that period of time.

2/Make sure the shock return path is properly isolated  from the amplifier ground.  

Cheers

François

Ramki,

The electrical environments on the exterior and the interior of an axon bathed in a volume conductor are quite different. The interior of an axon is a quasi-closed electrical system. An intracellular electrode measures true transmembrane potential at all times. A resting axonal membrane potential is dominated by the potassium ion battery, as the membrane is far more permeable to potassium ions than it is to sodium ions; on the other hand, the driving force for sodium ions across the resting membrane is large, and so even the very low permeability of the membrane to sodium ions at rest somewhat compromises the potassium ion battery; hence, the resting membrane potential is a hybrid of about -70 millivolts instead of -80 millivolts (E_K⁺). As an action potential approaches an intracellular electrode along an axon, the longitudinal currents flowing ahead of the AP discharge the membrane capacitance, causing a depolarization that begins to rapidly change the sodium ion conductance to a high state; at this point the membrane potential is dominated by the sodium ion battery, close to +45 millivolts (E_Na⁺), and this is the value at the peak of the AP, characterized by sodium ion influx. The greatly depolarized condition of the axonal membrane now inactivates the sodium ion conductance while at the same time greatly increasing the membrane conductance to  potassium ions, a period characterized by repolarization of the axonal membrane during potassium ion efflux. During this phase of the AP the membrane potential is even more dominated by the potassium ion battery, and the membrane potential in the vicinity of the intracellular electrode attains the value of about -80 millivolts, the hyperpolarized afterpotential of the AP, before the potassium conductance is reduced to resting values and the membrane potential settles back to -70 millivolts.

An extracellular recording electrode positioned on an axon in a volume conductor sees a very different electrical environment. With respect to a distant, 'indifferent' electrode in the medium, an active electrode adjacent to the axon and that is well insulated from the medium will record a fraction of the currents leaving and entering the membrane at that recording point as the AP passes. Initially, as an AP approaches the recording point, the transmembrane  currents are outward, the "local circuits" discoverd by Alan Hodgkin, and the electrode records a positive potential with respect to the distant indifferent electrode. As the AP passes under the active electrode the transmembrane current rapidly changes from outward to inward (due to local sodium ion influx) and the active electrode registers a large negative shift in potential with respect to the indifferent electrode. Finally, as the AP leaves the recording vicinity, traces of positive transmembrane current (from the receding sodium influx) are recorded by the active electrode. This sequence of current "source-sink-source" characterizing the membrane is always recorded when an AP approaches and then passes a fixed point on the axon.

In addition to the previous comments, to get simultaneous field and patch clamp recordings, you're going to have to set up a separate headstage for each recording pipette and configure the appropriate signals for each recording mode. 

Set the channel for your field headstage in I=0 to record fEPSPs, and your usual Vclamp settings for your whole cell recordings.

Thank you for your answer.

Are these concentrations valid for slice preparation or neuronal culture (dissociated primary neurons)?

Thanks a lot.

Hello Muthu!

Noise is noise, spread across wide frequency bandwidth. Grounding alone will not help. It needs to be PROPERLY GROUNDED and in addition to that PROPER SHIELDING is NECESSARY. I can help you in sorting out this issue if you are willing to share your experimental setup. Also note that you do not have control on the field conditions. If it is a NOISY environment, you need to take care in your electronics or measurement setup only. And if the noise still persists, you might even need to add some filtering also.

Looking forward to hear from you soon!

Best luck!

Between slice runs can vary more than 5%, due to dissection health, vibratome slicing protocol and slice number within sequence (discard first few ans last few), temperature of Acsf, age of the pefrusate etc... within a single slice once recording fEPSp measures started should vary less than 5% and baselines should be carried out for minimum of 30minutes prior to tetanization. Also need to be careful when running baseline that the number of sweeps recorded per minute does not exceed 6 (1/10s; and 4/min or 1/15s is preferable), as there is a little known phenomena known as low frequency potentiation, which can appear based upon other variables ie bath temperature Acsf composition etc... Can check out figures and details from Cliff Abraham's lab. Paper listed below can be downloaded from my research gate or my website "Eric L Hargreaves PageONeuroplasticity". Other papers from that lab around that era, should also be very consistent on methods and recordings etc. Particularly slope measurement and duration of slope measurement segment and whether segment is closer to inital roll off or slid closer to the peak. most canned packages should have rules of thumb for measurements, or reference a number of papers that utilized the system and its measurments Its pretty amazing what can be done with the same sweeps in different hands and thats why its important to document document document.

A DOUBLE DISSOCIATION WITHIN THE HIPPOCAMPUS OF DOPAMINE D1/D5 RECEPTOR AND b-ADRENERGIC RECEPTOR CONTRIBUTIONS TO THE PERSISTENCE OF LONG-TERM POTENTIATION. 

J. L. SWANSON-PARK,*† C. M. COUSSENS,† S. E. MASON-PARKER,† C. R. RAYMOND,†E. L. HARGREAVES,† M. DRAGUNOW,‡ A. S. COHEN†§ and W. C. ABRAHAM†  Neuroscience,  1999, Vol92, p485.

Can also checkout fEPSP as % of max I/O value and its impact upon degree of potentiation. Where you are on the I/O curve also impacts the variability of measures. the closer you are to max the less variable are the measures. The trade off of course the closer you are to max the less LTP you will observe (which is more complex than just playing with percentages versus absolute differences). Can check out Ambrose Au's thesis in Stan Leung's lab. Brain Research Bulletin in 1994 (when BRB was a reputable journal).

Long-term potentiation as a function of test pulse intensity: A study using input/output profilesOriginal Research Article

Brain research Bulletin, 1994 Vol33, Pages 453-460

L.Stan Leung, Ambrose S. Au

Thomas, that would be messed up. The stimulation parameters (usually 100 us pulse duration, at whatever voltage or current you set) during the baseline have to be the same AFTER the induction protocol. That is the only way to compare the same number of fibers activated before the induction with after the induction.

Make sure this is the case. Some people increase duration during LTP induction but it is imperative to bring it back to pre induction levels for the 3 hour post induction recording protocol.

Hello Anna, 

I don't know if you have solved your problems of stability. Just let me give you my contribution:

- I would let the slice rest at room temperature at least during one and half hour. Then, as you do, I let the slice rest in the recording chamber for another 30 minutes.

- I also had trouble with running down baselines. For me, it was very helpful to inmerse the electrodes and also let them "rest" during these 30 minutes with the slice (but not touching the slice, the electrodes just in the liquid). My rational to do so is some leakage of ACSF (or NaCl) form the pipette that may occur on the early minutes after placing the electrode.

- As others have mentioned, I have found critical the oxygenation of the ACSF. It is important to consider the death space you have in your setup. Try to use tubes as short as possible from the source of oxygenated ACSF to the recording chamber. Maybe you can increase the flow rate to 2.5 ml/min or even higher. Try always to keep the ACSF well bubbled with carbogen, until saturation. Slice health is highly dependent on good oxygenation.

- Also, don't forget to adjust your pH properly in a range between 7.3 - 7.4. For this purpose, don't forget that because of its content of CO2 (normally 5%) carbogen works as a buffer itself and it will modify the pH. So I recommend you to adjust the ACSF pH while bubbling it and saturating it with carbogen for at least 15 minutes.

- You can try high Magnesium ACSF for the resting period of the slices.

Hope this helps!

Best regards,

Diego

An important consideration here is whether you will be stimulating ChR2-positive presynaptic axons/neurons and measuring post-synaptic spiking, or whether you are measuring spikes in ChR2-positive neurons that are directly stimulated by light.

I think a before-and-after optical f-I curve as described above may work (and sounds neat), but I would caution against any comparison of f-I curves obtained via somatic current injection vs those obtained by optogenetic activation.

Another consideration: how will you modulate light? By duration? Or by intensity? 

Dear Shiva,

there are a couple of paper out describing manual voltage and current clamp recording from pluripotent stem cell-derived cardiomyocytes including the following:

Am J Physiol Heart Circ Physiol. 2011 Nov;301(5):H2006-17. doi: 10.1152/ajpheart.00694.2011. Epub 2011 Sep 2.

High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.

Ma J1, Guo L, Fiene SJ, Anson BD, Thomson JA, Kamp TJ, Kolaja KL, Swanson BJ, January CT.

Kind regards,

Ralf

How long do you incubate the slices before the recordings? Try to increase the incubation time, the metabolism will stabilize. Some people incubate the slices for 3-4 hours for the same reason for the late LTP recordings. Also, I would record the baseline for much longer time than 10 min. We usually record 30-60 min of the baseline. In some slices it is simply impossible to get the stable responses. How good is your perfusion and oxygenation? What chamber your are using, submerged or the interface? I have noticed that the responses are much stable in interface chambers. Also, try to reduce the tissue polarization as much as you can - use platinum or platinum/iridium electrodes, and try biphasic stimulation instead of the monophasic one. The stimulator could be tested by measuring the stability of the voltage (with the digital voltmeter or the scope) on the resistor connected to the stimulator output instead of the electrode.

Sometimes spreading depression occurs due to the

hypoxic condition especially in high potassium seizure model. It may be avoided by preparing slices with continuous provision of enough oxygen. You must also provide contin. oxygen at the time of recording in interface chamber, increase the oxygen level if you are already providing during both procedure. When you transfer the slices into the interface chamber, wait for at least one hour before recording.

it may also depend on the quality of slices, try to be as fast as possible.

You can also reduce the concentration of both KCL and MgSO4. I guess you already know that high K is a standard model for SD. Therefore reducing KCl may help. In compensation you can reduce the conc. of MgSO4 which increase the excitability.

I'm not certain if I am answering your question correctly, but I believe the answer you are looking for involves neurons' sodium potassium pump.

There are proteins in the cell membrane that use ATP to move sodium out and potassium in. This active transport does use energy (ATP) to help 'recharge' the cell. Because the net charge of the sodium (Na+) moving out of the cell during this process is greater than the net charge of the potassium (k+) moving into the cell, the inside of the cell becomes more negative than the extra-cellular space. This potential difference is akin to a battery and allows for subsequent action potentials.